Phospholipid trafficking and membrane biogenesis

Bioch

et Biophysica A~ta

ELSEVIER

Biochimica et Biophysica Acta 1197 (1994) 257-290

Phospholipid trafficking and membrane biogenesis P. Moreau, C. Cassagne * URA 1811 CNRS, IBGC, University of Bordeaux II, 1 rue Camille St-Sa6ns, 33077 Bordeaux-cedex, France Received 11 March 1994

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2. Phospholipid-binding proteins (PLBPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cellular localisation of PLBPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Roles of PLBPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. PLBP-mediated transfer of phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. The yeast P I / P C - b i n d i n g protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3. Emerging roles of PLBPs in cell biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

259 259 259 260 260 260 261

3. Phospholipid transfer to mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Mitochondrial lipid biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Phospholipid import into mitochondrial m e m b r a n e s . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. The PS-PE system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. PS synthesis and export originates in a specialized domain of the E R closely associated with mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. PE formation in the inner m e m b r a n e of the mitochondria . . . . . . . . . . . . . . . . . . . 3.3.3. The PS to be exported and decarboxylated is compartmentalized . . . . . . . . . . . . . . . 3.4. Pending questions: the early and late steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Early steps and the A T P requirement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Late steps and the effect on the PS transfer of drugs affecting protein import into mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

262 262 262 263

4. Vesicular transfer of phospholipids to the plasma m e m b r a n e . . . . . . . . . . . . . . . . . . . . . . . 4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Phospholipid composition of the m e m b r a n e s of the exocytic pathway . . . . . . . . . . . . . . . . 4.3. Vesicular transport of phospholipids to the plasma m e m b r a n e . . . . . . . . . . . . . . . . . . . . 4.3.1. Fluorescent lipid analogs as markers of lipid transport . . . . . . . . . . . . . . . . . . . . . 4.3.2. Half-times and mechanisms of transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Potential existence of fast vesicular processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1. Existence of multiple vesicular carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2. Experimental data supporting fast vesicular processes . . . . . . . . . . . . . . . . . . . . . 4.4.3. Vesicular transfer and the cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

266 266 267 268 269 269 270 270 270 271

263 264 264 265 265 266 266

Abbreviations: C6-NBD, N-6(7-nitro-2,1,3-benzoxadiazole-4-yl)aminohexanoyl; Cer, ceramide; DG, diacylglycerol; GPI, glycosylphosphatidylinositol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PLBP, phospholipidbinding protein; PS, phosphatidylserine; SM, sphingomyelin. * Corresponding author. Fax: + 33 56 966267. 0304-4157/94/$26.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 3 0 4 - 4 1 5 7 ( 9 4 ) 0 0 0 0 9 - 3

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t{ Moreau, ( . (assagne / Biochimica et Biophysica A c t a 1197 (1994) 2 5 7 - 2 9 0 4.4.4. The effect of t e m p e r a t u r e on vesicular transfer . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.5. E n e r g y r e q u i r e m e n t of vesicular t r a n s f e r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. O t h e r pathways to the p l a s m a m e m b r a n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

27 I 271 272

5. V e s i c u l a r endocytic and r e t r o g r a d e routes and the recycling of p h o s p h o l i p i d s . . . . . . . . . . . . . . 5.1. The i n t e r n a l i z a t i o n of p h o s p h o l i p i d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Recycling of p h o s p h o l i p i d s b e t w e e n the G o l g i a p p a r a t u s a n d the E R . . . . . . . . . . . . . . . . 5.3. Exocytic and endocytic c o u n t e r b a l a n c e of p h o s p h o l i p i d flow . . . . . . . . . . . . . . . . . . . . .

273 273 274 275

6. Lipid m e t a b o l i s m , lipid m o v e m e n t a n d v e s i c u l a r trafficking . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Lipid m e t a b o l i s m and vesicle b u d d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. A T P - d e p e n d e n c y of lipid t r a n s f e r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Low t e m p e r a t u r e s and v e s i c u l a r trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. M e m b r a n e lipid d o m a i n s and the sorting of lipids . . . . . . . . . . . . . . . . . . . . . . . . . . .

275 275 277 279 28()

7. P h o s p h o l i p i d s and biological m e m b r a n e fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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8. C o n c l u s i o n and p e r s p e c t i v e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

284

Acknowledgements .................................................

285

References ......................................................

285

1. Introduction The metabolic pathways and cellular sites of phospholipid biosynthesis have been largely described in the literature [1-3] and the endoplasmic reticulum, the Golgi apparatus and the mitochondria can be considered as the major sites of these events. However, some uncertainties still persist. For example, the cytoplasmic or lumenal localization of the base-exchange synthesis of PS in the endoplasmic reticulum is still being debated ([4], see chapter 3). More intriguing is the recent description of the soluble CTP: phosphocholine cytidyltransferase as an intranuclear protein [5]. The various subcellular membranes can be labeled to different extents in vivo with phospholipids synthesized via distinct biosynthetic pathways [6]. Consequently, it is likely that phospholipid assembly into the various subcellular membranes implicates not only different biosynthetic routes, but also multiple transfer pathways or mechanisms. As pointed out by Van Meet [7], Voelker [8] and Allan and Kallen [4], there are numerous difficulties in studying lipid transfer pathways. These include the existence of a large number of molecular species of all the phospholipids, the existence of two leaflets and numerous domains in biological membranes, the relatively broad distribution of lipid-remodeling activities, such as deacylation-reacylation and base-exchange processes, and the universal problem of the evaluation of the purity of subcellular membrane fractions and, consequently, the relevance of their reported phospholipid compositions, Of particular importance when studying the intracellular transport of phospholipids to the plasma membrane, for example, is the possible existence of phos-

pholipid base-exchange enzymes [9] and deacylationreacylation activities [10] in this membrane. The turnover of phospholipid acyt groups in different cellular membranes can be relatively important [11,12]. For example, 10-15% of hepatocyte phospholipids can be remodeled by deacylation-reacylation each hour [12]. Moreover, remodeling activities will not necessarily concern all the phospholipids to the same extent [13] and, for a given phospholipid, they can vary from one molecular species to another [14]. Hence, one has to keep in mind that experimental data concerning lipid transport will often, if not always, reflect the sum of a rather large number of cellular and membranous events. As depicted by Sleight [15], various mechanisms are likely to be involved in the intermembrane movement of phospholipids. In this context, spontaneous diffusion of monomers through the aqueous phase, or by membrane contact or collision, as well as facilitated movements mediated by phospholipid transfer or exchange proteins and vesicular transport, have to be considered. The spontaneous transfer of lipids between various donor and acceptor membrane vesicles has been reviewed recently [16] and will not be detailed here. Spontaneous transfer was considered not to be important in membrane biogenesis, chiefly on the basis of kinetic data. However, recent reports indicate that the spontaneous transfer rate of long chain diacyl PC molecules between phospholipid vesicles can be significantly increased by high concentrations of vesicles ([16] and references therein), or by a modification of the environment such as the addition of PE [17]. From a quantitative point of view, the participation of the spontaneous transfer of phospholipids in membrane

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biogenesis is probably not as important as other mechanisms (vesicular transfer, for example), but it will certainly have to be taken into account [16]. The other mechanisms and pathways believed to be implicated in phospholipid transport will be discussed in the light of many recent investigations. Special emphasis will be given to the intracellular vesicular pathways of phospholipid trafficking and to the potential roles that phospholipids themselves could play in these membranous events. In other words, we will discuss whether (and how) phospholipids are transported through vesicular pathways and how they could be involved in the molecular functioning of these pathways.

2. Phospholipid-binding proteins (PLBPs) 2.1. Introduction

Since the discovery by Wirtz and Zilversmit [18] of the first protein capable of exchanging phospholipids between liver mitochondria and microsomes in vitro, a great number of phospholipid-binding proteins have been isolated and characterized from many tissues in animal cells [19], plant cells [20] and micro-organisms [21]. All of them share in common the property of binding phospholipids and under certain experimental conditions of exchanging them. In animal cells, these proteins have been classed into three groups [22]. The first group is constituted by phospholipid-binding proteins highly specific for phosphatidylcholine (PCBPs). The members of the second group, characterized by a specificity for phosphatidylinositol (PIBPs), also transfer phosphatidylcholine, but to a lesser extent. The proteins of the third group, referred to as the nonspecific lipid binding proteins (nsLBPs), do not exhibit any specificity and are able to bind the major phospholipids and sterols. In Saccharomyces cereuisiae, three kinds of phospholipid transfer proteins have also been characterized. One has a greater specificity for PI than for PC and presents functional characteristics similar to the animal PIBPs [21]. Another class shows a higher specificity for PC, but will also transfer the other amino-phospholipids and PI [23]. Finally, the yeast PS-binding proteins [24] are rather similar to the nsLBPs identified in animal cells, but they lack the ability to transfer PI and PC. In plant cells, most lipid-binding proteins have a specificity for phospholipids similar to that observed for bovine nsLBPs, although a certain specificity for PC has been reported [20]. The in vitro properties of PLBPs and the PLBPmediated transfer of phospholipids have been reviewed many times [19-22], and it is not the aim of this review

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to focus on the innumerable in vitro experiments that have clearly established the efficiency of PLBPs to transfer lipid molecules. Instead, we will concentrate our attention on recent data concerning their localisations which have led to a rediscussion of their potential roles.

2.2. Cellular localisation o f PLBPs

For a long time, PLBPs have been considered as being exclusively cytosolic, because they were found in the 'soluble' fractions, but they could also be released from membrane compartments during the homogenisation of tissues and membrane isolation procedures [25]. In situ, PIBP has been found to be associated with Golgi membranes [26,27], but has also been detected in the cytoplasm of swiss mouse 3T3 fibroblast cells [27]. Whereas ns-LBPs have been found in the peroxisomal matrix, mitochondria and endoplasmic reticulum (25 and references herein), they were also detected in the cytoplasm of animal cells [25]. A maize PLBP has been localised, by immunocytochemistry, in the cytosol and its expression was organ-, time- and cell-specific [28]. The possibility that PLBPs could be transiently inserted into membranes, and could even have an extracellular location, was recently considered when it was shown that many PLBPs have leader sequences and are first synthesized as precursors. The nsLBP (sterolcarrier protein 2) is formed with a leader sequence of 20 amino acids and contains the C-terminal peroxisomal targeting tripeptide sequence Ala-Lys-Leu [25]; the nsLBP pre-sequence could be a targeting signal for the mitochondria [29], implying that nsLBP could contain targeting signals for both peroxisomes and mitochondria [25]. Numerous investigations have been devoted to the localization of nsLBPs to the peroxisomes and their hypothetical functions in a recent review by Ossendorp and Wirtz [25]. In plant ceils, a cDNA clone encoding a nsLBP from spinach was isolated and it was determined that a 26-amino acid sequence, presenting all the characteristics of a signal sequence, constituted the first amino acids of the open reading frame and were not present in the mature protein [30]. This protein was co-translationally inserted into the endoplasmic reticulum. Similarly, a lipid transfer protein from the aleurone grains of Barley contained a signal sequence and the protein was co-translationally and post-translationally transported across the membrane of the endoplasmic reticulum [31]. The first indication of an extracellular location of PLBPs came from work on carrot cells in culture, in which an extracellular protein (named EP2) was identified as a 'classical' lipid transfer protein [32]. More recently, a PLBP-protein A fusion protein was expressed in Arabidopsis and was localised, by im-

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muno-electron microscopy, in the cell wall and, particularly, in epidermal cells [33].

2.3. Roles of PLBPs 2.3.1. PLBP-mediated transfer ofphospholipids In vitro experiments have clearly established the efficiency of PLBPs to transfer lipid molecules. A PCBP, transferring PC from liposomes to the cytosolic leaflet of the outer envelope membrane of chloroplasts [34], was capable of using microsomes as donors [ 3 5 ] . In this case, PC transfer still existed, but was less efficient and required large amounts of PLBP [35]. PLBPs also have the ability to interact with other lipid molecules. For example, plant PLBPs may bind fatty acids and fatty acyl-CoAs [36], or even transfer some galactolipids [37]. In plant cells, the broad specificity of most PLBPs [36,38,39] has supported the proposal that these bifunctional proteins could be involved in the cooperation between the chloroplast and the ER in the biosynthesis of galactolipids [38,40]. However, it has been recently demonstrated that an oxidizing environment is required for an optimal function of maize PLBP [41], which is not in agreement with an active cytosolic PLBP in vivo maintained in the reduced environment of the cytosoi [42]. Several isoforms of PLBPs have, however, been found in various plants and these isoforms could have different cellular localisations and functions [41]. In rat liver, a nsLBP also exhibits the properties of the sterol carrier protein 2 [25]. These properties raise the question as to how to correlate a broad specificity in vitro with lipid traffic in vivo, even if nsLBPs have been proposed to transfer PE from intracellular membranes to the plasma membrane in vivo [43], or with the fact that mitotic cells, in which the vesicular transport of proteins is blocked, transport PE with the same efficiency as interphase cells [44]. However, lipid transfer activity in vitro is sometimes at variance with the observed transport kinetics of newly synthesized phospholipids in vivo [45]. This point could be less disturbing for the more specific lipid transfer proteins such as the PIBPs and PCBPs. Borror and Helmkamp [46] have reconstituted a PIBP-dependent transfer of PI from the endoplasmic reticulum to plasma membrane sheets with the correct orientation (i.e., cytoplasmic side exposed) isolated from rat liver. Although the conditions employed for these assays are closer to those encountered in the in vivo situation than those using liposomes, one can still wonder whether the in vitro property to transfer phospholipids between native cellular membranes reflects the function of the PLBP in vivo. The use of the yeast PIBP clearly showed that this protein cannot be responsible for the in vivo translocation of PI and PC from internal membranes to the plasma membrane

[47]. In addition, no correlation could be determined between the activity of a yeast PS-binding protein and the extent of membrane biogenesis during cell growth [48]. Finally, Sleight and Hopper [49] performed an assay to investigate a phospholipid transfer activity in vivo by using a phospholipid analog (N-lissamine rhodamine b sulfonyl-di CI6-PE, abbreviated as N-rh-PE) that has a lower rate of spontaneous diffusion than the classical acylchain-labeled fluorescent NBD analogs. The PE analog was inserted into liposomes, which were then injected into chinese hamster ovary (CHO)cells. The in vivo redistribution of this lipid analog into the nuclear envelope and mitochondria was in agreement with the intervention of a PLBP-facilitated PE movement in vivo. It is interesting that the redistribution of this PE analog was similar to that of NBD analogs [50]. The fact that non-diffusible (N-rh-PE) and diffusible (NBD) phospholipid analogs exhibited a similar redistribution can indicate that, to some extent, a control of this redistribution was achieved and that it could have been mediated by a PLBP.

2.3.2. The yeast PI/PC-bindingprotein The Saccharomyces cereuisiae SEC 14 gene product (SEC 14p) is a P I / P C binding protein whose function is essential for both the vesicular transport of proteins from the Golgi apparatus [51] and the cell viability [52]. The pioneering work of Bankaitis et al. [53] has established that SEC 14p is a P I / P C exchange protein (PIBP). Moreover, mutations in seven genes bypass the SEC 14p mutation and, among these, four concern the CDP-choline pathway for PC biosynthesis [26,54]. These results have led to consider the P I / P C ratio as critical for the physiological secretory function of the Golgi apparatus, perhaps in the budding of Golgi-derived secretory vesicles [55]. Recently, rat PIBP was expressed in yeast strains lacking their own functional PIBP (SEC 14p). This approach, based on the properties of rat and yeast PIBPs, which have similar catalytic activities, but distinct primary sequences, was used to investigate the putative role of the transfer activity of PIBP per se in the Golgi apparatus secretory function [56]. The expression of the rat PIBP complemented the yeast growth and Golgi secretory defects associated with the dysfunction of SEC 14p in temperature sensitive sec 14 t~ mutants but failed to rescue sec 14 null mutations [56]. These findings reveal that the in vitro phospholipid transfer activity is somehow relevant to the function of the protein in vivo, but that it is certainly not its unique activity [56]. Another interesting finding was that the localisation of rat PIBP in the Golgi apparatus was less stable than that of SEC 14p, which suggests that Golgi-binding is not related to the catalytic activity of the proteins [56]. An important question that remains unanswered is the relationship

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between SEC14p and the synthesis of PC via the CDP-choline pathway, It was proposed that the P I / P C ratio is crucial for the secretory function of the yeast Golgi apparatus and that high amounts of PC disrupt the normal behaviour of the Golgi apparatus. The P I / P C transfer protein would therefore have to specifically eliminate PC from the bilayer in order to bring the P I / P C ratio to its correct level. However, this theory fails in that PI, and not PC, is the best substrate in vitro [57]. Recent findings by Mc Gee et al. [58] propose that SEC 14p acts as a repressor of the CDP-choline pathway in yeast Golgi membranes. PI- and PC-bound forms of SEC 14p could serve as specific allosteric sensors of PC synthesis in the Golgi apparatus [58]. This model has the merit of providing an explanation for the suppression of SEC 14p dysfunction by the inactivation of the CDP-choline pathway while leaving the PE methylation pathway apparently intact. However, it is not known why the Golgi secretory function exhibits such a differential sensitivity to PC synthesis. The relevance of the yeast model to the situation in other eukaryotic cells, and particularly in mammalian cells, is open to discussion. The mammalian PIBP has been found to associate with the Golgi apparatus of rodent fibroblasts [27]. In plant cells, PLBPs can also be associated with microsomal membranes [59]. Recently, a mammalian PIBP has been shown to correspond to PEP3, a 'priming in exocytosis protein' [60]. The authors also determined that the yeast PIBP can substitute for PEP 3 in the priming reactions involved in the regulated secretion [60]. A PI/PC-binding protein homologous to the SEC14p has also been identified in the dimorphic yeast Yarrowia lipolytica [61], where it associates with cytoplasmic structures thought to be Golgi bodies. However, this protein was not essential for cell growth, nor for the secretory pathway, but it was found to be required for any event associated with the yeast-mycelia transition of Yarrowia lipolytica [61]. Hence, it is probable that distinct potential functions are associated with PI/PC-binding proteins in different cells. 2.3.3. Emerging roles o f PLBPs in cell biology

Besides the potential role of PLBPs in lipid transfer and/or secretion, other possibilities are beginning to be explored, emphasizing new intracellular and extracellular functions of these proteins. Madrid and von Wettstein [42] have proposed that some PLTPs with a signal sequence could stay in the lumen of the ER to serve as lipid translocators (in membrane domain formation?) and that lipid translocation to the cytosolic leaflet would be due to PLBPs similar to the yeast PIBP. The secreted plant PLBPs have been suggested to contribute to cutin formation [32]. Molina et al. [ 6 2 ] proposed a role of defense for nsLBPs in plant cells

26l

following the discovery of two proteins that inhibit growth of bacterial pathogens and that present some homology with previously described nsLBPs from plant cells. This role would be in agreement with the localisation of some nsLBPs in the epidermal cells of aerial parts of plants [28,32] and with the finding that a nsLBP gene could be induced by abiotic stress [63]. Antipathogenicity has recently been shown for PLBPs of Arabidopsis and spinach leaves [64]. In animal cells, the rat liver fatty acid-binding protein (FABP) also binds lyso-PC [65] and lyso-PA [66], supporting the hypothesis that this protein participates in the lipid metabolism of mitochondrial and microsomal membranes. It has also been observed that a PC transfer protein from rat liver could be involved in the regulation of PC metabolism in mammalian tissues [67]. The expression of a bovine synthetic acyl-CoAbinding protein gene in yeast [68] has led to the proposal that its role would be multiple; regulation of acyl-CoA pools, protection of membranes and enzymes against free acyl-CoAs, protection of acyl-CoA esters against hydrolysis by cytosolic and membrane-associated hydrolases, delivery of acyl-moieties for protein acylation [68]. Cells involved in secretion and other transport processes are rich in such proteins [69] and acyl-CoAs have been shown to stimulate the budding and fusion of transport vesicles at the Golgi apparatus [70,71]. In heart tissue, FABPs could transport acyl-Lcarnitine and protect against the toxic effects of high levels of fatty acid derivatives that arise during ischemia [72]. In view of their homology with growth inhibitors, such proteins could also be growth regulators [73]. The nsLBPs from animal cells have been proposed to be involved in steroidogenesis and /3-oxydation in mitochondria, but contradictory information has been presented [25]. The physiological function of nsLTPs remains unclear. On another hand, important findings have been recently obtained on the physiological role of the microsomal triacylglycerol transfer protein (MTP) from liver [74]. It has been determined that a defect in the gene for the large subunit of MTP is the cause of abetalipoproteinaemia, and that MTP is involved in the secretion of apolipoprotein B-containing plasma lipoproteins [74]. Finally, the PIBP is supposed to regulate the level and the intracellular distribution of PI molecules [53,75], and could also participate in PI metabolism [46]; this potentially connects the PIBP to the regulation of signalling pathways involving PI derivatives. It has been recently found that PIBP could be essential for phospholipase C-mediated signalling [76]. These results provide the first evidence for the involvement of a PIBP in cellular signalling. It has also been recently proposed that PIBP distribution could be regulated by the phosphorylation of the protein [77].

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Hence, it appears that PLBPs, nsLBPs and FABPs are important molecules issued from a large family of proteins with a wide range of fundamental functions, and one can propose that the identification of a specific role for one of these proteins will be the consequence of a specific location and environment of this protein that is carefully controlled by the cell. The putative functions of these proteins also appear to vary considerably between animal, yeast and plant cells,

3. Phospholipid transfer to mitoehrondria

3.1. Mitochondrial lipid biosynthesis So far, most of the mitochondria that have been purified and studied exhibit a limited capability for synthesizing the phospholipids of their inner and outer membranes. Mitochondria are able to carry out the de novo synthesis of cardiolipin, which is more or less restricted to this organelle [78]. They also possess an active phosphatidylserine decarboxylase capable of forming p h o s p h a t i d y l e t h a n o l a m i n e from phosphatidylserine [79]. The biosynthesis of phosphatidylglycerol in submitochondrial membranes may take place within mitochondria and relies absolutely on the delivery of CDP-diglyceride from microsomal membranes [80]. A consequence of this reduced autonomy of the mitochondria with regard to the biosynthesis of phospholipids, is the necessity of a massive import of lipids: PC, PS, and PI have to be transferred from their site of biosynthesis (chiefly, if not uniquely, the endoplasmic retieulum) to the mitochondrial membranes,

3.2. Phospholipid import into mitochondrial membranes The vesicle-mediated transfer of proteins within the endomembrane system has been established. The prorein traffic from the endoplasmic reticulum to the Golgi apparatus, the lysosomes and the plasma merebranes, as well as that involved in protein secretion, stems from the synthesis of proteins on ribosomes attached to the endoplasmic reticulum, and their subsequent processing and targeting through the membrane flow [81]. Though these mechanisms are still under active investigation, their existence has already been demonstrated and it is of no surprise that these events require a massive cotransport of lipids, not only to accompany the proteins in the vesicular structure, but also to meet the lipid-specific requirements for the correct functional activities of the various membranes. In good agreement, Wieland et al. [82] estimated that, every 10 min, roughly 50% of the E R phospholipids are exported through the membrane flow. It is therefore not unexpected that evidence has accumulated

concerning vesicle-mediated lipid transfer among endomembranes. The situation is completely different for mitochondria: the proteins, synthesized outside the mitochondria, in the cytosol, are formed on free ribosomes and no evidence of any vesicular transport into the mitochondria has been presented so far. This observation, of course, does not rule out the possibility that lipids are transferred from the E R to mitochondria via vesicular transport, but it does imply that, if this pathway were operative, the vesicles would most probably be devoid of proteins. Besides the vesicular transfer, other mechanisms have been proposed to account for phospholipid iraport into mitochondria. These include: (a) the use of phospholipid-exchange or transfer proreins; (b) the involvement of a collision-based mechanism involving the E R - or a specialized domain of the ER - and the mitochondria, or specialized domains of the mitochondrial membranes; (c) the transient fusion between donor (ER) and acceptor (mitochondrial) membranes, or between mitochondrial outer and inner membranes. Although the transfer of PC to mitochondria is of major interest, it has not yet been analyzed to any large extent. The transfer of phospholipids from microsomes to the mitochondrial inner membrane (IM) of rat hepatocytes was investigated by pulse-labeling in vivo. Microsomal PC and PE were rapidly labeled (30 min after [3H]glycerol injection), whereas the maximal incorporation into the IM occurred only after 5 h [83]. Though no data on the labeling of the outer mitochondrial membrane (OM) was presented [83], this process was so remarkably slow that it questioned, in that case, the involvement of phospholipid transfer proteins in vivo [84,85]. In another approach [45], the rates of translocation in vivo of newly synthesized PC and the analogue phosphatidyt-PDME (N-propyl-N,N-dimethylethanolamine-(2-hydroxyethyl)dimethylpropylammonium hydroxide) to the mitochondria of rat hepatocytes and baby hamster kidney cells were compared to the in vitro translocation activity of the purified corresponding PLBPs. It was observed that, in vitro, the partially purified PC binding protein isolated from baby hamster kidney cells transferred PC but not at all phosphatidyl-PDME, whereas both molecules were efficiently translocated in vivo. Therefore, it can be postulated that PLBPs are not the sole mechanism responsible for the translocation of phospholipids to the mitochondria [45]. As PC is largely represented in both the OM and the IM, the question of its transfer from the OM to the IM has been investigated. Using a number of fluorescent, or radiolabeled PC analogues added to the OM of highly purified rat liver mitochondria, Nicolay et al.

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[86] demonstrated that PC remained exclusively localized in the OM and suggested that additional extramitochondrial factors were required to initiate the transfer of PC to the IM.

3.3. The PS-PE system Phospholipids have to be - and are - transported to mitochondria, but the mechanisms by which they are transferred are just beginning to be understood. A great improvement of the basic knowledge in this field has resulted in the past few years from the development of the PS-PE model. In most cases, the phosphatidylserine synthase is microsomal, whereas the PS decarboxylase is a mitochondrial enzyme. Thus, as pointed out by Voelker [87], the metabolism of PS to PE is a discrete chemical event that demonstrates that a translocation has occurred from the ER to mitochondria, without requiring any further purification of the subcellular fraction, whereas in the case of PC, mitochondria and endoplasmic reticulum had to be physically separated and purified after the transfer experiments in order to establish that a phospholipid transfer had occurred, This elegant method has recently allowed important breakthroughs and this part of the review will focus now on the major points that have been elucidated,

3.3.1. PS synthesis and export originates in a specialized domain of the ER closely associated with mitochondria (a) Using Guinea pig liver mitochondria, StuhneSekalec and Stanacev [88] concluded that the spontaneous transfer of phospholipids from microsomal to mitochondrial membranes needed a close contact between these membranes, excluding a mechanism of phospholipid transfer by release into the aqueous medium. It was postulated that a short-lived complex was established between the donor and acceptor merebranes, thus allowing the lipid transfer, (b) In yeast, Zinser et al. [89] demonstrated that PS synthase is located in a microsomal fraction distinct from heavier microsomes. This major point was established by using a combination of differential and density gradient centrifugations. The fraction responsible for PS synthesis was devoid of other phospholipidsynthesizing activity and of the classical microsomal marker proteins, (c) Recent evidence indicates that there is a subfraction of the endoplasmic reticulum that is specifically associated with rat liver mitochondria [90]. This fraction, which was obtained following the fractionation of a crude preparation of rat liver mitochondria, had many similarities with microsomes, but exhibited higher PS synthase and glucose-6-phosphate phosphatase specific activities than those observed in the microsomes; the specific activities of CTP:phos-

263

phocholine cytidyltransferase and NADPH:cytochrome c reductase were much lower than in microsomes, and there was little, if any, contamination by mitochondria, peroxisomes, lysosomes, Golgi apparatus, or plasma membrane. This ER subfraction bears some resemblance to a previously observed domain of endoplasmic reticulum closely associated with mitochondria from rat liver [91], which had also been visualized by electron microscopy [92]. The combination of these microsome-derived membranes with the mitochondria could constitute a functional unit capable of synthesizing PS, PE and PC from labeled serine via specific interactions between a specialized ER domain and the mitochondria. In this scheme [90], PS would be synthesized in the mitochondria-associated ER domain, transferred to the mitochondria, where it would be decarboxylated, and the nascent PE could either return to the adjacent ER, where it could be transformed into PC by successive methylations, or be methylated there by a novel phosphatidylethanolamine methyl transferase that was found to be present in the vicinity of mitochondria [93]. The implications of this structural and functional organization of the mitochondria and of a specialized ER domain are 2-fold: (i) if this organization is lost, the capability of exchange between the membranes would be abolished, or greatly diminished; (ii) this structuration should lead to a compartmentalization of the PS (and PE) pools. (d) An important observation gave additional, indirect, support to the suggested existence of a specialized domain of the ER responsible for the synthesis and export of phospholipids destined to mitochondria. Voelker [94] disrupted CHO-K1 cells by shearing and demonstrated that, in this system, as in permeabilized cells, PS synthesis requires ATP and is blocked by preventing Ca 2+ sequestration. Moreover, mixing experiments using heterologous donor and acceptor cells showed that the transport of nascent PS is restricted to the mitochondria of autologous cells [94]. Though there are many plausible interpretations for these data, they suggest that there is some structural channeling of the nascent PS to autologous mitochondria, and support the suggestion of a close link between mitochondria and a specialized ER domain [90,94]. (e) In permeabilized CHO-K1 cells, the translocation of PS to the mitochondria is unaffected by dilution [95]. This could mean that there is a close association between the acceptor and donor membranes (which would be insensitive to dilution) a n d / o r that the translocation intermediate - if any - has a limited free diffusion capacity, as would be the case in a vesiclebased transport. The lack of any stimulatory effect of GTP (and the inhibiting effect of GTPTS) and the absence of any requirement for soluble factors other than ATP, Mg 2+ and Ca 2+ in the translocation of PS,

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were strong arguments against a vesicle-mediated transfer out of the endoplasmic reticulum [95].

3.3.2. PE formation in the inner membrane of the mitochondria Various methods have been used to subfractionate mitochondria, giving access to outer membranes and inner membranes and allowing to study the localization of the PS decarboxylase. So far, the results have repeatedly shown that this enzyme is localized in the inner membrane and, most probably, at the outer aspect of the latter [96-101]. This submitochondrial localization of the PS decarboxylase, observed in both yeast and mammals, raised the crucial question concerning the mechanism of translocation of PS between the outer and inner mitochondrial membranes, and the participation of contact sites was suggested [102,103]. The further subfractionation of rat liver mitochondria yielded a membrane fraction enriched in contact sites [100,101,104,105], which had been described for the first time by Hackenbrock [106]. Contact site-enriched membrane fractions have also been obtained from mouse liver [107]. Simbeni et al. [100] showed that the PS decarboxylase activity was present in contact sites, where PE and cardiolipin accounted for 50% of the total lipids. This presence of PS decarboxylase at the contact sites was confirmed by independent methods [101], but the PS decarboxylase was not specifically located at the contact sites [101]. It was also shown that treatment with 1,4-dinitrophenol (DNP), a reagent known to diminish the contacts between the inner and outer membranes [108,109], led to a decrease of PE formation. The uncoupling of the oxidative phosphorylation induced by DNP was not responsible for the inhibition of PE formation [101] and it was hypothesized that the DNP treatment resulted in an increase of the intermembrane space, thereby pulling the contact sites apart [101]. The involvement of contact sites in PS import into rat liver mitochondria was also inferred from a kinetic study [104]. In the presence of hydroxylamine, an inhibitor of PS decarboxylase, the authors observed an accumulation of PS in the contact site-enriched fractions and suggested the existence of a micro-cornpartmentation near the contact sites in which PS translocation and decarboxylation are linked [104]. A direct demonstration of this was obtained when contact site-enriched fractions, incubated with an endoplasmic reticulum-rich fraction from mouse liver, yielded a concerted synthesis of phospholipids from [14C]serine; the restoration of the functional interactions between the E R and the contact sites was also observed by electron microscopy [107]. The accumulation of PS at the contact sites in the presence of hydroxylamine was

confirmed [105] and the addition of 50 mM Ca 2+ was found to increase the phenomenon [110]. This observation, added to the fact that about one half of the lipids at the contact sites are PE and cardiolipin, strongly supported the hypothesis that lipid polymorphism (in this case, a calcium-induced transition of the lipids to HH-like phases) could play a role in lipid import and raised the question of the participation of non-bilayerforming lipids in the intramitochondrial transfer of PS. A recent study of intramitochondrial PS distribution and transport was carried out with pyrene-labeled lipid species [111]. Three different models were discussed; one involved semi-fusion sites ( = contact sites) bridging the two membranes; the second model implicated a phospholipid transfer protein, or spontaneous diffusion, in PS movement; the third model implied that the decarboxylation takes place while PS remains in the inner leaflet of the outer membrane. The results mainly supported the first model, whereas the second model was contradicted by several findings and was considered to be unlikely[ill].

3.3.3. The PS to be exported and decarboxylated is compartmentalized Increasing amounts of evidence are available indicating that the lipids (and especially PS) are compartmentalized in microsomes and, consequently, that there is a discrimination between the exchangeable and non-exchangeable lipids [103,112,113]. Using two reconstituted cell-free systems consisting of rat liver mitochondria and microsomes, Vance [ l l 4 ] d e m o n s t r a t e d the functional compartmentalization of PS and PE and obtained clear-cut evidence that newly synthesized PS is preferentially translocated from the E R to mitochondria, apparently without mixing with the PS already present in the membrane ('old PS'). Similar indications were obtained for the PE formed in the mitochondria and subsequently exported to the E R for methylation, in both liver [114] and yeast [103]. The existence of PS pools in microsomes was also established in the brain [115,116]. By measuring the mass and the radioactivity of the PS transferred to mitochondria, the authors showed that the newly synthesized radioactive PS is more efficiently exported than the bulk of the microsomal PS. It was further suggested that not all the mitochondrial PS was likely to be decarboxylated, maybe because of a comparmentalization of the lipids within the mitochondrial merebranes [116]. The reason for this differential susceptibility to translocate the PS molecules is not clearly established. It has been proposed by Vance [114] that newly made PS and PE are located in the outer, or cytosolic, leaflet of the membranes, whereas older molecules having undergone a transbilayer movement would no longer

P. Moreau, C. Cassagne / Biochimica et Biophysica Acta 1197 (1994) 257-290

be available for translocation. This hypothesis is supported by several observations: (i) Corazzi et al. [113] obtained indications that the PS, newly synthesized in vitro by base-exchange reactions, is restricted to a small pool (5% of the total PS) located at the external surface of microsomal vesicles isolated from rat brain. That the bulk of PS was located in the inner membrane leaflet was suggested by the lack of reaction with trinitrobenzene sulfonic acid under non-penetrating conditions [113]; (ii) microsomes transfer more labeled (neosynthesized) PS (but probably not more PS molecules) than do liposomes made up with microsomal lipids [116], since the PE formation in mitochondria is almost identical with the two PS donors (microsomes or liposomes). These experiments also showed that, when the compartmentation is lost, the transferrable (neosynthesized) PS pool is 'diluted' in the bulk PS pool, which becomes partly exchangeable; (iii) the transfer of PS from rat brain microsomes to mitochondria is regulated by the microsomal lipid pattern; it is enhanced by negatively charged phospholipids, among which PS itself is the most active in fostering its own transfer to mitochondria [115]. If this phenomenon takes part in the overall transfer process in vivo, it is not meaningless that the PS to be exported from the ER is apparently accumulated in specialized domains of this membrane; (iv) the existence of PS pools is highly consistent with the observation that PS synthesis and export are restricted to a specialized domain of the ER [90].

3.4. Pending questions: the early and late steps The reconstitution of phosphatidylserine import into mitochondria has recently allowed important information to emerge, some of which has been discussed in the preceding sections. However, it is probable that not all of the different steps involved in the process have been brought to light, so that many facets of this sophisticated pattern require further analysis. This is the case of the presumed early and late steps, which will now be briefly examined,

3.4.1. Early steps and the A T P requirement, A large amount of converging evidence has suggested that the movement of PS between microsomes and mitochondria does not require cytosolic proteins (see above sections). As an alternative to protein-mediated phospholipid transfer between the ER and mitochondria, a vesicle-mediated mechanism has been widely hypothesized. This mechanism, if operative, was considered to require a supply of energy, by analogy

265

with the situation in the endomembrane vesicle-mediated transfers. In fact, though it is now likely that it is not vesiclemediated, PS transfer to mitochondria has repeatedly been shown to rely on ATP in both intact and permeabilized cells [95,117]. This ATP-dependent translocation is also observed in disrupted cells [94]. However, neither ATP, nor any other nucleotide triphosphates, have any effect on the transfer of PS to mitochondria in vitro. Voelker [118] hypothesized that the ATP-dependent step in the cells may precede the events that are reconstructed using isolated fractions. For example, ATP could be required to place the microsomal PS in a position allowing its transfer to mitochondria (e.g., in the outer cytoplasmic leaflet of the membranes, if the PS formation takes place on the luminal side), or simply because it plays a role in the calcium-dependent PS synthesis. That the transfer of PS (and PE) between rat liver mitochondria and ER in vitro did not depend specifically on the hydrolysis of ATP was further shown in a study conducted by Vance [114]. Neither exogenous ATP, nor the nine other nucleotides tested, significantly stimulated the transfer and decarboxylation of PS in vitro [114]. These data supported a collision-based mechanism rather than a vesicle - or protein - mediated transfer of PS from the ER to mitochondria [114]. A recent study of the influence of various effectors on PS synthesis in rat liver microsomal fractions provided additional information concerning the potential role of ATP [119]. (i) the stimulatory effect of ATP-Mg 2+ on PS synthesis was reduced upon addition of a calcium ionophore (A23187) and by thapsigargin, a specific inhibitor of Ca 2+ and ATP-binding to the Ca 2+ATPase [120]; (ii) modulators of Ca 2+ release by receptor channels also modulated the ATP-dependent PS synthesis; (iii) when freeze-thawed and sonicated (i.e., fully disrupted) microsomes were used, ATP and Mg 2+ did not stimulate PS synthesis. These data are consistent with the hypothesis that PS synthesis occurs in the luminal leaflet of the ER [119,121],in marked contrast to the proposal discussed above. Furthermore, PS biosynthesis coud also be regulated by events responsible for Ca 2+ movement across the membranes. Besides its role in PS formation, Ca 2÷ could also play a role in protein-dependent lipid (and particularly PS) scrambling. There is strong evidence that, under near-physiological conditions (i.e., by moderately increasing the Ca 2+ concentration), the lipid asymmetry may be lost, leading to a rapid Ca 2+-dependent reorientation. Whether this phenomenon, which has been shown chiefly for plasma membranes, is also operative in endomembranes, remains to be elucidated (for review [122]).

2~(~

P. Moreau, C. Cassagne/ Biochimica et Biophysica Acta 1197 (1994) 257-290

A n y mechanistic proposal should also take into ac-

count the fact that, in the endoplasmic reticulum isolated from rat liver, PS seems to be chiefly located in the cytoplasmic leaflet, whereas PC is distributed randomly [123]. Though the difficulty in obtaining reliable data may explain a few diverging results (for review [122]), it is remarkable that, in the case of the ER from chick brain, PC has been located to the cytoplasmic leaflet, whereas PS was randomly distributed [124]. These points illustrate the dramatic need for a further examination of the location of PS synthesis and distribution in a variety of cell types and cell membranes, and preclude any firm conclusion to be drawn before new evidence is gathered. 3.4.2. Late steps and the effect on the PS transfer of drugs affecting protein import into mitochondria

The study of the ATP-dependent steps of PS transfer to mitochondria may help to shed some light on the early stages of the process. In order to gain new insights into the late steps of PS import, a new strategy was recently used. The effect of the antineoplastic drug, adriamycin (a potent inhibitor of protein import into mitochondria), on the ATP-dependent PS transfer was investigated in permeabilized CHO-K1 cells [125]. Adriamycin had no significant effect on ATP-dependent PS synthesis, did not inhibit PS decarboxylase, but blocked the time- and translocation-dependent PS decarboxylation, probably near, or at, the mitochondrial inner membrane of the permeabilized cells. By using NBD-PS analogs, which partition freely between membranes, it was shown that adriamycin (which completely inhibited the decarboxylation of NBD-PS) probably acted on a step taking place between the outer and inner mitochondrial membranes [125]. Whether the effect of adriamycin on PS translocation bears some similarity with its action on protein import awaits further examination, as do the effects of other inhibitors of protein import on PS transfer/decarboxylation. However, it is remarkable that adriamycin, in addition to its well-known effect on protein import, exhibits a high affinity for anionic phospholipids [126]. Given the potential importance of lipid polymorphism (and particularly of the non-lamellar organization) at the level of the contact sites for the PS intramitochondrial transfer (see above), it is highly significant that the antineoplastic drug, adriamycin, inhibits the formation of nonlamellar phases in cardiolipin/PC (1:1) model membranes, but induces H n phase formation in cardiolipin/PE (1:2)and P E / P S (1:1)mixtures (reviewed in [ 127]). 3.5. Concluding remarks Phospholipid import into mitochondria emerges as an original process, differing greatly from those re-

Mitoehondria Associated ER

Mitochondria OM IM

/\

-~ PSm

S~e a P

r

/ /

iv hi C

$

c /iPEer i 11 lid[

PEru ( ~

/*1 ]PC~- . . . .

~, ~C

II Fig. 1. The PS-PE-PC cascade. Serine is incorporated into PSer in the ER and transferred to the mitochondria(a); mitochondrialPSm is then decarboxylated(b) and mitochondrial PEm is partly transferred to the ER (c), where it may be methylated(d). Drawn from data of Vance [90,114], Voelker [94,95], Ardail et al. [104,105,107] and Jasisnka et al. [111]. ER, endoplasmic reticulum; OM, outer membrane; IM, inner membrane; CS, contact sites. Solid arrows refer to established movements and/or metabolisms, dotted arrow refers to a postulated movement in the PS-PE-PC cascade between the mitochondria-associatedER domains and the mitochondria. ported (or postulated) for the endomembrane system (Fig. 1). Over the last few years, a tremendous improvement of our understanding of the mechanisms underlying the important sequence of events allowing the mitochondria to acquire (and eventually metabolize) the phospholipids required for the formation of functional outer and inner membranes has been achieved. The various steps involved in, or required by, the transfer from the ER to mitochondria are better defined, the interrelations between ER domains and organelles are delineated and the general pattern of this still intriguing phenomenon will probably emerge during the next decade.

4. Vesicular transfer of phospholipids to the plasma membrane 4.1. Introduction

The transport of membrane components to the plasma membrane by vesicular carriers [81,128] has been extensively studied and illustrated in the literature for glycoproteins [129], for glycosphingolipids in animal cells [7] and Saccharomyces cerevisiae [130] and for sterols [131]. The transport of secretory and membrane proteins and glycolipids through the ER-Golgi apparatus-plasma membrane pathway, studied by following the intracellular fate of a tripeptide with the signal sequence for glycosylation and a water-soluble eeramide analog, has led to the proposal that forward transport to the plasma

P. Moreau, C. Cassagne /Biochimica et Biophysica Acta 1197 (1994) 257-290

membrane is achieved by 'bulk flow', implicating no special signal for molecules which follow the vesicular pathway to the plasma membrane [82,132,133]. Similar conclusions were obtained for glycosphingolipid transport to the plasma membrane in CHO cells [134], where the rate of transfer to the plasma membrane was consistent with bulk flow kinetics and was as fast as that reported for many proteins [135]. However, the bulk flow model was recently questioned in yeast [136] by the finding that glycosylation acceptor tripeptides were not bulk flow markers in yeast. Instead, their transport to the plasma membrane was ensured by ATP-driven pumps located in both the ER and the plasma membrane. In the ER, these pumps could be involved in the release, into the cytosol, of short peptides resulting from protein degradation occuring in the ER [136]. The secretion of peptides and proteins which lack specific signal sequences could be under the control of ATP-driven membrane translocators, both in prokaryotes and eukaryotes (reviewed by Kuchler and Thorner [137]). The extent to which the bulk flow hypothesis is compatible with the recent finding that soluble secretory proteins are concentrated in the ER before their delivery to the Golgi apparatus [138] awaits further examination, Whether the bulk flow model is valid or not, it is obvious that many secretory and membrane proteins and lipids are transported to the plasma membrane via vesicular membrane intermediates, It has been clearly determined that the transport of cholesterol to the plasma membrane can be vesiclemediated [139,140]. Moreover, it has been postulated that cholesterol and the VSV-G protein are transported to the plasma membrane via distinct lipid-rich vesicular carriers, since brefeldin A blocks the transport of the VSV-G protein, but not that of cholesterol [141]. ER-derived transition vesicles produced in a

267

cell-free system from rat liver contain cholesterol molecules, as determined by lipid analysis (see Table 1) and digitonin-shift experiments [142]. This ER-derived vesicle fraction could contain the two types of lipid-rich vesicles postulated by Urbani and Simoni [141]. However, the presence of cholesterol in transition vesicles involved in the transport between the ER and the Golgi apparatus cannot be ruled out, since only one population of vesicles was isolated in both studies [141,142].At this point, it must be noticed, however, that cholesterol can also be efficiently transferred between membranes according to an activation-collisionbased mechanism [143]. Anyway, the existence of various vesicles as intermediate carriers for protein, glycolipid and cholesterol transport to the plasma membrane has been clearly and extensively demonstrated. What, then, is the situation for membrane phospholipids? The existence of intracellular vesicular carriers and the very low probability that these vesicular carriers are totally devoid of phospholipids, suggest that phospholipids are most probably transferred by such vesicles.

4.2. Phospholipid composition of the membranes of the exocytic pathway The phospholipid compositions of the different membranes involved in the exocytic pathway in rat liver are shown in the Table 1. The phospholipid composition of the transitional elements of the ER membranes (TER) shown is very similar to that described earlier by Zambrano et al. [144] for the whole endoplasmic reticulum. The phospholipid composition of Golgi membranes is intermediate between those of TER membranes and plasma membranes, in good agreement with the previous data of Zambrano et al. [144]. The fractionation of the Golgi apparatus by free-flow

Table 1 Phospholipid composition (mass% of total phospholipids) and cholesterol and ceramide content of rat liver subcellular membranes ER 1

TER 2

T.V. 2

Golgi 2

cis-Golgi 3

med. Golgi 3

trans-Golgi 3

PM 3

SM PC PS PI PE other a Chol/PL 3

2.5 58.4 2.9 10.1 21.8

4.6 58.7 2.7 10.4 19.0 4.6

71.4 7.2 5.4 16.0 -

8.8 56.1 4.8 5.4 21.8 -

7.8 58.2 4.8 5.4 21.0 2.8

8.1 56.9 4.1 5.9 21.5 3.5

10.6 53.1 5.5 4.8 23.0 3.0

15.3 52.3 10.6 5.8 10.3 5.7

(mol/mol) Ceramides 2 (/xg//a.mol phospholipids)

0.08 5.2 _+ 0.6 4 11.1 _+ 1.6 4

+ 1.4 + 1.3 +0.8 +0.2 + 1.6 _+ 1.2

0.13 9.6 _+ 1.1

+2.9 + 1.6 +0.1 _+3.0

0.27 0.7 _+ 0.03

___0.5 +2.1 +0.6 +0.4 _+0.8

0.17 7.5 -+ 2.0

0.12

+ 2.6 +4.3 _+0.5 +0.3 _+4.3 _+ 0.4

0.16

+ 2.3 +2.8 + 1.0 +0.5 + 1.7 _+ 0.7

0.27

+ 3.3 + 1.1 5:0.6 + 1.0 _+ 1.3 _+ 0.5

+ 1.4 +3.6 _+ 1.0 _+ 1.5 _+0.9 _+ 0.8 b

0.49 11.6

a This lane includes predominantly lysophosphatidylcholine and phosphatidic acid as the other phospholipids. Phosphatidic acid was found to account for most of this value and consequently to be higher in the plasma membrane. (1) Data from Zambrano et al. [144]. (2) Data from Moreau et al. [189]. (3) Unpublished data from Dr. D.J. Mort6 (Purdue University, West Lafayette, Indiana) and the authors. (4) Data from two distincts ER fractions of different densities reported from Moreau et al. [189].ER, endoplasmic reticulum; TER, transitional elements of the ER; TV, transition vesicles operating between the ER and the Golgi apparatus; PM, plasma membrane.

2~

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electrophoresis [145,146] reveals a concentration gradient of sphingomyelin and that the amount of cholesterol increases only in the trans-Golgi and plasma membranes (Table 1). Cholesterol in the Golgi cisternae has already been described by measuring the density of filipin-cholesterol complexes [147]. In view of the large amounts of cholesterol present in the transGolgi-enriched fraction, it is probable that the cholesterol is associated with the trans-Golgi membranes themselves, rather than with any contaminating membrane types (4 and references therein). The enrichment of sphingomyelin is observed already at the level of the cis-Golgi apparatus (Table 1). Futerman et al. [148] have shown strong evidence that the major intracellular site of sphingomyelin synthesis in rat liver is 1ocalised in the cis and medial cisternae of the Golgi apparatus. The presence of sphingomeylin in T E R membranes could be due to membrane contaminants, or to the partial recycling of the lipid from the Golgi apparatus, but this second alternative is less likely [7]. There is a remarkable enrichment of PS and cholesterol in the transition vesicles (Table 1). PS is synthesized in the cndoplasmic reticulum [1,2], but is enriched in the plasma membrane ([7], Table 1). AIthough there is no experimental evidence, PS transport to the plasma membrane was suspected to be mediated by vesicular carriers [7] and its increased concentration in the transition vesicles (Table 1) can support this hypothesis, Besides the increasing amounts of sphingomyelin, PS and cholesterol, we observe a continuous decrease of the percentage of PC in the intracellular membranes as they progress along the exocytic pathway (Table 1). Finally, we found less PI and PE in the plasma membrane than in the whole ER, while Zambrano et al. [144] observed similar amounts of PE in the two membranes, The differences between the lipid compositions of the various subcellular compartments and the intracellular distribution of phospholipid synthesizing activities, imply that an important intracellular traffic of phospholipids must exist,

4.3. Vesicular transport of phospholipids to the plasma membrane." the controuersy Many in vivo studies have led to contradictory results concerning the mechanisms by which phospholipids are transferred to the plasma membrane and, in particular, with respect to the putative requirement of vesicular carriers and whether this mode of transport is quantitatively significant for phospholipids. This controversy has been largely illustrated for animal cells and as much experimental data for than against the involvement of vesicular carriers in the intracellular transport of phospholipids have been described

[4,7,8,149-151]. Some of the points raised by PC and PE trafficking have been already analysed, the reader is referred to previous reviews for the details concerning for example the controversial effects of energy poisons, monensin, brefeldin A and colchicine on PC and PE vesicular trafficking in animal ceils [4,7,8,149151]. In plant cells, a lower amount of information is available. However, the biogenesis of peribacteroid membranes in mature legume root nodules has been suggested to involve the intracellular traffic of merebrane material through smooth and coated vesicles [152,153]. The transport of labeled phosphatidylcholine from the ER to the peribacteroid membrane is likely to be vesicle-mediated [154]. In leek cells, a vesicle-mediated transport of lipids (including phospholipids)from intracellular membranes to the plasma membrane has been demonstrated [155-158]. Recently, Gnamusch et al. [47] studied the in vivo transport of phospholipids in wild-type yeast cells and in the yeast secretory mutant Sec 14, whose phenotype results in a defect in the PI transfer protein at the level of the Golgi apparatus [53]. It was found that an energy-linked process is involved in the transport of PS, PE and PC to the plasma membrane. With Sec 14, they confirmed the preceding results obtained with Sec 18 and Sec 7 [159], indicating that the transfer of PI and PC to the plasma membrane was not dependent on the secretory pathway. Moreover, their results also demonstrated that the P I / P C binding protein was not required for the transport of PI and PC to the plama membrane. The fact that the transport of PS, PE and PC to the plasma membrane was energy-dependent in wild-type yeast cells is either in favor of the existence of another type of vesicular process, or argues for the involvement of a membrane collision-based transfer [94,114]. It is inevitable that different methodologies (use of fluorescent lipid analogs, de novo labeling, membrane isolation, derivatization of plasma membrane lipids) and the use of different cell types (animal, plant, yeast, sec mutant cells) can lead to conflicting results. For example, Sleight and Pagano [160] analysed the internalization of a fluorescent PC analog in chinese hamster V79 fibroblasts and its recycling from the Golgi apparatus back to the plasma membrane. It appeared that the tl/2 of this transfer was 20 min, in agreement with the involvement of a vesicular process. These results differ markedly from those obtained by Kaplan and Simoni [161] for the transfer of newly synthesized PC from the ER to the plasma membrane (t¿/2 of 1-2 min). One explanation [8] is that the membrane location of these PC molecules was different (de novo synthesized PC is in the cytosolic leaflet of the E R and fluorescent PC analogs are always in the luminal leaflet

P. Moreau, C. Cassagne /Biochimica et Biophysica Acta 1197 (1994) 257-290

of intracellular membranes). Sleight and Abanto [162] observed different intracellular fates of a fluorescent analog of PC for various cell types, using various conditions (defect in glycoprotein processing, or virus transformation) for a given cell type. The differences noticed in lipid movement strongly suggest not only that lipid transport involves several mechanisms, but also that the regulation of a given pathway could be highly sensitive to the experimental conditions employed to investigate them. In that respect, the use of fluorescent lipid analogs and the signifcance of half-times of transfer need special comments.

4.3.1. Fluorescent lipid analogs as markers of lipid transport Pagano and Sleight [163] have summarized the properties and the intracellular movements of various fluorescent lipid analogs (C6-NBD derivatives of phospholipids and glycolipids and the N-rh-PE), which have undoubtedly allowed the development of a new approach to the study of lipid transport and have provided important new information. That these fluorescent lipid analogs could reflect the behaviour of endogenous lipids has been convincingly shown by several lines of evidence [163]: (i) the nature of the fluorescent group (dansyl instead of NBD) does not change the behavior of the analog, suggesting that the non-physiological parts of the molecules do not influence the (natural ?) intracellular fate of these lipids. This does not mean, however, that only the polar head plays a prominent role in the process. This is well illustrated by the fact that the chain length of pyrenyl fatty acids was determinant in the metabolism and incorporation of these fluorescent fatty acid analogs into the cellular phospholipids and neutral lipids [164]. Moreover, Colleau et al. [165] have shown that the position of the NBD (close to the glycerol or to the methyl terminal of the acyl chain) considerably influenced the transmembrane diffusion properties of the lipid analogs. As stated by Naylor et al. [166], a careful selection of fluorescent lipid analogs, based on comparisons with the natural molecules, would be required for each cell line being investigated; (ii) the metabolism of C6-NBD derivatives of PA and ceramides (Cer) is not abnormal in that it leads to classical end products of the metabolic pathways. Moreover, C6-NBD-Cer and [3H]palmitoylsphingosine are metabolized in a similar manner by membrane fractions isolated from chinese hamster V79 fibroblasts ceils [167]. However, whether C 6NBD derivatives are metabolized in vivo at the same rate as their natural counterparts is not clear since the rate of metabolism of short-chain analogs,

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such as hexanoyl-Cer, is very high [148,168] compared to that of natural exogeneous substrates [169]; (iii) the intracellular movements and sorting of C 6NBD derivatives are consistent with those observed for their natural counterparts [7,163] and there is convincing evidence that, even if their metabolism differs slightly from that of their natural counterparts, they mimick them closely enough to allow their use for the study of existing lipid pathways.

4.3.2. Half-times and mechanisms of transfer As summarized by Vance et al. [151], mean tl/2 values of transfer of proteins, cholesterol, PE and PC, at 37°C, have been determined in various systems. The significance of tl/2 values calls for further comments, which are rendered necessary by the large differences observed between those reported for the transport of cholesterol to the plasma membrane, for example [139,170]; De Grella and Simoni [170] found a t~/2 of 10 min, whereas Lange and Matthies [139] determined a tl/2 of 60-120 min. De Grella and Simoni [170] incubated CHO cells for 1.5 min with the labeled precursor, whereas Lange and Matthies [139] incubated human fibroblasts for 20 min with the same labeled precursor. In these experiments, both the cells and the experimental conditions were different, and it therefore appears that these factors can profoundly influence the value of the t~/2. This was also observed for the transfer of phospholipids in etiolated leek seedlings. By using pulse-labeling periods of 120 min, approximative tl/2 values of 30-45 min were determined [156,171], whereas a shorter pulse-labeling period of 60 min gave a shorter tl/2 of about 15 min [156]. Therefore, values of t~/2 have to be considered with care. Sleight and Pagano [43] have determined a tl/2 of 22 min for the transfer of PE to the plasma membrane of chinese hamster V79 lung cell fibroblasts. This value appears, at first sight, compatible with those (>__30 min) found for the vesicular transport of proteins [135,172].However, the fact that PE was detected in the plasma membrane in as little as 2 min, led the authors to conclude that the transport of PE to the cell surface was fast, and consequently, could not be accounted for by the classical vesicular pathway used by glycoproteins. In contrast with the situation observed for proteins, the significance of t~/2 values is questionable when considering the existence of various pools of transferrable lipids in membranes. There is no reason that phospholipids will be transferred from each pool with the same rate and that the same amount of phospholipids will be concerned in each case. Different types of mechanism of transfer have been

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proposed to be associated with the different ranges of values of t~/: values, tl/2 of 20-30 rain, representative of vesicular transport, are well documented for proteins [135,172] and for glycosphingolipids [7]. tl/2 below 10 rain and as short as 1 rain are associated with the transfer of other lipid species, maybe via a vesicular process bypassing the Golgi apparatus for the transport of cholesterol [141], or other putative mechanisms for the transfer of phospholipids. One of these is described by the concept of collision-based transfer, which has found good experimental support for the transfer of PS between the ER and the mitochondria [94,114] and was also suggested for the transfer of phospholipids in the mutant SEC 14 of yeast [47]. A close proximity between the donor and the acceptor membranes would be required [90,114]. For example, some electron microscopic studies have revealed the possible existence of membrane contacts between the ER and the plasma membrane [173]. Another possibility would be that fast vesicular processes also exist in cells and some arguments can be advanced for the existence of multiple types of vesicles and their eventual rapid transfer,

4.4. Potential existence of fast l,esicular processes 4.4.1. Existence of multiple cesicular carriers The existence of different vesicular pathways to the plasma membrane has been demonstrated for secretory and membrane glycoproteins in animal cells [174]. It has also been shown that distinct transport vesicles are responsible for the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells [175]. Similarly, Atkinson [176] has suggested the possibility of separate secretory and membrane-growth pathways in Saccharomyces cerecisiae. Membrane growth and secretion could be differentially blocked by genetic mutations of lipid metabolism, implying that the membrane lipid requirements for membrane growth and secretion differed. However, in vitro, exocytotic transport vesicles produced from the trans-Golgi network carry both secretory and plasma membrane proteins [177]. Both secretion and membrane growth were blocked in fatty acid-starved cells, whereas secretion did not stop in inositol-starved cells, even when membrahe growth had stopped [176]. The distinction would originate from the fact that secretory vesicles are highly conserved entities that do not blend into the plasma membrane, or get degraded rapidly, whereas the production of constitutive membrane-growth vesicles will stop as soon as lipid synthesis stops. The divergence of both pathways could be at the level of the Golgi apparatus [176], but another alternative must be considered: the ER could produce constitutive vesicles that would be directly transferred to the plasma merebrane. This has been proposed to explain the differ-

ence between the transport of cholesterol and proteins in animal cells [141]. In the same respect, evidence is available for a secretory pathway bypassing the Golgi apparatus of monensin-treated cells [178]. Different intracellular vesicular pathways have been suggested for secretory components in the perfused rat liver following monensin treatment [179]. In leek seedlings, the transport to the plasma membrane of C20-C24 lipids is blocked by monensin, whereas that of CI6-Cls lipids is not [158], suggesting that C16-C1~ lipids are transferred to the plasma membrane, either directly from the ER, or were synthesized in the Golgi apparatus and transferred to the plasma membrane, or both. It is therefore likely that multiple vesicle types are involved in intraceilular membrane traffic. Numerous exocytic and endocytic carrier vesicles implicated in the intracellularvesicular pathways have been isolated from several sources [177,180-189]. Although the proteins of most of these vesicles have been characterized, their lipid composition has been rarely analysed. The phospholipid content of a population of ER-derived vesicles from rat liver has been determined [188,189] and that of endocytic coated vesicles is also available (see chapter 5). The presence of phospholipids in exocytic and endocytic vesicles obviously implies that some of these molecules are transported by these vesicular carriers. However, the transfer of phospholipids through the classical vesicular pathway does not appear to be an important process for the delivery of phospholipids to the plasma membrane. This could be partly due to the balance between the exocytic and endocytic pathways, which is discussed in another part of this review (see chapter 5.3).

4.4.2. Experimental data supporting fast t,esicular processes To reconcile all the data of the literature with the notion of vesicular transport of phospholipids, the existence of fast vesicular transfers of phospholipids can be considered. Even if some results of the literature have been taken to indicate the implication of a non-vesicular transfer, there is in fact no experimental proof that a fast transfer cannot be associated with a vesicle-mediated transfer. A good indication that some vesicular processes may be rapid was provided by studies of the transport of the VSV G protein between two populations of Golgi membranes in vitro. The G protein originated from donor Golgi membranes prepared from VSV-infected CHO cells, which lack N-acetylglucosamine transferase I, thereby producing a G protein without that sugar. Acceptor Golgi membranes issued from wild-type CHO cells were used to monitor the transport of G protein from donor to acceptor Golgi membranes by measuring the acquisition of Nacetylglucosamine by G protein. Hence, the measure of

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glycosylation is indicative of the transfer process. This transport is thought to be mediated by COP-coated vesicles [190]. COPs are a family of coat proteins from which many distinct proteins have been identified and are involved in the intra Golgi vesicular transport [190]. Interestingly, Hiebsch and Wattenberg [191] have reassessed the kinetics of the transfer of the G protein and they showed that the limiting step is the glycosylation itself and that it is likely that vesicle budding, transport and fusion are somewhat faster processes, However, it must be pointed out that the existence of these COP-coated vesicles as free vesicles has recently been questioned [192]. In another cell-free system from rat liver, it was found that the production of ER- derived vesicles is detectable within 5 min of incubation [193] and that their transfer to Golgi acceptor membranes is also a rapid process [183]. In the same system, the ATP-dependent transfer of PC is extremely fast [188,194] and tl/2 values of 5 min, or less, have been estimated, Both in vivo and in vitro studies have also shown that lipid transfer via ER-derived vesicles could be quite fast in leek seedlings [155,156,158,195]. Effectively, tl/2 values of less than 5 min can be proposed for the in vivo formation of ER-derived lipid-rich vesicles [156,158], and an in vitro transfer of lipids between the ER and the Golgi has been detected after only 5 min [195]. However, the hypothesis of the existence of fast vesicular processes implies that they are insensitive, or less sensitive, to temperature, energy inhibitors and drugs, such as monensin, brefeldin A and cytoskeleton-disrupting agents [151].

4.4.3. Vesicular transfer and the cytoskeleton It has been established that vesicle movement in animal cells occurs via microtubules [196], one of the best examples being the axonal transport of vesicles and organelles in neurons [197]. However, it has also been observed that microtubule disruption of cultured human embryonic skin fibroblasts did not stop the intracellular transport and secretion of fibronectin [198]. In MDCK and CaCo-2 cells, it has been shown that microtubule disruption slowed protein transport to the apical surface, but not to the basolateral surface (199 and references therein). Similarly, glucosylceramide and sphingomyelin transport to the basolateral membrane of MDCK cells was not blocked by nocodazole treatment [199]. In plant cells, it has been suggested that actin microfilaments, rather than microtubules, would direct vesicular transport [200] and an inhibition of secretory vesicle movement by cytochalasin 13 has been observed in root tips of maize [201]. The situation in yeast is thought to resemble that described for plant cells [202]. However, it does not appear that cytoskeletal ele-

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ments are always required for vesicular transport. Consequently, the absence of inhibition of a transport process by disrupting agents of the cytoskeleton is not sufficient by itself to rule out another possible vesicular mechanism.

4.4.4. The effect of temperature on vesicular transfer It is well established that low temperatures, comprised between 15°C and 20-22°C, block the in vivo transport of membrane constituents at different levels along the ER-Golgi apparatus-plasma membrane pathway [203-206]. This effect is also observed in vitro with cell-free systems [142,188,207-209]. The low temperature block between the ER and the Golgi apparatus has been observed in vitro in a cell-free system from rat liver, for both proteins [209] and phospholipids [142,188].Based on current knowledge, it is not obvious that vesicle budding and fusion reactions are insensitive to low temperatures. However, it has recently been observed that the transport of C16-C18 lipids (lipids with fatty acyl chains having 16 and 18 carbon atoms) in leek ceils is 'insensitive' to low temperatures which block the classical ER-Golgi apparatus-plasma membrane pathway followed by C20-C24 lipids (lipids with fatty acyl chains of more than 20 carbon atoms) [210]. It is likely that either another vesicular transfer occurred for C16-C~8 lipids, or that another mechanism, such as a collision-based transfer, was involved. Whatever the mechanism(s) involved in the transfer of C~6-C~8lipids at low temperature, it is tempting to suggest that specific membrane microdomains [211213] having different phospholipid compositions are involved as sources of transferrable phospholipids used by the different lipid transfer mechanisms (see chapters 6.3 and 6.4).

4.4.5. Energy requirement of vesicular transfer Two exocytic pathways have been observed in permeabilized mast cells; one ATP-dependent and one ATP-independent, depending on whether NaCl-based, or glutamate-based electrolyte solutions were used [214]. It was suggested that either protein phosphorylation (ATP-dependent), or protein dephosphorylation (ATP-independent) were at the origin of the differences. This example of differential regulation of exocytosis in permeabilized mast cells illustrates how experimental conditions can modify the conclusions on ATP-dependency. A recent investigation on the transport of sphingomyelin to the cell surface in rat hepatocytes [215] gave some credit to the potential existence of vesicular processes requiring less energy than that normally observed for the well known vesicular transport of secretory glycoproteins. The transport of sphingomyelin to the cell surface was less sensitive to energy depletors than that of a secretory protein, since a 61%

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depletion of the ATP level did not stop sphingomyelin transport, whereas that of the secretory protein was abolished. Assuming that the transport of sphingomyelin is vesicle-mediated [7], it has been questioned whether low ATP levels were sufficient for the formation and fusion of sphingomyelin-carrying vesicles [215]. Consequently, the existence of vesicular processes requiring less energy (illustrated by sphingomyelin transport [215]) and fast vesicular processes (for exampie for phospholipids) could constitute new possibilities which will have to be investigated in parallel to that of membrane-contact based transfer [47,94,114]. That lipid transport can involve several types of vesicles imply the existence of multiple pathways to the plasma membrahe,

4.5. Other pathways to the plasma membrane The possibility that a direct cis-medial-Golgi-toplasma membrane route exists for the transport of sphingomyelin was raised by the work of Shiao and Vance [215]. These authors found that neither monensin, nor brefeldin A, blocked the transport of sphingomyelin. Monensin is known to block essentially the post-medial progression of membrane flow by exerting its most profound effects on the trans cisternae of the Golgi apparatus stacks [178]. A direct transfer of sphingomyelin from the cis-medial cisternae of the Golgi apparatus to the plasma membrane is expected to be insensitive to monensin. Secretory pathways bypassing the monensin block have been suggested for glycoproteins in the literature ([178] and references therein), Brefeldin A is known to induce a redistribution of Golgi enzymes back to the ER [216] and to block protein transport. Transport of cholesterol was not blocked by brefeldin A and was assumed to be achieved by a direct ER-plasma membrane route [141]. However, Kallen et al. [217] have observed a brefeldin A block of sphingomyelin transport. These discrepancies, that are probably partly due to the different methodological approaches employed, are interesting in that they illustrate the difficulty in investigating lipid traffic and interpreting experimental data. One has also to keep in mind that the drug treatment itself can trigger cellular reaction(s), that will eventually complicate the interpretation of the experiments, For example, monensin does not inhibit protein and phospholipid synthesis in retinas of leopard frogs and does not inhibit (and sometimes even stimulates) the transfer of phospholipids to the rod outer segment membrane [218]. Similarly, brefeldin A stimulated both the synthesis and transfer of PE synthesized from [3H]serine, to the plasma membrane [151]. It must also be pointed out that the nature of the target membrane has its importance. Van Meer and Van 't Hof [199] have found that the transport of

glucosylceramide and sphingomyelin to the apical domain in MDCK cells is affected by monensin, whereas their transport to the basal domain was less sensitive to the drug. In contrast, brefeldin A did not block glycosphingolipid transport to the apical domain of Caco 2 cells, but considerably slowed the transport of sphingomyelin to the basal domain of these cells [199]. The relative polarity of transport between glucosylceramide and sphingomyelin was, therefore, abolished [199]. This could be partly due to the existence and, therefore, the involvement of other transport pathways for both membrane domains. Allan and Kallen [4] postulated that the synthesis of sphingomyelin could also occur in recycling endosomes and that sphingomyelin is transported to the plasma membrane concomitantly with cholesterol via the exocytic part of the endocytic recycling pathway. This model could explain why cholesterol transport is brefeldin A-insensitive (no Golgirequirement), but only when the recycling pathway is brefeldin A-insensitive. It is not easy to explain how brefeldin A blocked sphingomyelin transport [217]. Allan and Kallen [4] suggested that a vesicle-mediated transport delivers Cer to the cell surface and that it would then be endocytosed for sphingomyelin synthesis in the endosomal compartment. The brefeldin A sensitivity of the vesicle-mediated transport of ceramides would explain the decrease induced by brefeldin A of sphingomyelin synthesis and maybe also its effect on sphingomyelin transport. One has to keep in mind, however, that recent reports have questioned the classical vesicular transport of Cer [189,219] and suggested that nonvesicular processes might be operative in this case. Again, it is not excluded that ceramides can be transferred by different transport pathways. Consequently, it can be postulated that sphingomyelin follows various vesicular pathways differing in their sensitivity to brefeldin A and the existence of a direct cis-medial Golgi-to-plasma membrane route is likely. A weak point that remains in the postulate for a direct cis-medial-Golgi apparatus-to-plasma membrane route is that few, or no, free vesicles are observed near the medialGolgi cisternae [192]. The cis-Golgi network would probably have to be considered as the escape level from the classical exocytotic route, rather than the medial-Golgi cisternae. The direct ER-to-plasma membrane and cismedial-Golgi apparatus-to-plasma membrane routes are attractive hypotheses for sphingomyelin and phospholipid transport. Recent data on the transport of C20-C24phospholipids and Cl6-C18 phospholipids to the plasma membrane of leek cells revealed the possible existence of distinct pathways for phospholipids [158,210]. Assuming that a population of ER-derived vesicles is directly targeted to the plasma membrane, phospholipids could be transferred to the plasma mem-

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brane via vesicular carriers within 5 min, or less. The existence of protein-free lipid vesicles, or specific lipid-protein microvesicles, are possibilities that could be exploited. For example, specific microvesicles have been isolated from bean cotyledons and are thought to be implicated in removing degraded phospholipids from the membrane into the cytosol [220,221]. In conclusion, it is likely that different types of vesicles and multiple transport pathways to the plasma membrane exist in cells and that, as a consequence, the complexity of the regulation of their respective functions exceeds our means of investigation. This field of research constitutes a challenge for the future years,

5. Vesicular endocytic and retrograde routes and the recycling of phospholipids

5.1. The internalization ofphospholipids Lipid traffic in this particular case has been essentially studied in cultured cells by following the fate of externally applied fluorescent analogs of various phospholipids and glycosphingolipids. The internalization of fluorescent analogs of PC and PE has been investigated in chinese hamster V79 lung fibroblasts [160,222]. The internalization of PC was energy-dependent and was thought to be dependent on endocytosis, and the PC analog reached essentially the Golgi apparatus [160]. The fate of the fluorescent analog of PE was more complex. A similar energy-dependent pathway leading to the Golgi apparatus was observed, but the nuclear envelope and mitochondria were also labeled, via a pathway requiring a lower amount of energy [222]. This transfer is certainly composed of two successive steps; an energy-dependent, or energy-independent translocation of PE from the external leaflet to the internal leaflet of the plasma membrane, followed by its transfer through the cytoplasm to other intracellular membranes by a process that is restricted to some membranous compartments (nuclear envelope and mitochondria). The transbilayer step can be achieved by translocating activities which have been extensively characterized [122,223-226]. The internalization could theoretically be achieved by specialized proteins, such as PLBPs, or by the lateral diffusion of the analogs within cytoplasmic membranes in close apposition to, or continuous with the plasma membrane [173]. If the latter mechanism is operative, it is curious that no labeling of the ER was observed, since connections of the plasma membrane with the ER have also been reported [173]. This situation probably reflects the fact that only specific membrane domains can interact with each other, As shown in the case of PE, the internalization observed for C6-NBD-DG , derived from the hydrolysis

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of C6-NBD-PA incorporated into the plasma membrane, was endocytosis-independent [227]. C6-NBD-PA was not internalized as such and needed to be hydrolysed to C6-NBD-DG prior to internalization [227]. Recently, this process was confirmed when a phospholipase C located at the surface of Swiss 3T3 cells was shown to hydrolyze C6-NBD-PI to C6-NBD-DG, which was subsequently internalized [228]. The internalization of PS was investigated and demonstrated by several authors using either natural molecules [229,230], or fluorescent analogs [231]. Exogenous PS seems to be so efficiently internalized that it abolishes endogenous PS biosynthesis [230], supports the growth of mutant cells defective in PS biosynthesis [229] and leads to the formation of PE by the mitochondrial PS decarboxylase [8,229]. The mechanism of internalization of PS was recently investigated by studying the fate of fluorescent analogs of PS in CHO-K1 fibroblasts [231]. First, PS translocation from the outer leaflet to the inner leaflet of the plasma membrane was achieved by a translocase [122]. Secondly, it was observed that (palmitoyl-C~2-NBD)-PS was more efficiently internalized into the Golgi apparatus than Nrh-PE, which has recently been proposed as a membrane traffic marker of the endocytic pathway [232]. The uptake of the PS analog was still observed in mitotic cells, in which endocytosis is blocked [233], whereas that of N-rh-PE was no longer observed. Moreover, the uptake of the PS analog was not blocked at 18°C, temperature at which the endocytosis of proteins is blocked [234]. Hence, it is unlikely that the classical endocytic pathway was followed. The requirement of a PLBP was proposed to explain the facilitated transport of the PS analog, which has a low solubility in water, through the cytoplasm. On the other hand, transfer of this lipid analog through membrane interconnections has to be considered, as for other phospholipids. Interestingly, Kok et al. [232] have not only shown that N-rh-PE and C6-NBD-PC internalization were both blocked by energy poisons (in agreement with the implication of endocytosis), but also that the final destinations were different (Golgi apparatus for C 6NBD-PC and lysosomes for N-rh-PE), implying that the two analogs follow distinct endocytic pathways. Moreover, C6-NBD-PC endocytosis was far less efficient than that of N-rh-PE [232]. N-rh-PE was found to reach the outer leaflet of the plasma membrane as small aggregates, whereas C 6NBD-PC seemed to be homogeneously distributed in the plane of the bilayer [232]. These different behaviours could be at the origin of the different intracellular destinations of the two analogs and the different endocytic pathways followed by them. It was suggested that C6-NBD-PC was internalized via receptor-mediated endocytosis (because of its similarity with transfer-

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rin endocytosis) and the colocalization of N-rh-PE with lucifer yellow (a fluid phase endocytic marker) strongly suggested that N-rh-PE followed the fluid phase endocytosis route [232]. Interestingly, the form under which the lipid analog was incorporated into the outer leaflet of the plasma membrane was apparently correlated with the endocytic pathway followed, Sleight and Abanto [162] have shown that the final destinations and the potential mechanisms of the internalization of a PC analog were dependent on the cell lines used. Do different fluorescent lipid analogs undergo different pathways of internalization in a given cell line? Several new elements of information may help to answer this question: (i) the internalization of C6-NBD-SM was studied in CHO-K1 fibroblasts [235]. C6-NBD-SM was internalized via intracellular endocytic vesicles which accumulated near the centriole and were proposed to correspond to a perinuclear endosomal compartment distinct from the more peripheral endosomal compartment [235]; (ii) recently, Mayor et al. [236] analysed the internalization and recycling of C6-NBD-SM, C6-NBD-PC and C6-NBD-galactosyl ceramide in CHO cell lines and compared their results with those obtained for the internalization and recycling of fluorescently labeled transferrin. These authors found that the endocytosis of the fluorescent lipid analogs occurred in a manner that was both kinetically and morphologically similar to that observed for the fluorescently labeled transferrin. They concluded that there exists a default pathway (bulk flow process) for recycling membrane components, as well as transferrin and lipid analogs. Moreover, these molecules would have to be sorted from lysosome-targeted molecules and would not reach the lysosomal compartment; (iii) in agreement, Koval and Pagano [237] found that less than 5% of C6-NBD-SM was delivered to late endosomes and lysosomes. Mayor et al. [236] proposed that recycled molecules (chiefly membrane components) and lysosome-targeted molecules (chiefly luminal components) are sorted in an early 'sorting endosomal compartment' and these molecules are then targeted to late 'recycling endosomal' and 'pre-lysosomal endosomal' compartments, before reaching the cell surface and the lysosomes; (iv) it has been suggested that aggregation may be a general mechanism for the targeting of molecules to lysosomes ([236] and references therein). N-rhPE, which was directed to lysosomes, was incorporated into the plasma membrane in the form of small aggregates [232]. This rapid survey of the literature reveals that phospholipids such as PC and PE can undergo internaliza-

tion, either via endocytosis, like glycosphingolipid analogs, or via other mechanisms consuming less energy. In contrast, the internalization of PS analogs was shown not to follow a vesicular endocytic pathway. This is somewhat surprising, since endocytic vesicles have been found to contain rather high amounts of PS (see below). The internalization of phospholipids insensitive to energy poisons nevertheless required the prior translocation of the fluorescent analogs, or natural lipids (in the case of PS), from the outer leaflet to the inner leaflet of the plasma membrane. This translocation step is probably catalyzed by ATP-dependent translocases located in the plasma membrane [122]. If this is the case (which is likely, because a passive flip-flop would be too slow to account for the translocation rate [122]), it implies that translocation consumes less energy than vesicle budding a n d / o r other molecular events in the early phase of endocytosis. It has been determined that the stoichiometry of the ATP-dependent transport of aminophospholipids is one molecule of ATP consumed for one molecule of aminophospholipid translocated [238]. 5.2. Recycling o f phospholipids between the Golgi apparatus and the E R

The bulk flow theory presented by Wieland et al. [82] assumes that every 10 min half the phospholipid content of the E R will leave the E R to account for the vesicle flow out of this compartment. However, the rate of phospholipid biosynthesis is not sufficient to counterbalance the lipid export from the ER. Consequently, it was suggested that an important and fast recycling of phospholipids from the Golgi apparatus back to the E R must exist [239] and the involvement of phospholipid transfer proteins in this recycling was proposed [53,82,239]. The relationship between membrane traffic and organelle structure was investigated using brefeldin A ([240] for a review). The use of this drug has led to the reconsideration of the organization and dynamics of intracellular organelles and membranes. It was proposed that a retrograde transport of membrane material back to the E R could be achieved via tubular membrane extensions [241] originating from the intermediate recycling compartment involved in protein seggregation [242,243], rather than from the Golgi apparatus. This phenomenon was found to be energy-dependent, with many requirements similar to those of vesicular flow, and was microtubule-stimulated [241]. Hoffmann and Pagano [244] used fluorescent analogs of PE to demontrate an energy-dependent recycling of PE between the Golgi apparatus and the E R of CHOK1 cells and human skin fibroblasts. These authors

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pre-loaded the Golgi apparatus of perforated cells with non-exchangeable fluorescent PE analogs, by fusing liposomes containing the analogs with the Golgi apparatus of these cells [245]. They then demonstrated a time-dependent redistribution of the fluorescent analogs to the ER and the mechanism responsible for this recycling was energy-, temperature- and cytosoldependent and was inhibited by GTPyS [244]. These characteristics resemble those required for the vesicular transport of proteins [129,190,246] and glycosphingolipids in intra-Golgi transport [247] and that of phospholipids at the ER-Golgi apparatus level [142]. The exact nature of the membrane structures involved in this lipid recycling (tubular membranes, or vesicles) is not clear. Since most sphingolipids are thought to be synthesized in the Golgi apparatus [149], specific Golgi lipids would have to escape from this recycling pathway [7]. Lipid sorting events by recycling processes must therefore be considered in parallel with the sorting events occurring during the forward transfer of molecules. This concept of membrane lipid trafficking implies that numerous specific membrane lipid microdomains exist in cellular membranes,

5.3. Exocytic and endocytic counterbalance ofphospholipid flow The balance between the exocytic and endocytic vesicular pathways could explain why, in many cases, no clear contribution of the bulk vesicular flow was observed for the transfer of phospholipids to the plasma membrane. It is obvious that a balance will be effective only if the endocytic flow of phospholipids is high enough compared to the exocytic flow, the ratio of both depending on the cellular growth rate. Many exocytic and endocytic vesicles have been isolated from various sources, but unfortunately, the lipid composition of these vesicles has rarely been determined. The few lipid compositions available concern those of the transition vesicles operating between the ER and the Golgi apparatus of rat liver [188,189] and those of coated vesicles from pig brain [248], bovine brain [249] and adreno-cortical coated vesicles [250]. The phospholipid compositions of these vesicular carriers give some credit to a balance phenomenon and suggest a particular behavior and role for PE and possibly PS. Pearse [248] and Altstiel and Branton [249] determined that coated vesicles isolated from pig and bovine brains have similar phospholipid compositions. The PE and PS contents (expressed as percentages of total phospholipids) of coated vesicles were found to be higher than those habitually observed for plasma membranes. In bovine brain coated vesicles, PE and PS reach 27% and 17% of the total phospholipids respec-

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tively [249]. Moreover, these phospholipids were found to be preferentially located in the outer leaflet of the coated vesicle membrane and this localisation was suggested to be important for the fusion of the coated vesicles with lysosomes [249]. Bomsel et al. [250] also observed an enrichment of PE in plasma membrane-derived coated vesicles isolated from adreno-cortical cells. Endocytic coated vesicles also have a high amount of cholesterol [180,250] and sphingomyelin [249,250]. Finally, the lipid composition of endosomes isolated from chinese hamster V 79-UF cells revealed a high content of PE and an enrichment in PS and sphingomyelin when compared to the plasma membrane [251]. Recently, this specific increase in PE and PS was also observed in transition vesicles isolated following their budding from the transitional elements of the ER [188,189]. These data indicate that PE and PS are important constituents of various carrier vesicles. The extent to which these lipids play a role in vesicle fusion will be discussed later in this review. However, an interesting possibility emerges from these results: PE could cycle between exocytic and endocytic pathways due to its possible universal requirement in vesicular traffic. A direct consequence of this would be that a net vesicular transfer of PE to the plasma membrane might not be systematically detected. In the same respect, other phospholipids are also expected to cycle between exocytic and endocytic pathways. In this case, one can expect that the vesicular flow of phospholipids will frequently be difficult to demonstrate.

6. Lipid metabolism, lipid movement and vesicular trafficking Many cell-free reconstitutions of the vesicular transfer of glycoproteins and the use of yeast sec mutants have allowed the discovery of a considerable number of cytosolic and membrane-bound proteins involved in distinct steps of the budding and fusion processes [129,190,246,252-256]. Far less is known about the role of lipids and particularly of phospholipids in the vesicular transport of membrane components. We will discuss the putative role of phospholipid metabolism in the budding and fusion of vesicles and, in relation to these phenomena, the problem of phospholipid sorting and formation of membrane microdomains.

6.1. Lipid metabolism and vesicle budding Many years ago, Jamieson and Palade [257] determined that the movement of secretory proteins from the ER to zymogen granules in guinea pig pancreatic exocrine cells was dissociated from the synthesis of

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these proteins. A 98% inhibition of protein synthesis by cycloheximide did not stop protein transport to the granules. In other words, these experiments demonstrated that protein transport did not depend upon the continued input of protein into the ER. On the other hand, we can wonder whether lipid synthesis is associated with the vesicular transport of membrane material and secretory products. It is not revolutionary to think that lipid synthesis and vesicle transport are implicated in cell growth, but the question will be more difficult to answer in the case of non-growing secretory cells, in which a balance has to be maintained between the exocytic and endocytic events. Sphingolipids have been shown to be essential for CHO cell growth [258,259]. Similarly, Harel and Futerman [260] have observed that the inhibition of dihydroceramide synthesis by fumonisin B 1 affected axonal outgrowth in cultured hippocampal neurons. Rosenwald et al. [261] have provided evidence that proteins and sphingolipid transport in various cultured cells was slowed by PDMP (1-phenyl-2-(decanoyl-amino)-3-morpholino-l-propanol), which is an inhibitor of glucosylceramide synthesis. At high concentrations of PDMP (75-100 /xM), the delay in VSV G protein and sphingolipid transport to the cell surface of CHO-K1 cells was related to a maximal inhibition of glucosylceramide synthesis and an appropriate 50% decrease in sphingomyelin synthesis. At this point, it can be argued that sphingolipid synthesis is required for vesicle formation by the Golgi apparatus, or in any related event, or that sphingolipids are necessary for the targeting a n d / o r fusion of Golgi-derived vesicles with the plasma membrane. At low concentrations of PDMP (10 /.~M), there was already a maximal inhibition of glucosylceramide synthesis, but a slight increase in sphingomyelin synthesis and no significant change in the half-time of sialylation of the VSV G protein was observed [261]. Hence, the transport of VSV G protein appeared to be impaired only at high concentrations of PDMP. Since glucosytceramide synthesis was already repressed at low concentrations of PDMP, it can be wondered whether the retardation of protein and lipid transport was correlated with the inhibition of glucosylceramide synthesis. Another alternative is that the PDMP-induced alterations in the amounts of glucosylceramide and the increased levels of dimethylsphingosine could modulate the activity of protein kinase C [261]. The shutdown of sphingomyelin synthesis at high concentrations of PDMP [261] should correlate with a variation in the amount of diacylglycerol at the level of the Golgi apparatus. Also, a long-term depletion of glycosphingolipids in PDMP-treated cells can lead to a release of myo-inositol-l,4,5,-triphosphate [262]. More recently, Rosenwald and Pagano [263] have shown that high concentrations (> 20 mM) of exter-

nally-added ceramides (that are probably more concentrated in the distal cisternae of the Golgi apparatus than in the proximal ones [264,265]) inhibit the VSV G protein traffic in CHO-K1 cells. I_x)w concentrations of Co-NBD-Cer corrected the block of axonal growth induced by fumonisin BI [260]. Cer has been shown to stimulate a cytosolic protein-phosphatase activity [266], that is inhibitable by okadaic acid, which leads to the fragmentation of the Golgi apparatus and to the arrest of intracellular transport [267]. Moreover, ceramide has also been shown to activate a membrane-bound protein kinase [268,269]. Furthermore, high amounts of Cer concentrated in the trans-Golgi apparatus could alter the physical properties of this membrane [263] and inhibit the formation of Golgi-derived vesicles. All these features tend to suggest that either vesicular trafficking is coupled to sphingolipid biosynthesis and is impaired when the latter is blocked, or that 'sphingolipid-related cell signalling' could also be involved in a precise regulation of vesicular transport or both. Hence, it is still difficult to evaluate the role of sphingolipid biosynthesis per se in vesicular trafficking insofar as it is not possible to distinguish any effects on other cell functions. Nevertheless, these studies support the view that vesicular trafficking is coupled with sphingolipid metabolism. If there is still a debate concerning the importance of in vivo vesicular flow for phospholipids, it is obvious that phospholipids are present throughout the exocytic pathway(s) and, for example, in ER-derived vesicles [188,189]. The ATP-dependent transport of phospholipids (assumed to be vesicular) has been reconstituted between the ER and the Golgi apparatus of rat liver [142,188,189,194], spinach leaves [270] and leek seedlings [195]. Recently, the question whether phospholipid synthesis can be considered as a driving force for vesicular transport from the ER to the cell surface has been addressed in rat gastric mucosal tissues [271-273]. The ER-to-Golgi apparatus transport of Apomucin was studied in this system. Although the amount of cytosolic proteins was surprisingly high ( x 12 to 60 compared to that of ER membrane proteins present in the assays), it was found that the vesicular transport of Apomucin was dependent on phospholipid synthesizing enzyme activities. The authors proposed that CTPphosphocholine cytidyltransferase and 1,2-diacyl-snglycerol:CDP-choline phosphotransferase activities were associated with the ER-derived transport vesicles [272]. Moreover, these activities could be transiently associated with the vesicles and they have been suggested to be part of the coat material of the vesicles [272]. These new findings are relevant to the hypothesis of coupling between vesicular transport and phospholipid biosynthesis. In good agreement, 80% of PC synthetized de novo is recovered in the ER-derived transport vesicles [271]. That phospholipid synthesizing en-

P. Moreau, C. Cassagne / Biochimica et Biophysica Acta 1197 (1994) 257-290

zymes participate in the dynamics and physical events involved in providing the driving force of vesicle formation is likely [4], but that these enzymes are part of the vesicle coat needs further experimental support under conditions avoiding a too large excess of cytosolic proteins [272]. Moreover, the observation that 1,2-diacylsn-glycerol:CDP-choline phosphotransferase is part of the coat material of the vesicles implies that this enzyme could also be cytosolic, a possibility that does not yet have any experimental support, Two processes of membrane budding have been confronted: the membrane-mediated budding hypothesis and the coat-mediated budding hypothesis [240,274]. The first implies that budding is an intrinsic property of Golgi membranes, not dependent on cytosolic coat proteins and that the latter will only be required for pinching off vesicles. One can consider, at this point, that lipid synthesis is a possible active component of the membrane-mediated budding hypothesis. The coat-mediated budding hypothesis, proposed by Orci et al. [274], suggests that lipid synthesis will eventually furnish the amount of lipid needed, but that both vesicle budding and the pinching off of the vesicles will only be achieved in the presence of coat proteins. The analysis is complicated by the fact that a functional in vitro cell-free system does not necessarily require the addition of the cofactors required for a sustained lipid synthesis. Therefore, we can wonder whether endogenous lipid synthesis is sufficient (or even required), or whether vesicle coat formation is the principal driving force. However, it must be underlined that, in the presence of brefeldin A (which stops the production of coated vesicles), the intercisternal Golgi transport of the VSV G protein is not stopped in vitro [275]; this was explained by a lateral diffusion of the viral protein through tubule networks. As for coated vesicle formation, the formation of these tubule networks is also dependent on ATP and cytosol, but is insentive to GTPy-S [275]. The formation of these tubular merebranes has been suggested to be an intrinsic property of Golgi membranes [276] and was proposed to precede vesiculation controlled by coat formation. It has been proposed that these tubular structures are involved in the retrograde transport to the ER [275], or that both pathways (vesicular and tubular) are involved in intercisternal Golgi transport [277]. In this context, brefeldin A was shown to affect phospholipid and sphingolipid synthesis in a cell-free system from rat gastric mucosa and the inhibition of the lipid synthesizing activities was correlated with a reduction in the production of ER-derived and Golgiderived vesicles [273]. These effects of brefeldin A could support the view that both lipid synthesis and coat proteins are required for vesicle formation. This assumption, however, should not be too rapidly generalized, since the effects of

277

brefeldin A are multiple and sometimes contradictory: for example brefeldin A has been shown both to inhibit sphingomyelin and glycosphingolipid synthesis [273,278] and to stimulate their synthesis [217,279]. Brefeldin A could also promote sphingomyelin hydrolysis in a specific 'brefeldin A-sensitive' pool [280]. Consequently, it is difficult to interpret the results obtained following drug treatment and the conclusions that can be drawn concerning the requirement of lipid synthesizing enzymes in transport vesicle formation are still speculative. Interestingly, when an acute phase response of plasma protein synthesis is induced by turpentinemediated experimental inflammation in rat liver, the tl/2 values for secretion of various proteins was unchanged whatever their rate of synthesis [281]. Therefore, the increase of the amount of protein secreted can either be explained by an increase of the number of transport vesicles (carrying a constant amount of proteins), or by an increase of the amount of proteins present in these vesicles. In the first case, the model of acute phase response should allow to investigate whether an increase of the number of transport vesicles is coupled with a stimulation of lipid synthesis and, therefore, whether lipid synthesis is a possible driving force for membrane budding. Besides the eventual participation of lipid synthesizing enzymes as a driving force in transport vesicle formation, a role of a phospholipid binding-protein in the regulation of the vesicular exocytic route has also been pointed out in Saccharomyces cerevisiae (see section 2.3.2). Another set of experiments has suggested a role for fatty acyl-CoAs in the intra-Golgi transport of the VSV G protein [282]. It has been suggested that the acylation/deacylation of components of the transport machinery would regulate their action. The requirement for acyl-CoAs in both vesicle budding and vesicle fusion has been argued [70,71]. Altogether, these various investigations tend to closely correlate lipid metabolism and the formation of transport vesicles.

6.2. ATP-dependency of lipid transfer ATP-dependency is an important characteristic of the vesicular exocytic and endocytic pathways. Many years ago, Jamieson and Palade [283] determined the metabolic requirements of the intracellular transport of secretory proteins in pancreatic exocrine cells. Many steps of the exocytic pathway(s) and endocytic pathway(s) have since been characterized both in vivo and in vitro. In vitro, various cell-free systems have been employed and have demonstrated the energy requirement of many intracellular transport steps. The ERto-Golgi apparatus transport of proteins has been stud-

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ied in various cell-free systems and was shown to be ATP-dependent [183,193,253,284,285]. The same dependence is observed for the intra-Golgi transport of the VSV G protein [190]. The budding of trans-Golgi network-derived exocytic vesicles is also ATP-dependent [177,286]. Recently, Davidson and Balch [287] reconstituted the transport of the VSV G protein from the ER to the trans-Golgi network and showed that the movement between each compartment was strikingly dependent on ATP and distinct cytosolic proteins, The endocytic traffic has also been shown to be ATPdependent [288-292]. Similarly, the in vivo vesicular transport of sphingolipids [293] has been reconstituted in vitro between Golgi cisternae [247] and from the trans-Golgi network to the apical surface of permeabilized MDCK cells [294]. In both cases, the ATP- and cytosol-dependencies of vesicular sphingolipid transport were similar to those observed for protein transport, Various cell-flee systems have shown that an in vitro ATP-dependent transfer of phospholipids can be reconstituted between the ER and the Golgi apparatus in rat liver [142,188,189,194], spinach leaves [270] and leek seedlings [195]. An ATP-dependent cell-free transfer of lipids has also been observed between nuclei and Golgi apparatus fractions of germinating axes of garden pea [295]. A cell-flee transfer of envelope monogalactosylglycerides to the thylakoids of garden pea and spinach has also been shown to require ATP [296]. However, it was not possible to observe any ATP-dependent transfer of phospholipids between the trans-Golgi apparatus and the plasma membrane of rat liver [297], but, in this case, NADH in combination with ascorbate free radicals stimulated the transfer [297]. These findings were consistent with an involvement of electron transfer reactions described for the trans-Golgi apparatus [298] and for coated vesicles [299] in the dynamics of transport between the Golgi apparatus and the plasma membrane, With the exception of this last case, all the assays developped so far have shown a requirement for ATP. It must be kept in mind that lower amounts of energy can be required ([215], see paragraph 4.4.5). Besides the requirement of energy for protein synthesis, folding and assembly in the ER, ATP is needed for the vesicular transfer per se of membrane material, It has been found that small GTP-binding proteins are involved in the vesicular exocytic and endocytic pathways [300] and that they could participate in the control and regulation of the formation of vesicles (Sar 1 p), the assembly and retrieval of coat proteins (ARF), the targeting of vesicles (Ypt l p / S e c 4 p) and possibly in the fusion itself [252,255]. It has recently been determined that heterotrimeric G proteins are also involved in intracellular membrane traffic [301]. The activity of these proteins is determined by their conformation, the

latter depending on whether they bind GDP or GTP. An obvious explanation for the requirement of ATP would be to control the amount of GTP available. ATP is involved at various stages of the de novo synthesis of phospholipids [3]. Another possibility would be that ATP-dependent aminophospholipid translocation is involved in the triggering of endocytosis and intracellular membrane budding events [302]. The idea is that invaginations of the plasma membrane (for endocytosis) and budding from intracellular organelles (for exocytosis) could be initiated via phospholipid translocation from one leaflet of the membrane to the other. This implies that a small redistribution of phospholipids should be able to induce the phenomenon, whatever the mechanism involved in the phospholipid redistribution. In excellent agreement with this, giant unilamellar egg PC-egg PG (100:1) vesicles (originally at pH 5.5), when submitted to an external pH of 9.5, undergo spectacular shape transformations due to the redistribution of less than 0.1% of the total phospholipids of the membrane [303]. There are arguments that translocating activities are higher in cells where endocytosis is important [302]. A translocase with the proper orientation has been identified in secretory granules [304]. However, a sphingomyelin translocase has been observed in the Golgi apparatus, but with a membrane orientation that is incompatible with a role in vesiclebudding (A. Zachowsky, personal communication). The putative involvement of a translocating activity in ER budding is also controversial due to the existence of the ER flippases [3], which should eliminate any lipid gradient involved in membrane bending. However, a fine regulation of the lipid synthesizing enzymes, translocases and flippases could permit budding towards the cytosol [302]. To conclude on the problem of lipid metabolism and movement in relation to membrane budding, a theoretical model has recently been discussed in which a flat, or weakly curved membrane domain, growing subsequently to the aggregation of molecules which diffuse laterally within the membrane, will become unstable enough upon reaching a certain limiting size to undergo a spontaneous budding, or invagination process [305]. Hence, phospholipid synthesis, which occurs predominantly, but not exclusively, in the cytosolic leaflet of the ER, as well as the transbilayer movement of phospholipids and their lateral diffusion within a membrane, could both take part in the budding of biological membranes. These shape transformations are certainly induced by lipid redistribution and pinching off could be stabilized by a cytosolic protein coating around membrane buds which, by increasing the lateral tension within the membrane [306], would facilitate the fission of the budding vesicle. As pointed out by Devaux [302], coat-

P. Moreau, C. Cassagne/ Biochimica et Biophysica Acta 1197 (1994) 257-290

ing could be induced by membrane invagination or budding, rather than causing it. This model would be more in agreement with the membrane-mediated budding hypothesis exposed earlier, than with the coatmediated budding hypothesis presented by Orci et al. [274]. More experimental investigations will have to be conducted to unravel all the mechanisms involved in membrane budding at the different steps of intracellular membrane traffic. Drugs such as brefeldin A, primaquine, which blocks the formation of membrane buds and vesicles [307] and ilimaquinone, which has recently been shown to vesiculate the Golgi apparatus without effecting ER-to-Golgi apparatus transport [308], could be useful tools. On another hand, more physiological disturbances, such as the use of low temperatures, are also promissing possibilities for investigating in vivo and in vitro mechanisms of membrane traffic, 6.3. L o w temperatures and vesicular trafficking

Low temperatures have an important effect on the vesicular transport of proteins and sphingolipids along the secretory pathway [7,8,203-206]. Similarly, the low temperature block of the ER-to-Golgi apparatus transport of phospholipids has been studied in vitro in a cell-free system from rat liver [142,188]. We have also described that the synthesis and transfer of C20-C24 phospholipids and C16-Cls-phospholipids to the plasma membrane of leek seedlings vary in response to temperature [210]. Our findings suggest the existence of distinct pathways for the transfer of phospholipids with various chain lengths to the plasma membrane. One of these pathways is the conventional ER-Golgi apparatus-plasma membrane pathway, which is highly sensitive to temperature and specifically mediates the transfer of C20-C24-containing phospholipids, In contrast, the transfer of the C16 and C18 fatty acid-containing phospholipids to the plasma membrane is not sensitive to low temperatures. It is rather well documented that 'glycolipid rafts' are part of membrane domains in epithelial cells where lipid and protein sorting can be managed [211-213]. The phase transition temperatures of distinct membrane domains could be different enough to explain - or provoke large variations in the temperature sensitivity of lipid transport originating from these domains, Low temperatures also inhibit the possible vesicular transfer of membrane material between the inner membrane of the chloroplast envelope and the thylakoids of mature chloroplasts in expanding leaves of garden pea, soybean, spinach and tobacco [ 3 0 9 ] . The effect of temperatures ranging between 8°C and 16°C on the ER-to-Golgi apparatus transfer has received special attention in both animal and plant cells.

279

In animal cells, lowering the temperature to 16°C and below leads to an accumulation of transition vesicles and smooth tubular transitional membranes near the cis-Golgi apparatus [206]. Similarly, temperatures of 12°C cause an accumulation of transition vesicles between the ER and the cis-Golgi apparatus in plant cells [210]. The 12°C block in plant cells therefore seems to resemble that of animal ceils at 16°C. In vitro, the low temperature block of the ER-toGolgi apparatus transfer of both proteins [209] and lipids [142,188] has been reconstituted in a cell-free system from rat liver. Similar reconstitutions have been obtained for the ER-to-Golgi apparatus transfer of phospholipids in leek seedlings [195]. Since transition vesicles accumulate at low temperatures [206,210], it is tempting to speculate that these temperatures affect the fusion of ER-derived vesicles with the Golgi apparatus more than they affect vesicle budding and release. In fact, it has been observed that the fusion process itself is not highly sensitive to low temperatures. It has been found that once endocytic markers have passed an unidentified pre-fusion step (a rate-limiting step sensitive to low temperature), the fusion of endosomes with lysosomes can continue even at 16°C [310]. Similady, using a cell-free system from rat liver that reconstitutes ER-to-Golgi apparatus transport, it has been observed that, once formed at 37°C, ER-derived vesicles can fuse with the Golgi apparatus at 20°C, whereas when they are formed at 16°C-20°C, ER-derived vesicles are no longer able to fuse with the Golgi apparatus [188]. We have determined that, at 16°C-20°C, the amount of transition vesicles formed represented around 80-85% of the amount formed at 37°C [188]. Therefore, neither the emission of ER-derived vesicles, nor the fusion of ER-derived vesicles formed at 37°C with the Golgi apparatus, appeared to be directly affected by lowering the temperature to 16°C-20°C. We therefore wondered whether the transition vesicles produced at low temperature were similar to those formed at 37°C and we determined that the phospholipid composition of the vesicles formed at low temperature was significantly different from that of the vesicles formed at 37°C. The amounts of PE and, to a lesser extent, PS were decreased in the vesicles formed at 16°C-20°C, whereas the proportion of PI increased [188]. Insofar as PE and PS are considered as 'fusioncompetent phospholipids' [127], their decrease in the ER-derived vesicles formed at low temperature could explain why these vesicles did not fuse with the Golgi apparatus at 37°C [188]. Recently, Veit et al. [311] analysed the effect of ilimaquinone on the Golgi apparatus at various ternperatures. Ilimaquinone disassembles the Golgi apparatus into small vesiculated Golgi membranes (VGM~ [308]).Veit et al. [311] found that, while VGM forma-

2~(~

P. Moreau, ('. Cassagne /Biochimica et Biophysica Acta 1197 (1994) 257-290

tion is inhibited at 16°C, the VGM S are able to fuse and reassemble into stacks of Golgi cisternae at 16°C. That the fusion of VGM S is not affected at 16°C confirms preceding results obtained for endosomelysosome fusion and ER-derived vesicle fusion with the Golgi apparatus. However, the production of VGM~ by ilimaquinone appeared to be stopped at 16°C [311], whereas the formation of ER-derived vesicles is not dramatically decreased at this temperature [188]. The discrepancy between these results could be due to the use of different experimental approaches, or simply results from the fact that the situation observed at the level of the Golgi apparatus is not necessarily the same as that observed for the ER. Moreover, it has also been shown that, after brefeldin A treatment, there is no reversibility of Golgi assembly at 16°C as was observed with ilimaquinone [311]. The big difference between brefeldin A and ilimaquinone is that the first drug blocks the ER-toGolgi apparatus transport, whereas ilimaquinone does not [308]. In addition, Golgi reassembly after ilimaquinone treatment does not depend on the ER-toGolgi transport, as it does after brefeldin A treatment, Consequently, and in agreement with our findings [188], it is not surprising that Golgi reassembly is sensitive to low temperature in one case, but not in the other, Our findings [188] raise the question of the role of the lipid and protein compositions of the vesicles produced at low temperature and introduce the possibility that membrane domain formation and the lateral sorting of membrane phospholipids and other constituents could be sufficiently altered at low temperature to result in the production of abnormal vesicles,

6.4. Membrane lipid domains and the sorting of lipids Sorting has been defined as a process by which the ratio between two membrane components in a transport vesicle becomes different from that observed in the starting compartment [312]. The differences in the phospholipid compositions of the various intracellular compartments and the plasma membrane (see for example Table 1) could mean that some phospholipid sorting, or at least some specific partitioning of these molecules, occurs, or that the turnover rates are different for each lipid, For example, the PS gradient observed between the ER and the plasma membrane of rat liver (Table 1) and the enrichment of PS in the apical and basolateral domains of the plasma membrane of epithelial cells [313], strongly suggest a specific targeting of PS to the plasma membrane but does this necessarily mean that there is sorting? Effectively, the same situation is observed for sphingomyelin (Table 1). However, sphingomyelin is not sorted between the apical and basolateral domains of the plasma membrane in epithelial

cells, as is the case for glucosylceramide molecules [314]. At first sight, it does not appear that there is any sorting of PS between the two domains [313]. In vitro, with a cell-free system from leek seedlings reconstituting the vesicular ATP-dependent ER-to-Golgi apparatus transfer of phospholipids, we observed a selectivity of transfer whereby PI seemed to be 'sorted' from the other phospholipids so that it was not included in the transport vesicles [195]. Therefore, we have to consider the possible existence of different levels of 'sorting'; one, at least, which would control the amount of a given lipid that will enter the transport vesicle for a given destination and one that would determine the target membranes of the different vesicular carriers of specific compositions. Epithelial cells, whose apical and basolateral plasma membrane domains have different lipid and protein compositions, constitute a particularly appropriate systern to address the problem of the sorting of membrane constituents [211,315]. They have been especially useful in studying the polarity of glycosphingolipids and the potential mechanisms of their sorting [211]. The tendency of glycosphingolipids to undergo hydrogen-bonding leads them to self-aggregate, which could result in the formation of membrane microdomains [211]. The formation of specific microdomains in the sorting of glycosphingolipids, glycoproteins and GPI-anchored glycoproteins, has been studied in various systems [212,213,316]. Phospholipids do not have the ability to form interlipid hydrogen bonds with the sphingolipids and, hence, could be excluded from the sphingolipid microdomains of the exoplasmic leaflet [211]. PC is enriched in the basolateral membrane as compared to the apical membrane and, therefore, would be concentrated in domains of the trans-Golgi membranes other than those destined for the apical membrane [211]. The transbilayer asymmetry of glycosphingolipids [7] and phospholipids [122,302], as well as their lateral mobility in the plane of the membrane, most probably contribute to the segregation of the lipid molecules into specific domains. The lateral diffusion of phospholipids is largely dependent on the composition of the merebrane [317]. Interestingly, it has also been shown that the lateral diffusion of fluorescent analogs of various phospholipids is much faster in the cytosolic leaflet than in the exoplasmic leaflet of the plasma membrane in fibroblasts [318] and bovine aortic endothelial cells [319]. Epithelial cells provide the opportunity to investigate the sorting of phospholipids, as well as that of glycosphingolipids. The phospholipid compositions of the apical and basolateral domains of polarized MDCK cells have been determined [313]. It is clear that, whereas phospholipids are more abundant in the basolateral membrane [211], only slight differences between

P. Moreau, C. Cassagne / Biochimica et Biophysica Acta 1197 (1994) 257-290

the amounts of PE and PS are observed in the two domains [211,313] and, in particular PC is found mostly in the basolateral membrane of polarized cells [313]. This situation disappears in non-polarized BHK cells [313]. Insofar as phospholipids are free to diffuse in the cytoplasmic leaflet of the plasma membrane of polarized epithelial cells, but not in the exoplasmic leaflet (due to the tight junctions, 320), and that phospholipids (like sphingolipids) have an asymmetric distribution [313], the following points can be discussed with respect to PE and PC: (i) due to the cytoplasmic leaflet localization of PE (65-90%, [313]) and its free lateral diffusion between both apical and basolateral membrane domains, it can be expected that PE will be randomly distributed in both domains and, thus, it is likely that no sorting of PE between the two domains is required; (ii) assuming that PC (more abundant in the basolateral membrane) is largely found in the exoplasmic leaflet of the basolateral domain (75-90%, [313]) and is not allowed to diffuse into the apical domain, it follows that either PC is more abundant in Golgi-derived vesicles destined for delivery to the basolateral membrane than in those destined for delivery to the apical membrane, or that the transport pathway to the basolateral membrane is more efficient in delivering PC than is the pathway to the apical membrane; (iii) the polarity of the transport of glucosylceramide is interpreted as indicating a sorting of this molecule

APICALDOMAIN(HIGHGSL/PCRATIO)

~ " ?

~ APICAL VESICLES Pc s0 PE/PS 20 ~

BASOLATERAL GsLVESICLES25

CHOL 30

PC PE/PS CHOL ----> (~) __> (~)

30 15 30 ----> ....>

~ > ~ ~

~ -~ ~ ~ ~ "~

Fig. 2. Transport and sorting of glycosphingolipids and phospholipids to the apical and basolateral domains of plasma membrane epithelial cells. An oversimplified lipid composition (mol%) of the apical and basolateral transport vesicles is proposed. The thick membranes have a high G S L / P C ratio. Adapted in part from Simons and van Meer [211], van Meer and Simons [313] and van Meer et al. [320]. The difference in PC content of both plasma membrane domains could also be caused by the regulation of local synthetic and degradative activities [150,321]. GSL, glycosphingolipids+sphingomyelin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phos-

phatidylserine;CHOL, cholesterol,

281

between the two domains [211,314]. It is tempting to suggest that there is also an opposite and concomitant 'sorting' of PC [211]. It must be pointed out, however, that the difference in the content of PC between both plasma membrane domains could also be caused by the regulation of a local synthesis or degradation, thus moderating the requirement of a selective intracellular lipid transport [150,321]; (iv) the lipid compositions of the Golgi microdomains and of the transport vesicles issued from the latter, could mimick those of the respective plasma merebrahe target domains ([211], Fig. 2); (v) the presence of similar, non-negligible amounts of PE (and to a lesser extent of PS) in both the transport vesicles and their respective target merebrane domains, could indicate the importance of these phospholipids in membrane fusion (see next chapter). Another important question that arises concerns the determination of which parts of the lipid molecules are determinant for the sorting process. Recently, it was observed that the efficiency of sphingolipid sorting in MDCK and CaCo 2 cells was not qualitatively affected by modifications of the sphingosine backbone, or by decreasing the fatty acyl chain length of these lipids [322]. However, it must be pointed out that glycosphingolipids frequently possess very long-chain fatty acids of up to 24-26 carbon atoms [323]. The fatty acyl chains of these lipids may cross the midplane of the bilayer, thereby modifying the lateral diffusion properties of these lipids, a n d / o r enhancing specific interactions with other membrane components (such as cholesterol, or receptor proteins), which could also contribute to their clustering in specific domains [323]. For phospholipids, the possibility of sorting based on the fatty acyl chain length has been suggested using two different systems. Longmuir and Haynes [324] determined that the lamellar body material from type II cells, i s o l a t e d f r o m c u l t u r e d r a b b i t lung tissue, was highly enriched in species of PC and (to a lesser extent) PE, containing fatty acids with 14 and 16 carbon atoms and was poor in PC species containing fatty acids with 18 carbon atoms. These results led the authors to propose that a cellular phospholipid-sorting process which selects PC species according to their fatty acid chain length (and not their saturation or unsaturation) w a s responsible for the enrichment of specific phospholipids in the pulmonary surfactant [324]. I n a similar fashion, it was observed that C20-C24 phospholipids in leek seedlings are routed to the plasma membrane through the ER-Golgi apparatus-plasma membrane pathway and that their transport is blocked, or slowed, by monensin and low temperatures [158,210]. On the other hand, C16-C18 phospholipids are still transferred to the plasma membrane [158,210]. It was postulated

P. Moreau, C Cassagne / Biochimica et Biophysica Acta 1197 (1994) 257-290

282

CI6-C18PL ) O

ER "~ C20-C24PL

~

) 0w " ~

20 ~

!C'2

L

cis Med T . . . . ) O C16-C~SPL

l~

vesicles effectively retain cholesterol in proportion to the amount of PC molecules present, but these authors

)

v-~'c2°92

M

M PLASMA

• ~

P~L MEMBRANE

~

o - 4

TGN

)

Fig. 3. In vivo transfer pathways of phospholipids with various fatty acid chain length to the plasma m e m b r a n e of leek seedlings. C20-C24 PL: phospholipids having at least one very long chain fatty acid of 20 to 24 carbon atoms are routed through the E R to Golgi apparatus to plasma m e m b r a n e pathway and their transfer to the plasma membrane is blocked by monensin and at low temperatures (below 12°C). C16-C18 PL: phospholipids without very long chain fatty acid can

follow the ER-Golgi-plasma membrane pathway but in the presence of monensin or at low temperatures, their transport to the plasma

membrane is not arrested, suggesting that other routes such as a direct ER to plasma membrane transfer and/or a direct cis-medial Golgi to plasma membrane transfer can occur. ER, endoplasmic reticulum; Cis, Med and Trans correspond to the cis, medial and trans cisternae of the Golgi apparatus; TGN, trans Golgi netwotk; M. monensin (the possible localizations of monensin blocks are indicated); T°, temperatures below 12°C (at 12°C the vesicles between the ER and the Golgi apparatus accumulate whereas the number of secretory vesicles is highly decreased and the surface of the T G N is increased as compared to 24°C). Results adapted from Bertho et al. [158] and Moreau et al. [155,210].

that the phospholipids could be targeted (sorted?) to different intracellular pathways according to their fatty acyl chain length ([158], Fig. 3). It can be suggested that a particular form of phospholipid 'clustering' prior to exportation could initiate the formation of C20-C24 and C16-Cts lipid domains in intracellularmembranes, as was proposed for the selective endocytosis of lipids [325] and for the export of glycosphingolipids and glycoproteins [212,213,316,326]. The interactions of the polar head groups of phospholipids with other membrane constituents and, for example, with cholesterol must also be considered. It is well known in model systems that sphingomyelin and PC have a higher affinity for cholesterol than do PE or the anionic phospholipids. These properties are used to explain the heterogeneous distribution of cholesterol in the plasma membrane [327]. It has also been determined that 75-80% of cholesterol is located in the inner leaflet of the human erythrocyte membrane [328], whereas PC and sphingomyelin are predominantly located in the outer leaflet [122,302]. Therefore, it would be too simple to explain the transmembrane distribution of cholesterol in erythrocytes only on the basis of specific interactions with sphingomyelin or PC. Jacobsohn et al. [329] also observed that mixed phospholipid

also showed that the length and unsaturation of the fatty acyl chains, the degree of hydratation of the head-groups and the interactions between juxtaposed charged groups as a function of the pH, had significant effects on the interactions ofphospholipids with sterols. It is clear that lipid-protein interactions and their lipid selectivity [330] have to be included among the parameters that modulate the behaviour of the phospholipids. Consequently, we can imagine that specific microenvironments (with specific pH domains, for example) will determine specific interactions between various merebrane components. These interactions will result in the formation of specific membrane microdomains, n o t only for glycoproteins and glycosphingolipids, but also f o r phospholipids. A clustering of polyunsaturated species of phospholipids (PC and PE) has been proposed to occur in Golgi-derived coated vesicles and plasma membranerelated coated pits in adrenocortical cells [331]. Such results indicate that, in order to understand microdomain formation and the eventual sorting of phospholipids, we will have to consider and analyse the molecular species of phospholipids [324].

7. Phospholipids and biological membrane fusion Membrane fusion has been extensively studied in the case of model membranes [127,332,333] and, more recently, in that of biological membranes [190,334,335] and common features have emerged from various systerns studying viral, cell-cell, intracellular and liposomal fusion events [334]. The understanding of the role of cytosolic and membrane proteins in membrane fusion has progressed rapidly over the last years [190,334-336] and numerous models for biomembrane fusion have been described [332, 334-336]. Lipid polymorphism and its role in model membrane fusion has been largely studied [127,333,337,338]. Artificial model membranes are relatively homogeneous and the results obtained with these systems cannot necessarily be extrapolated to natural biological membranes, characterized by an asymmetry and the existence of membrane domains [337]. Progress has recently been made with respect to the role of phospholipids and their metabolism in biomembrane fusion and we will rapidly discuss some aspects of these findings here. First, it has been shown recently that the fluidity (lateral diffusion) of the inner leaflet of the plasma membrane is higher than that of the outer leaflet [318,319]. This would be in agreement with a competency of the cytosolic leaflet of the plasma membrane for fusion with secretory vesicles. It is not known

P. Moreau, C. Cassagne / Biochimica et Biophysica Acta 1197 (1994) 257-290

whether this is also true for the cytosolic leaflet of intracellular vesicles and membranes engaged in fusion. Phospholipid asymmetry has also been suggested to play a major role in membrane fusion. Insofar as it has been established that PE and PS in model membranes are more fusogenic than PC and sphingomyelin [127], it is relevant to consider that PE and PS are predominantly found in the cytoplasmic leaflet of the plasma membrane and some organelle membranes and are randomly distributed between the two leaflets of intracellular membranes [122]. This indicates that sufficient amounts of PE and PS, compatible with intracellular membrane fusion, will be present on the cytoplasmic interfaces. The lipid asymmetry created by the aminophospholipid translocases has also been proposed to induce, or maintain, a surface tension of small carrier vesicles, avoiding surface undulations that would be incompatible with their fusion [339]. The redistribution of PS to the exoplasmic leaflet is associated with the CaZ+-phosphate-induced and osmotically-induced fusion of human erythrocytes [340342]. On the other hand, the fusion of Influenza virus with erythrocytes does not appear to depend on the transbilayer phospholipid distribution [343]. It is generally assumed that membrane fusion includes at least three distinct steps: membrane aggregation, bilayer destabilization and merging of the membrane components (and aqueous ones for complete fusion) [127]. For the close apposition of two merebranes to occur before fusion, strong repulsive hydration forces must be overcome [127]. It has been suggested that weakly hydrated lipids, such as PE, could concentrate in the fusion region [344] and contribute to lower the hydration repulsion. Moreover, PE could participate in destabilizing the membrane bilayer due to its tendancy to adopt a non-bilayer conformation [127]. Indeed, nonbilayer phase-forming lipids such as PE [127,337,338] have been proposed to induce the formation of inverted micellar intermediate structures leading to fusion [345]. These structures do not correspond to H n phases [345,346]. It has also been observed that physiological amounts of diacylglycerols, produced for example during the PI cycle, could stabilize these nonbilayer phases and promote membrane fusion [347]. Moreover, phospholipase C-induced fusion of PC/PE/cholesterol vesicles indicated that fusion was stimulated by diacylglycerol molecules only within a certain range of concentrations (below and above these concentrations, fusion was inhibited), suggesting a possible physiological control of the fusion process by this system [348]. The central role of PE in membrane fusion has been observed in various studies. Liposomes and cytochrome

283

c oxidase proteoliposomes made with cardiolipin, PC and PE, undergo calcium-induced fusion only for PE contents greater than 10% [349], suggesting the requirement of sufficient amounts of such nonbilayer phase-forming lipids. The delay in myoblast fusion induced by cesium has been correlated to changes in the phospholipid composition of myoblast membranes [350,351]. These changes indicated that, during the fusion process, a decrease of bilayer-stabilizing phospholipids (such as PC) and an increase of bilayer-destabilizing phospholipids (such as PE and PA) occur [350,351].Studying the in vitro fusion of rabbit liver Golgi membranes with small and large unilamellar vesicles, Kagiwada et al. [352] have shown that integral Golgi membrane proteins were involved in fusion and that the addition of PE or PS resulted in a two-fold increase in fusion. Recently, using a cell-free system from rat liver reconstituting the ER-to-Golgi apparatus transfer of lipids [142,194], we investigated the potential role of PE and PS in the fusion of ER-derived vesicles with the Golgi apparatus [188]. We found that fusion-incompetent ER-derived vesicles produced at low temperatures (16°-20°C) have a much lower content of PE than fusion-competent ER-derived vesicles produced at 37°C [188]. Moreover, we observed that only the addition of dioleoyl-PE (and not dipalmitoyl-PE) and, to a lesser extent, the addition of bovine brain PS, restored the fusion capacity of ER-derived vesicles formed at low temperatures [188]. Besides the effect of low temperatures on the phase transition of lipids [127], low temperatures must also have disturbed the formation of the ER-derived vesicles, as judged by their phospholipid composition. Altogether, these studies argue in favor of a key role of PE in membrane fusion. Besides the proposed roles of PE and PS in biological membrane fusion, the participation of phospholipid metabolism has also been described. Evidence for an important role of inositol phospholipid breakdown in myoblast membrane fusion has been obtained [351,353,354].Jolicoeur et al. [355] observed that the GTP-stimulated fusion of RER membranes could be sustained in the absence of GTP when the RER merebranes were incubated with cofactors (particularly CTP) required for the synthesis of PI. Further investigations led the authors to consider that a CTP-dependent formation of diacylglycerol and an accumulation of polyunsaturated free fatty acids [356,357], which are known to be fusogenic [347,358,359], could have been involved. Other aspects of the role of phospholipid and fatty acid metabolism in membrane fusion include the putative role of phospholipases A 2 and the products of their activity (unsaturated free fatty acids and lysophospholipids), the fatty acylation of proteins and the requirement of lipid deacylation-reacylation cycles.

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Initiation and Progress of membrane fusion

Involvement of nonbilayer intermediates structures with nonbilayer forming lipids such as PE present in the ER-derived vesicles and the cytosolic leaflet of the cis Golgi membrane. ~L

Lale stens in membrane Fusion .Step 1: Conversion of nonbilayer structures to lamellar structures by the addition to PE of lysoPC formed by a Golgi lumenal or cytosolic phospholipase A-mediated hydrolysis of PC. Step 2: Stabilisation of the membrane bilayer by reacylation of lysoPC to PC by an acyl-CoA-lysoPC acyltransferase localised in the cis-Golgi membrane, Scheme 1. Working hypothesis for the involvement of PE, lysoPC and deacylation-reacylation cycles involving phospholipase A and acyI-CoA-lysoPC acyltransferase in the fusion of ER-derived vesicles with the cis-Golgi apparatus in a cell-free system from rat liver, From Moreau and Morr~ [194], Moreau et al. [371] and Lawrence et al.

[371].

Fatty acylation of one (or more) component of the transport machinery has been suggested to be required in the cell-free transport of glycoproteins from the cis to medial Golgi cisternae [71]. In intact pancreatic acini, the glycerophospholipids undergo a deacylation-reacylation process activated by secretagogues [360]. Membrane-bound phospholipase A 2 and acyltransferase activities have been identified in pancreatic zymogen granules [361]. A direct or indirect role of phospholipase A 2 activity in membrane transport (and potentially membrane fusion) has been suggested in various systems, such as the fusion of chromaffin granules with the plasma membrane [362], the cell-free transport of glycoproteins from cis to medial cisternae of the Go[gi apparatus [363], the cell-free transfer of apomucin from the ER to the Golgi apparatus of rat gastric mucosa [364] and endosome fusion [365]. Lyso-PC has long been considered as a putative biological fusogene [366]. However, recent findings concluded that lyso-PC and lyso-derivatives in general were clearly inhibitors of cortical granule exocytosis, granule-granule fusion, GTP-dependent fusion of rat liver microsomal membranes and baculovirus infected cell-cell fusion [367]. Lyso-PC and other lyso-derivatines were suspected, due to their cone-like shape (with a larger cross-sectional area at the headgroup-water interface than at the extremity of the hydrocarbon tail [337]), to restrict the formation of a highly curved stalk, an early fusion intermediate connecting the two membranes [332]. Therefore, these compounds inhibited

membrane fusion at a stage preceding fusion-pore formation [335,367]. Lyso-PC has recently been used to dissect the subsequent steps of exocytosis and viral fusion [368]. On the other hand, it was postulated that, in terms of the stalk hypothesis, it could be considered that lyso-PC might participate at a later stage, by causing the transition of monolayer fusion to complete fusion [332]. In a cell-free system from rat liver reconstituting the transfer of lipids from the ER to the Golgi apparatus, we observed that the ATP-dependent vesicular transfer of membrane lipids was accompagnied by the appearance of lyso-PC in the Golgi apparatus [194]. Moreover, the formation of lyso-PC was only observed under conditions optimal for the vesicular transfer and did not occur in the absence of ATP, or at low temperature [194]. We also determined, using this cell-free system, the importance of the amount of PE in the transfer vesicles for their fusion efficiency [188]. Due to the well known opposite cone-like shapes of PE and lyso-PC [332,337], w e p r o p o s e the following s e q u e n c e involving PE and lyso-PC at a late stage of the fusion of ER-derived v e s i c l e s w i t h t h e cis-Golgi apparatus in rat liver (Scheme 1): (i) After fusion has progressed via the formation of nonbilayer intermediate structures requiring PE, the production of [yso-PC would contribute to recover a bilayer conformation at the end of the fusion process. (ii) The acylation of [yso-PC to form PC would then definitively stabilize the bilayer. This hypothesis is further supported by the fact that a phospholipase A activity has been shown to be present predominantly in the cis-Golgi apparatus of rat liver [194]. However, the phospholipase could also be recruited from the cytosol [369,370]. A lyso-PC:acy]CoA acyltransferase activity has been recently observed in the cis- and medial-, but not the trans-cisternae of the Golgi apparatus [371]. This activity was inhibited by manoalide, an inhibitor of phospholipase A 2 activity [363]. Lastly, it is tempting to suggest that the formation of nonbilayer intermediate structures in membrane fusion would induce either the recruitment of a cytosolic phospholipase A modulated by the ratio of non-bilayer- to bilayer-forming lipids [369,370,372, 373], or the activity of a membrane-bound phospholipase a activity [372,374]. This model could be 'relatively specific' to the ERto-Golgi apparatus step due to the restricted ]ocalization of deacylating and reacylating activities. of events

8. Conclusion and perspeetivies Due to the recent interest in lipid trafficking which has arisen from different in situ and in vitro ap-

P. Moreau, C. Cassagne / Biochimica et Biophysica Acta 1197 (1994) 257-290

proaches, our knowledge of the intracellular transfer of lipids, the formation of m e m b r a n e lipid domains and lipid sorting is evolving rapidly. The existence of multiple vesicular pathways and distinct mechanisms for phospholipid transfer, and lipid transfer in general, between distinct subcellular compartments can be suspected. Moreover, studies dealing with the transfer of phospholipids from the E R to the mitochondria and those analysing the ER-Golgi apparatus-plasma m e m b r a n e pathway strongly suggest that the spatial organization of the E R and the Golgi apparatus, as well as the existence of specific m e m b r a n e domains, are of particular relevance to the understanding of the specificity of the transfer of phospholipids and m e m b r a n e material in general. The higher efficiency of homologous transfer between the E R and the mitochondria [94] and between the E R and the Golgi apparatus in intact heterokaryons [375], as compared to heterologous transfer, is a good support for an important role of the spatial organisation of the E R and the existence of a 'specific proximity' between various organelles. As pointed out by Valtersson et al. [375], intracellular organelle interactions could be spatially restricted, or exhibit some cell-type specificity, or both. It is certain that an important progress can be expected in this area of research in the future years. Another area of research which will be developped concerns the formation of specific m e m b r a n e domains and the sorting of lipids between different domains of the same m e m b r a n e and between various subcellular compartments. In this respect, it will be necessary to discriminate between the numerous molecular species of phospholipids and other lipids. Finally, a great improvement of our knowledge o n the role(s) of phospholipids and other lipids in the events governing and regulating the various intracellular transfer pathways of m e m b r a n e traffic can be expected in the near future.

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Recherche et de l'Espace' and the 'Minist~re de l'Industrie et du C o m m e r c e Ext6rieur' (France).

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