Bridging gaps in phospholipid transport

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Vol.30 No.7 July 2005

Bridging gaps in phospholipid transport Dennis R. Voelker Program in Cell Biology, Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206, USA

Phospholipid transport between membranes is a fundamental aspect of organelle biogenesis in eukaryotes; however, little is know about this process. A significant body of data demonstrates that newly synthesized phospholipids can move between membranes by routes that are independent of the vesicular traffic that carries membrane proteins. Evidence continues to accumulate in support of a system for phospholipid transport that occurs at zones of apposition and contact between donor membranes – the source of specific phospholipids – and acceptor membranes that are unable to synthesize the necessary lipids. Recent findings identify some of the lipids and proteins that must be present on membranes for inter-organelle phospholipid transport to occur between the endoplasmic reticulum and mitochondria or Golgi. These data suggest that protein and lipid assemblies on donors and acceptors promote membrane docking and facilitate lipid movement.

Introduction The maturation of organelles within eukaryotic cells primarily requires the selective transport of specific proteins and lipids to the limiting membrane and interior of the developing structures. In the past two decades, large amounts of information and fine mechanistic detail about protein sorting to many organelles has been obtained [1,2]. By contrast, our understanding about the process of phospholipid transport for the purpose of new organelle assembly remains small. However, recent advances, especially the identification of mutant strains of yeast and mammalian cells, are now providing the tools to begin to unravel some of the complexities of phospholipid transport. This discipline is in the early stages of identifying some of the molecules involved in transport and experimenting to test their mechanisms-of-action. These endeavors are providing a framework for how a few of these polar lipids can be transported. One feature of intermembrane phospholipid transport that is not widely appreciated is the resistance of the process to inhibitors that affect membrane protein and secretory protein transport. This resistance is surprising from several perspectives. It is well established that many membrane and secreted proteins travel between organelles in vesicles whose structure is primarily defined by Corresponding author: Voelker, D.R. ([email protected]). Available online 13 June 2005

phospholipid [3]. Thus, some phospholipid movement between organelles must occur via vesicles. When the production of these vesicles is arrested by mutation [2] or intoxication with poisons such as brefeldin A [4,5], neither the lipid in the vesicle nor the membrane proteins or encapsulated cargo proteins are delivered to their destinations. However, in several reports the traffic of biosynthetically radiolabeled phospholipids between organelles proceeds unabated under the same conditions that arrest membrane or secreted protein transport [6–9]. In addition, reconstitution studies with permeabilized cells and isolated organelles in many cases fail to demonstrate any dependence of newly synthesized phospholipid transport upon cytosolic factors, ATP or GTP [10–16]. These findings strongly support a mechanism for phospholipid transport between many organelles that can proceed via routes that are independent of vesicle formation, migration and fusion. It is not clear why there should be a non-vesicular mechanism to move phospholipids between membranes or why it should be so predominant. One possibility is that non-vesicular phospholipid transport might be an evolutionarily primitive system for moving components between membranes that preceded the development of vesicular mechanisms. The efficiency and efficacy of the non-vesicular transport could be such that it has been retained as a default mechanism for the majority of phospholipid transport. Progress in addressing the mechanisms of intracellular phospholipid transport in eukaryotes has been hampered by the lack of strong genetic selections and screens, and also convenient biochemical methods for measuring the processes. Despite these impediments, modest progress continues to be made and a growing number of mutants have been isolated from mammalian cells and yeast that now implicate specific genes and their products in the processes [17–21]. Most importantly, the genetic advances provide crucially important raw materials for reconstitution studies that can be used to probe the mechanisms of phospholipid transport. Organelle specific metabolism of aminoglycerophospholipids One combined biochemical and genetic approach for examining phospholipid transport makes use of the organelle specific metabolism of the aminoglycerophospholipids (Box 1), phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn) and phosphatidylcholine

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Box 1. Aminoglycerophospholipids In many eukaryotic cells, including those from mammals and the yeast Saccharomyces cerevisiae, the aminoglycerophospholipids comprise w70–80% of the total phospholipids present in cell membranes. The structures of these lipids are shown in Figure I. On average, intracellular membranes in many eukaryotes have a phospholipid composition of 50% PtdCho, 10–25% PtdEtn and 1–10% PtdSer. Yeast can synthesize the

Phosphatidylserine H 2C HC H 2C

full complement of their aminoglycerophospholipids by decarboxylating PtdSer and methylating PtdEtn. The methyl groups are transferred to PtdEtn from S-adenosylmethionine. Nucleated mammalian cells also synthesize PtdSer and decarboxylate it to form PtdEtn. However, with the exception of hepatocytes, mammalian cells do not synthesize significant amounts of PtdCho from PtdEtn.

Phosphatidylethanolamine

O R1

H 2C

O R2 CO2 O O P O CH 2 CH NH3 O–

HC H 2C

O R1

Phosphatidylcholine H 2C

O R2 O O P O CH 2 CH2 NH 3 O–

HC H 2C

O R1 O R2 CH 3 O O P O CH 2 CH2 N CH 3 O– CH 3

Figure I. Phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) comprise a family of lipids known as the aminoglycerophospholipids. These lipids are closely related structurally and linked metabolically. PtdSer is decarboxylated to form PtdEtn, which is subsequently methylated to form PtdCho. The CO2 moiety (red) of PtdSer that is removed to form PtdEtn and the methyl groups (green) that are added to PtdEtn to form PtdCho are shown.

(PtdCho), as shown schematically in Figure 1. In the yeast Saccharomyces cerevisiae, PtdSer is synthesized in the endoplasmic reticulum (ER) and a subdomain of this organelle known as the mitochondria-associated membrane (MAM) [15]. After its synthesis, PtdSer is transported from the ER and MAM to numerous organelles including the plasma membrane, mitochondria and Golgi. Upon arrival of PtdSer at the mitochondria, it is imported to the inner membrane where it becomes a substrate for PtdSer decarboxylase 1 (Psd1p; GenBank accession number: NP_014230) [22], which catalyzes the conversion to PtdEtn. Transport of PtdSer to the Golgi also provides the substrate for PtdSer decarboxylase 2 (Psd2p; NP_011686) and generates PtdEtn in this locale [23,24]. In either case, the formation of PtdEtn constitutes a chemical reporter for the transport of PtdSer to the respective organelles. The subsequent transport of PtdEtn out of the mitochondria or Golgi to the ER results in the further metabolism of a significant portion (but not all) of

this lipid to PtdCho by the combined actions of PtdEtn methyltransferases 1 and 2 (Pem1p and Pem2p; NP_011673 and NP_012607, respectively) [25,26]. This synthesis of PtdCho (from what was originally PtdSer) constitutes another chemical reporter for the transport of PtdEtn from the loci of Psd1p and Psd2p to the ER. Mammalian cells also synthesize PtdSer in a MAM fraction [27] and transport the lipid to the mitochondria where it is decarboxylated by Psd1p [28]. However, mammalian cells have not been reported to contain a Psd2p enzyme and, with the exception of hepatocytes, do not methylate the resultant PtdEtn to form PtdCho. Consequently, the majority of studies with mammalian cells use the action of Psd1p to examine PtdSer transport to the mitochondria [29]. Genetic screens for phospholipid transport mutants The organelle specific metabolism of PtdSer and PtdEtn has been used to develop genetic screens in yeast to

Mitochondria Psd1p PtdSer PtdEtn Pss1p Ser PtdSer ER/MAM

PtdEtn

PtdSer

Psd2p PtdEtn

Pem1p Pem2p

PtdCho

ER

Golgi Ti BS

Figure 1. The transport and metabolic itinerary of the aminoglycerophospholipids. PtdSer is synthesized in the endoplasmic reticulum (ER) and closely related mitochondriaassociated membrane (MAM) by the action of PtdSer synthase (Pss1p). After synthesis, the PtdSer is transported to other organelles. Upon arrival at the mitochondria, PtdSer is imported to the inner membrane and decarboxylated by the Psd1p to form PtdEtn. Likewise, when PtdSer arrives at the Golgi it is decarboxylated by Psd2p to form PtdEtn at this location. The PtdEtn is exported from either organelle to the ER, where it is sequentially methylated in three reactions by PtdEtn methyltansferases 1 and 2 to form PtdCho. The arrows between the organelles define major transport steps for these phospholipids. All steps shown occur in yeast. In mammalian cells, PtdSer is transported to Psd1p and the PtdEtn is subsequently transported out of the mitochondria. In mammals, only hepatocytes express significant levels of PtdEtn methyltransferase. www.sciencedirect.com

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(a) PSTA and PEEA pathways Mitochondria Ser

PtdSer

PtdSer

ER

PtdEtn

PtdEtn

Etn

MAM

pstA mutations

peeA mutations

PtdCho

(b) PSTB and PEEB pathways Golgi Ser

PtdSer

PtdSer

ER

PtdEtn

PtdEtn

Etn

ER

pstB mutations

peeB mutations

PtdCho

Ti BS

Figure 2. Genetic screening in yeast for phospholipid transport mutants. (a) In psd2D mutant strains lacking Psd2p, aminoglycerophospholipid synthesis proceeds along the PtdSer transport A and PtdEtn export A pathways (PSTA and PEEA), which involve lipid movement into and out of the mitochondria. The psd2D strains can be mutagenized and then screened for Etn auxotrophs. Among the Etn auxotrophs will be new strains with defects in PtdSer transport (pstA mutants) and PtdEtn export (peeA mutants). The PSTA portion of the pathway is highly active in mammalian cells and in yeast. (b). In psd1D mutant strains lacking Psd1p, aminoglycerophospholipid synthesis proceeds along the PtdSer transport B and PtdEtn export B pathways (PSTB and PEEB), which involve lipid movement into and out of the Golgi in yeast. The psd1D strains can be mutagenized and then screened for Etn auxotrophs. Among the Etn auxotrophs will be new strains with defects in PtdSer transport (pstB mutants) and PtdEtn export (peeB mutants). Abbreviations: ER, endoplasmic reticulum; MAM, mitochondria-associated membrane.

identify mutations, genes and proteins that participate in lipid transport. The general features of this screening are outlined in Figure 2. In yeast that have been manipulated to delete the PSD1gene encoding Psd1p (i.e. strains with a psd1D mutation), or to delete the PSD2 gene encoding Psd2p (i.e. strains with a psd2D mutation), growth is relatively normal in minimal media [22–24]. By contrast, when doubly mutated psd1D psd2D strains are constructed, they are unable to synthesize sufficient PtdEtn for survival in minimal media [23]. However, like many eukaryotes, yeasts possess multiple pathways for the synthesis of PtdEtn [30]. One of these pathways, usually called the Kennedy pathway in recognition of the seminal contributions of Eugene P. Kennedy, enables yeast to use ethanolamine (Etn) to synthesize PtdEtn. Empirically, one finds that the psd1D psd2D double mutant strains of yeast can be rescued by Etn supplementation [23]. From these findings, Trotter and Voelker postulated that if Etn can rescue PtdEtn deficiency due to inactive Psd1p and Psd2p activity, then it might also rescue PtdEtn deficiency due to a defect in transport of PtdSer to the loci of either Psd1p or Psd2p [18]. From a theoretical standpoint and for simplicity, the transport of PtdSer to Psd1p was named the PtdSer transport A (PSTA) pathway and the transport of PtdSer to Psd2p was named the PtdSer transport B (PSTB) pathway. Likewise, newly formed PtdEtn must be exported from the mitochondria and Golgi back to the ER for the synthesis of PtdCho and these pathways are named PEEA and PEEB. www.sciencedirect.com

In execution, the screen for yeast mutants defective in PtdSer transport to Psd1p uses strains harboring a psd2D mutation that are mutagenized and analyzed for Etn auxotrophy [20]. The psd2D genetic background forces almost all PtdEtn synthesis to proceed via transport of PtdSer to Psd1p, as shown in Figure 2a. The mutation of psd2D strains and screening for Etn auxotrophs enables the identification of new strains defective in the transport of PtdSer to Psd1p, in addition to strains with defects in the activity of Psd1p. In yeast, this screen has produced a mutant named pstA1 (described in more detail later) and its corresponding complementing gene. Similar types of analyses can be performed with strains that have a psd1D mutation, which are dependent on the transport of PtdSer to Psd2p for the synthesis of the majority of their PtdEtn. Mutation and screening for Etn auxotrophs in strains with a psd1D mutation enables the identification of new strains that are defective in transport of PtdSer to Psd2p, or that are defective in the activity of this enzyme, as illustrated in Figure 2b. Thus far, this second screen has yielded two mutants (pstB1 and pstB2) and their corresponding complementing genes, in addition to a variant of Psd2p, that have provided important new insight into the process of PtdSer transport [16,18,19,21]. In principle, both of these screens should also yield new mutants (peeA and peeB) in the export of PtdEtn from the mitochondria and Golgi back to the ER. A screen related to that described previously for the PSTA pathway has also been applied to mammalian cells.

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This screen was designed to identify strains that have a plasma membrane deficiency in PtdEtn and are resistant to the effects of a toxin that binds the lipid. The toxin is the cyclic peptide Ro09–0198, which recognizes PtdEtn and causes cytolysis. This screen produced a variant of CHO-K1 cells (R41) that is defective in PtdSer transport to the locus of Psd1p and results in a deficiency in cellular PtdEtn [17]. Biochemical studies In addition to the genetic approach, biochemical studies with intact cells, permeabilized cells and isolated organelles have used the action of Psd1p and Psd2p to examine and characterize PtdSer transport. For lipid transport to Psd1p, yeast and mammalian cells exhibit some differences, if the process is dissected spatially and temporally. The biosynthetic machinery for PtdSer is also different between yeast and mammalian cells. Yeast uses a PtdSer synthase (NP_010943), which requires CDP-diacylglycerol as the source of the phosphatidic acid moiety that condenses with serine to make PtdSer [30]. This PtdSer is preferentially synthesized in the MAM compartment of the ER and transferred to the mitochondria in an ATP-independent reaction [14,15,31]. Morphological evidence indicates that PtdSer transfer occurs at the zones of apposition between the MAM and the outer mitochondrial membrane. The machinery that affects the transfer of PtdSer between the organelles remains unknown but genetic studies (described later) are providing some clues about the regulation of the process. Once the PtdSer is incorporated into the outer mitochondrial membrane, there are no demonstrable energetic requirements for it to gain access to Psd1p present in the inner membrane [11,15]. The mitochondrial outer membrane can also be loaded with a PtdSer analog (1-acyl, 2-(NBD-aminocaproyl)-PtdSer, and this lipid is also imported to the locus of Psd1p in an ATP-independent manner [20,32]. In mammalian systems, studies using metabolic inhibitors with intact cells reveal that there is an ATP requirement for PtdSer transport to the locus of Psd1p [28]. Evidence for this ATP requirement is also produced in transport studies using permeabilized cells [13,33]. However, experiments with liposomal or isolated microsomal donors and purified mitochondria do not demonstrate an ATP requirement for PtdSer transport between the membranes [12]. These results strongly suggest that the terminal event of lipid transfer from a donor membrane to the outer mitochondrial membrane does not require ATP in either mammalian cells or yeast (as described earlier). However, unlike yeast, mammalian cells require ATP at multiple steps preceding the final lipid transfer between the membranes. Mammalian cells synthesize PtdSer by an exchange reaction in which the amino alcohol substituents (Etn and Cho) present in PtdEtn and PtdCho are exchanged for Ser [34]. Thus, in contrast to yeast, the phosphatidic acid moiety of mammalian PtdSer is derived from PtdEtn and PtdCho. The mammalian reaction requires Ca2C and in intact and permeabilized cells is coupled to the ability of the ER and MAM to sequester the ion via a Ca2C-ATPase [13,32]. After PtdSer is formed, there is another ATP requirement www.sciencedirect.com

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that precedes the final lipid transfer to mitochondria. This step is known to be separate from the ATP requirement necessary for synthesis of PtdSer because it is possible to pulse-radiolabel the PtdSer pool and subsequently arrest further synthesis of this lipid. Even under conditions of arrested PtdSer synthesis, the previously synthesized radioactive lipid is still transported to the mitochondria. The ATP requirement that follows the synthesis of PtdSer is not well understood because isolated mitochondria readily import PtdSer when incubated with liposomes containing this lipid in the absence of ATP [12]. The current assessment of these data is that the newly synthesized PtdSer requires ATP to enter a portion of the MAM that enables it to interact directly with the mitochondria [35]. Although it is tempting to speculate that the nascent PtdSer is lumenally sequestered and requires the ATP-dependent action of a transbilayer translocase to move it to the cytosolic surface of the bilayer, to date, there is no experimental evidence to support this idea. The reconstitution of PtdSer synthesis and transport in permeabilized mammalian cells has also been used to screen for macromolecular factors that augment the transport reaction. A successful application of this approach led to the identification in brain cytosol of an EF-hand protein capable of increasing the rate of transport of PtdSer from the ER and MAM to the mitochondria [36]. The protein, named S100B (NP_006236), increases the rate of lipid transport approximately threefold. It is not known whether this protein actually participates in the transport reaction or promotes the stability and/or assembly of interactions between the ER or MAM and mitochondria. Studies with transport mutants Examination of the biochemical characteristics of PtdSer transport using the mutants identified in the screens described previously has provided important insights into the types of molecules involved in the process and their mechanisms-of-action (Figure 3). The yeast mutant pstA1 is an Etn auxotroph that shows a defect in the rate of conversion of nascent PtdSer to PtdEtn in intact cells [20]. The mitochondria purified from the mutant strain have a markedly reduced level of PtdEtn and examination of these mitochondria on sucrose density gradients reveals that they are much denser than the organelles from wildtype cells. This is expected if the phospholipid:protein ratio is reduced in mutant mitochondria as a consequence of reduced phospholipid transport to the organelle. PtdSer transport between the outer and inner mitochondrial membranes is normal in the pstA1 mutant when measured using the analog 1-acyl, 2-(NBD-aminocaproyl)-PtdSer. Reconstitution experiments using MAM and mitochondria reveal a defect in the transport of PtdSer between the organelles. When MAM and mitochondria are purified from wild-type and mutant cells and recombined in different combinations as donors and acceptors, the lesion in pstA1 maps to both of the membranes. This finding means that the MAM from the pstA1 strain are defective as donor membranes, and the mitochondria from the pstA1 strain are defective as acceptor membranes. The gene that rectifies all the

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Met30p Mitochondria X

MAM

Y Psd1p

PtdSer

PtdSer

PtdSer

PtdEtn

ATP Ser

PtdSer

S100B

R41p

PtdIns

Golgi Adapter proteins?

Stt4p PtdIns4P PtdSer domain

Ser

PtdOH

PstB2p PtdSer C2 domain

Psd2p PtdEtn

ER Ti BS

Figure 3. Molecular components and properties of PtdSer transport to mitochondria and the Golgi. PtdSer synthesized in the mitochondria-associated membrane (MAM) domain of the endoplasmic reticulum (ER) is transported to the outer mitochondrial membrane under the regulation of yeast Met30p, a ubiquitin ligase subunit. Met30p affects the properties of both the MAM and the mitochondria and this is proposed to occur via protein components (designated X and Y) on the respective organelles. It is not yet known if Met30p ubiquitinates X and Y directly on the MAM and mitochondria, or if its action regulates their degradation or transcription. The rate of transfer of PtdSer to mammalian mitochondria can be augmented several-fold by the action of mammalian S100B, an EF-hand-Ca2C binding protein. Genetic screening identifies a CHO-K1 mutant (R41) that is proposed to have a lesion in a putative protein, R41p, required for efficient transport of PtdSer between the outer (om) and inner (im) mitochondrial membranes. In mammalian cells, but not yeast, there is an ATP requirement for newly synthesized PtdSer to become competent for transport to the mitochondria. In yeast, PtdSer synthesized in the ER is transported to the Psd2p in the Golgi for the synthesis of PtdEtn. The Psd2p has not been identified in mammalian cells. Reconstitution studies with chemically defined donors demonstrate that PtdSer-rich domains are the preferred source of the lipid destined for transport to Psd2p, and PtdIns4P and PtdOH can augment the transport reaction. Genetic studies implicate Stt4p as a source of Ptd-Ins-4-P required for PtdSer transport but there is uncertainty how this occurs because the enzyme is primarily found in the plasma membrane. The presence of Psd2p and PstB2p on the Golgi membrane is required for intermembrane PtdSer transport to occur. The C2 domain of Psd2p is not required for catalysis by the enzyme but is necessary for the transport reaction. The C2 domain might function by recognizing anionic lipids in the donor membrane and promoting docking with the acceptor membranes. It is also likely that adaptor proteins have a role in promoting interactions among proteins and lipids between the membranes.

phospholipid synthesis and transport abnormalities of the pstA1 mutation was cloned by complementation and identified as MET30 (NP_012218). MET30 encodes a protein subunit (Met30p) of a multi-component E3 ubiquitin ligase [37]. The Met30p is an F box protein that dictates substrate specificity of the ubiquitin ligase [38]. This result is quite striking and provides unanticipated linkage between protein ubiquitination and phospholipid transport. A growing body of data now implicates protein ubiquitination in many membrane trafficking events, including viral budding at the cell surface, endosomal protein sorting and multivesicular body formation [39]. Ubiquitination is also known to regulate gene expression by controlling the half life and/or activity www.sciencedirect.com

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of transcription factors. One known substrate for E3 ubiquitin ligase containing Met30p is the transcription factor Met4p [40–42] (NP_014296). Upon ubiquitination, Met4p is inactivated. Biochemical studies reveal that the pstA1 mutant is defective in ubiquitination of Met4p [20]. Currently, it is not known how many proteins can serve as substrates for recognition by Met30p. At this stage, it is unclear how Met30p regulates PtdSer transport between the MAM and the mitochondria. Three possible explanations seem most likely. The first hypothesis – and perhaps the simplest – is that protein substrates present on both the MAM and mitochondria are recognized by Met30p. Ubiquitination of these putative target proteins (designated as X and Y in Figure 3) could have a role in either direct activation of transport molecules, or MAM-mitochondria recognition and docking for the purpose of lipid transport. A second hypothesis is that an inhibitor of PtdSer transport resides on both the MAM and mitochondria. In this case, Met30p directed ubiquitination of a protein substrate could lead to its degradation and alleviation of an inhibitory block upon transport. A third hypothesis is that the action of Met30p upon Met4p regulates transcription of a factor involved in lipid transport. Because ubiquitination directed by Met30p inactivates Met4p, it is likely that the factor would be a negative regulator of PtdSer transport. Biochemical studies in mammalian cells, with the R41 mutant cell-line that is resistant to the cytolytic effects of the toxin Ro09–0198, also reveal important information about the process of PtdSer transport within the mitochondria [17]. The R41 cells show a reduction in total cellular PtdEtn content and the metabolism of nascent PtdSer to PtdEtn. Enzymatic studies reveal that Psd1p activity is normal but the transport of 1-acyl, 2-(NBDaminocaproyl)-PtdSer between the outer and inner mitochondrial membranes is markedly reduced. Interestingly, this defect in lipid import into the mitochondria does not affect protein import into the organelle. These results provide significant evidence for the existence of specific proteins involved in the transport of phospholipids between the outer and inner mitochondrial membranes that are independent of the protein transport machinery. Thus far, the cDNA and gene required to complement the R41 strain have not been cloned. Examination of the mutants obtained in the PSTB pathway is also providing some new definition to the molecular requirements for the transport of PtdSer to Psd2p in yeast cells. The first mutant described in this pathway was pstB1 [18]. The pstB1 mutant shows a defect in the conversion of nascent PtdSer to PtdEtn by Psd2p. The reduced PtdEtn synthesis is not due to an alteration in Psd2p activity but in the access of the substrate to the enzyme. The gene that complements the pstB1 growth defect (Etn auxotrophy) and abnormality in lipid synthesis is STT4 (NP_013408), which encodes a phosphatidylinositol 4-kinase (PtdIns4-kinase; Stt4p) [43]. The Stt4p is one of three PtdIns4-kinases that have been described in yeast and shows both plasma membrane and microsomal distribution in subcellular fractions. The enzyme is tethered to the plasma membrane by a structural protein named Sfk1p [44] (NP_012873).

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There is reasonable evidence that the major pool of phosphatidylinositol 4-phosphate (PtdIns4P) produced by Stt4p resides in the plasma membrane [45]. However, it remains unclear whether the PtdIns4P generated by this enzyme can also be found in other intracellular membranes. Although the genetic experiments with pstB1 strains implicate PtdIns4P at some stage of PtdSer transport to Psd2p, much remains uncertain. It is unknown whether PtdIns4P or its downstream product, phosphatidylinositol (4,5)-bisphosphate PtdIns(4,5)P2, is important in the transport process. Analysis of the sequence of Psd2p reveals the presence of a C2 domain. Typically, C2 domains are involved in Ca2C binding, protein–lipid and protein–protein interactions [46]. Anionic lipids, including PtdSer and polyphosphoinositides, are recognized by C2 domains and this raises the possibility that Psd2p might participate directly in recognition of these lipids on the donor membrane as part of the transport reaction. To test the role of the C2 domain of Psd2p in PtdSer transport, deletion mutants lacking this domain (psd2-C2D) were constructed [21]. In strains lacking wild-type copies of Psd1p and Psd2p, the mutant protein, Psd2-C2Dp, is catalytically active and can be expressed at levels 10-fold higher than that normally required to produce the PtdEtn required for cell growth. The catalytic activity of Psd2-C2Dp can be measured using a 1-acyl, 2-(NBD-aminocaproyl)-PtdSer substrate that spontaneously inserts into membranes harboring the enzyme. Further analysis reveals that the Psd2-C2Dp undergoes the same post-translational processing as its wild-type counterpart and is localized correctly within the cell. Despite the high catalytic activity of the Psd2-C2Dp expressing strains, the cells fail to grow unless they are supplemented with Etn. Thus, the C2 domain of Psd2p has an important non-catalytic role in the action of the protein. Measurement of PtdSer transport in intact and permeabilized cells reveals that this lipid cannot be delivered to the enzyme lacking the C2 domain. Collectively, these results define a direct role for the C2 domain of Psd2p in the transport process. The mechanism of how the C2 domain is functioning remains to be elucidated. It could form a docking module that recognizes anionic phospholipids (e.g. PtdSer, PtdIns, PtdIns4P, PtdIns(4,5)P2) and perhaps proteins on the donor membrane. If the C2 domain only acts in docking, then other proteins would need to contribute to the assembly of a transport apparatus for moving PtdSer to the acceptor membrane. Alternatively, the C2 domain might bind to and physically participate in the transport of the lipid between the membranes. With either mechanism it seems probable that Psd2p can function in concert with other proteins present on the acceptor membrane to form a PtdSer transport complex. The genetic screening of the PSTB pathway identified another mutant strain (pstB2), and its corresponding gene (PSTB2) and encoded protein (PstB2p), as a component in the process of PtdSer transport to the locus of Psd2p [19]. The mutant strain displays a profound defect in the transport dependent metabolism of PtdSer to PtdEtn in intact and permeabilized cells and reconstitution www.sciencedirect.com

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reactions with isolated organelles. The PstB2p is a member of the phospholipid exchange and transfer protein family and is structurally similar to Sec14p, a PtdIns and PtdCho transfer protein that participates in protein trafficking events [47]. The gene complementing the pstB2 mutant strain has been named PDR17 (NP_014135) and SFH4 in other screens [48,49] but for consistency it is referred to as PSTB2 in this review. The PstB2p transfers PtdIns between liposomes and mitochondria in cell-free assays but does not transfer PtdSer. The protein is amphitropic and can be either soluble or membrane bound in yeast and in insect cell expression systems. The membrane bound form of the protein is highly resistant to removal by alteration of ionic conditions. Reconstitution assays with permeabilized cells and organelle preparations demonstrate that the protein must be present on the acceptor membrane for transfer of nascent PtdSer to Psd2p to occur [16]. The mechanism-ofaction of PstB2p is still unknown and many outstanding questions need to be resolved. The protein is capable of binding phosphatidylinositol (PtdIns) but it is not clear how this relates to PtdSer transport. This lipid binding could account for the amphitropic properties of PsdB2p and it could facilitate interactions between donor and acceptor membranes. The requirement for PstB2p on the acceptor membranes also suggests that it interacts with specific proteins (perhaps Psd2p) at that location but this has not yet been demonstrated. Additional studies with the PSTB pathway have provided further insights into the role of lipids in the process of PtdSer transport. In an effort to define the separate molecular requirements of the donor and acceptor membranes in lipid transport, a chemically defined donor membrane system was developed [50]. The defined donors exhibit some interesting and unexpected characteristics. The first unusual property is that membranes with a relatively planar geometry made from large unilamellar vesicles (with diameters of w400 nm) are superior donors compared with highly curved membranes made from small unilamellar vesicles. A second unusual property is that PtdSer-rich donors are the preferred substrate for transport. Pure PtdSer vesicles (400 nm diameter) transfer the lipid to acceptors at 20-times the rate of vesicles containing 50% PtdSer and 50% PtdCho. The decline in PtdSer transfer with change in liposome composition is exponential and, at 50% PtdSer, the transport reaction is negligible. Interestingly, phosphatidic acid (PtdOH) and PtdIns4P can reverse the inhibition caused by surface dilution with lipids such as PtdCho. The PtdOH completely reverses the inhibition, whereas the PtdIns4P partially reverses the inhibition. In reconstitution studies with chemically defined donors, the fidelity of the process to the properties of PtdSer transport observed in vivo and in permeabilized cells, was crucially tested using both pstB2D and psd2-C2D mutants. These experiments function as important tests to rule out spurious fusion of chemically defined donors with the acceptor membranes. The results reveal that the reconstitution of transport with chemically defined donors faithfully recapitulates the properties of PtdSer transport found in both intact and permeabilized cells. These

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Box 2. Emerging lipid and protein motifs implicated in phospholipid traffic Ubiquitin Ubiquitin is a 76-amino acid protein that is attached to target proteins via an isopeptide linkage at specific lysine residues. In many cases, ubiquitin serves as a modification that destines the modified target protein for degradation via proteasomes. Recently, ubiquitin has been recognized in many systems to also serve as an important signaling motif for either activating or inactivating proteins without degradation. This activation and inactivation can serve to regulate catalysis, transcription, subcellular trafficking and protein–protein interactions.

Ubiquitin ligases Ubiquitin ligases attach ubiquitin to target proteins. The ubiquitin ligase subunit of interest in phospholipid traffic, Met30p, is part of a large multiprotein complex. The role of Met30p is to bind to the holoenzyme and dictate the choice of target protein to be modified by ubiquitin.

S100B S100B is an EF-hand domain-Ca2C binding protein that seems to function as a regulator of lipid transport machinery on mammalian mitochondria-associated membrane (MAM) and/or mitochondria.

Phosphatidylinositol-4-kinase Phosphatidylinositol-4-kinase phosphorylates phosphatidylinositol to yield the product PtdIns4P. This modification can serve to mark membrane domains for interactions with specific binding proteins. Genetic and biochemical studies implicate the PtdIns4 kinase, Stt4p, in phospholipid transport. The PtdIns4P also functions as an important precursor to PtdIns(4,5)P2, which also acts as a membrane recognition domain for interaction with binding proteins. Cleavage of

findings raise the possibility that segregated domains of PtdSer, or PtdSer and PtdOH, or PtdSer and PtdIns4P within the donor membrane, could serve as specific local regions for intermembrane lipid transport. Such domains could arise as a consequence of specific proteins present in the donor membrane that corral PtdSer and other anionic lipids. Alternatively, the anionic lipid-rich domains might also be induced in the donor membrane by interactions with proteins in the acceptor membrane. Soluble adaptor proteins might also function to recognize specific lipid and protein elements in donor and acceptor membranes. These models are all highly speculative at this stage but they provide a provocative hypothetical framework for future experiments. Summary Phospholipid transport associated with new organelle formation is a fundamental process of biochemistry and cell biology whose mechanisms remain to be elucidated. Genetic and biochemical tools applied to the problem are implicating specific proteins and lipids in the transport and its regulation, as summarized in Figure 3 and Box 2. The proteins or protein motifs implicated thus far include ubiquitin, ubiquitin ligase (Met30p), PtdIns4-kinase (Stt4p), a phospholipid binding protein (PstB2p), a soluble EF-hand protein (S100B) and a C2 domain (present on Psd2p). The lipids implicated in the process include PtdSer, PtdIns, PtdIns4P and PtdOH. PtdSer serves not only as a substrate for transport and decarboxylation but also as a potential ligand for the C2 domain of Psd2p. PtdIns, PtdIns4P and PtdOH are also potential www.sciencedirect.com

PtdIns(4,5)P2 produces two important signaling molecules, diacylglycerol and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3).

Phospholipid exchange and transfer proteins Phospholipid exchange and transfer proteins can function to exchange lipids between membranes in vitro but their function in vivo remains uncertain. The exchange protein described in this report, PstB2p, recognizes and transfers PtdIns in vitro. However, in vivo it is an essential component in PtdSer transport to Psd2p. The property of PtdIns recognition might promote the attachment to membranes in vivo, where it functions as part of a larger molecular complex that transports PtdSer between membranes.

C2 domains C2 domains are Ca2C- and phospholipids-binding domains involved in protein–lipid interactions. A C2 domain that is present on Psd2p is not required for catalysis but is necessary for the transfer of PtdSer between donor and acceptor membranes.

PtdSer domains Reconstitution studies with liposomes as donor membranes demonstrate that PtdSer-rich domains are the preferred source of the lipid for transfer to Psd2p. Dilution of pure PtdSer domains with small amounts of PtdCho dramatically inhibits intermembrane transport of the lipid.

Acidic phospholipids Reconstitution studies show that dilution of PtdSer domains with phosphatidic acid does not disrupt transport like PtdCho but has a stimulatory effect on the process. PtdIns4P, the product of Stt4p, shows similar albeit weaker activity than phosphatidic acid.

recognition ligands for docking reaction between the donor and acceptor membranes. In addition, the anionic lipids could also function as nucleation sites for interaction with membrane proteins either in their resident or apposed membranes that can serve to assemble docking and transport machinery at regions of membrane contact. These molecules are likely to constitute an early list of components involved in non-vesicular polar lipid transport between organelles. From this current list of molecular participants, the focus now needs to move towards defining their precise roles in the transport reactions by addressing the questions: (i) what interacts with what? (ii) Do protein–protein and protein–lipid interactions between membranes introduce physical changes in local membrane environments (e.g. segregation of PtdSer) that are essential for transport? (iii) How are intermembrane docking and phospholipid transport interrupted to stop the process and disengage the donors from the acceptors? (iv) How applicable are the molecular motifs used for aminophospholipid transport to other types of polar lipids? (v) Do mammalian cells use the same molecular motifs for aminophospholipid transport as those uncovered in yeast? These current, challenging questions require new genetic, biochemical and physical approaches to the problem. The resolution of these issues is likely to provide new insights into the mechanisms of phospholipid transport during membrane biogenesis. Acknowledgements This work was supported by a grant from the National Institutes of Health 2R37 GM 32453.

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