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Internalization and trafficking of fluorescent-labeled phospholipids in yeast
seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 13, 2002: pp. 179–184 doi:10.1016/S1084–9521(02)00046-0, available online at http://www.idealibrary.com on
Internalization and trafficking of fluorescent-labeled phospholipids in yeast J. Wylie Nichols
establish and maintain this non-random distribution of lipids among and across cellular membranes are largely unknown. Thus, the questions of why and how phospholipids are sorted and transported between and across cellular membranes are among the most fundamental, unanswered questions in cell biology. Numerous approaches have been used to investigate the mechanisms of phospholipid trafficking and sorting in eukaryotic cells. These include the biochemical purification of soluble and membrane proteins that transport phospholipids between and across membranes, the development of in vitro and in situ reconstitution assays to investigate particular sorting or trafficking steps, and the use of short-chain reporter phospholipids to visualize and study sorting and trafficking in living cells. Each of these approaches has provided new insights into this difficult problem; however, future advances in this field are likely to depend on the insightful integration of information from all of the above mentioned approaches in addition to the development of novel techniques. This brief review will focus on insights gained by combining biochemical and cell biological approaches using fluorescent-labeled phospholipids with the genetic approaches available with the yeast, Saccharomyces cerevisiae.
Phospholipid reporter molecules, containing a fluorescent group attached to a short, acyl chain, spontaneously insert into the plasma membrane of yeast cells allowing retrograde trafficking to intracellular organelles as well as their metabolic fates to be monitored. This approach provides the framework for determining the dependence of particular phospholipid trafficking and metabolic steps on a wide range of genes known to be required for related membrane transport functions as well as for developing genetic screens to identify novel genes required for these processes. This review presents an overview of insights gained into phospholipid trafficking and metabolism using this approach. Key words: Saccharomyces cerevisiae / plasma membrane / NBD / flip–flop / vacuole © 2002 Elsevier Science Ltd. All rights reserved.
Introduction The organellar membranes in eukaryotic cells maintain unique compositions of lipids that include as many as 50 of the approximately 500 different cellular membrane lipids.1 Specific functions have been identified for many of these lipids. For example, membrane lipids serve as receptors, second messengers or precursors to second messengers, and anchors for membrane-associated proteins. Although a rationale for the complex lipid compositions of membranes has begun to emerge, the full significance for maintaining different lipid compositions between the membranes of cellular organelles as well as between both leaflets of the same membrane has not been elucidated. Furthermore, the molecular mechanisms that
Advantages and disadvantages of NBD-labeled phospholipids Of the numerous choices of fluorescence groups that are suitable for attachment to phospholipids, for studies in yeast, the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) moiety has been used almost exclusively. The molecule most commonly used is acylated with either a myristoyl or palmitoyl group in the sn-1 position of the glycerol backbone and with N-NBD aminocaproyl in the sn-2 position. Placing the label in the acyl chain allows for the synthesis of labeled phospholipids with each of the commonly occurring head groups. These
From the Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1084–9521 / 02 / $– see front matter
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molecules are commercially available and relatively inexpensive—a significant advantage when large amounts are required for genetic screens. The short acyl chain in the sn-2 position increases the water solubility sufficiently to allow rapid spontaneous equilibration between membranes while maintaining a strong preference for hydrophobic membranes.2 The NBD group has spectral properties that allow it to be visualized in a fluorescence microscope with a standard fluorescein filter set. The high environmental sensitivity of its fluorescence yield—the fluorescence of water soluble products is only ∼1% of the hydrophobic parent compound3 —can be exploited to monitor degradation rates in situ. One of the major disadvantages of the NBD fluorophore is its propensity to photobleach although this can be overcome by the use of a high sensitivity video camera and short exposure times. These properties allow one to introduce NBD-labeled phospholipids to the plasma membrane of living yeast cells and to monitor their fates during internalization in order to gain insight into the mechanisms of sorting and trafficking of phospholipids in yeast. Although NBD-labeled phospholipids and reporter phospholipids in general provide useful tools for the investigation of phospholipid trafficking and for the identification of phospholipid translocators, one must use caution in extrapolating their behavior to that of endogenous phospholipids. Demonstration of short-chain reporter phospholipid transport does not constitute proof of endogenous phospholipid transport in the absence of supporting evidence. However, this disadvantage is balanced by the lack of alternative approaches to address these problems and the fact, that when appropriate supporting evidence is available, the behavior of reporter phospholipids has been shown in many cases to reflect the behavior of their endogenous counterparts.4
Figure 1. Schematic of NBD-phospholipid traffic in yeast.
vesicle or DMSO suspension. Their internalization, distribution and metabolism were monitored and quantified by several different methods including fluorescence microscopy, flow cytometry, extraction and purification. Step 1 refers to internalization by inward-directed transmembrane transfer across the plasma membrane, commonly referred to as flip. Studies of the four NBD-phospholipids indicate that each is internalized predominantly by flip as opposed to endocytosis although the mechanistic details differ. NBD-PC, NBD-PS and NBD-PE are internalized into wild-type yeast cells from donor vesicles at 30 ◦ C, however their fates are distinctly different. NBD-PC is internalized predominantly to the vacuole (Figure 2) and degraded whereas a minor component is localized in the mitochondria and nuclear envelope/ER.5 On the other hand, NBD-PE and NBD-PS (not shown) are internalized to the nuclear envelope/ER and mitochondria (Figure 2) and are not transported to the vacuole. NBD-PE is not degraded intracellularly,6 however the metabolic fate of NBD-PS has not been determined. The exclusive transport and degradation of NBD-PC in the vacuole was initially interpreted to reflect that a significant fraction of that internalized was endocytosed from the plasma membrane and transported through the late endocytic pathway to the vacuole.5 However, subsequent investigations have led to the conclusion that NBD-PC, like NBD-PE and NBD-PS, is transported primarily across the plasma membrane by flip and that endocytosis is a minor contributor, if any. NBD-PC is transported to the vacuole (step 6) subsequent to its flip across the plasma
Mechanism of internalization across the plasma membrane A generalized scheme of the trafficking steps investigated with NBD-phospholipids is illustrated in Figure 1. The numbered mechanistic steps will be discussed below in relation to four NBD-labeled phospholipids (NBD-phosphatidylcholine, NBD-PC; NBD-phosphatidylserine, NBD-PS; NBD-phosphatidylethanolamine, NBD-PE; and NBD-phosphatidate, NBD-PA) studied to date. In all cases the NBD-phospholipids were spontaneously introduced to the exoplasmic surface of the plasma membrane from a 180
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Figure 2. Localization of NBD-PC and NBD-PE in yeast at 30 ◦ C. A diploid strain of S. cerevisiae was grown to mid-log phase at 30 ◦ C and incubated with vesicles containing either NBD-PC or NBD-PE. Panels A and C show cells incubated with NBD-PE and panels B and D show cells incubated with NBD-PC. Used with permission of The Journal of Cell Biology; Kean et al. (1997) JCB 138, 255–270.
membrane. These conclusions are based on several pieces of evidence. First, at 2 ◦ C, both NBD-PC, NBD-PE and NBD-PS are internalized similarly to the mitochondria and nuclear envelope/ER whereas an established morphological tracer of the endocytic pathway in yeast, FM 4-64,7, 8 remains at the plasma membrane and is not internalized. Thus, endocytosis is not required for their internalization at low temperature. At higher temperatures that block the early stages of endocytosis in the end4-1 strain, FM4-64 internalization is significantly inhibited while the internalization of NBD-PC and NBD-PE is unaffected. If the early stages of endocytosis requiring End4p contributed significantly to the total internalization of either NBD-PC or NBD-PE, a measurable decrease in the amount of internalized fluorescent phospholipid would be expected. A similar result was observed using the ‘back exchange’ assay. The end4-1 strain was labeled with NBD-PC or NBD-PS, washed, and the amount of internalization inferred from the fraction not removed with an albumin wash. Both NBD-PC and NBD-PS were internalized in the endocytosis deficient mutant.9 NBD-PA was also shown to be internalized predominantly by flip across the plasma membrane followed by its metabolism to numerous products including diacylglycerol, phosphatidylinositol, phos-
phatidylcholine, and phosphatidylethanolamine in decreasing order of magnitude.10 Although the four NBD-phospholipids appear to be flipped across the plasma membrane, the mechanisms of transport appear to differ. When measured by fluorescence microscopy and flow cytometry, flip of NBD-PC, NBD-PS, and NBD-PE are dramatically inhibited by ATP-depletion,4 NEM treatment,4 and collapse of the plasma membrane proton-electrochemical potential11 (Figure 3). These results support the conclusion that their flip occurs via a proton gradient coupled transporter susceptible to inhibition by sulfhydryl modification. However, results obtained in the end4-1 strain using the ‘back exchange’ assay demonstrated no ATP-dependence or NEM-sensitivity to the flip of NBD-PC and NBD-PS.9 This discrepancy may be the result of an interesting property of the end4-1 strain. ‘Back exchange’ experiments, in which only the NBD-phospholipid inserted into the exoplasmic surface of the plasma membrane is allowed to flip inward, are precluded in most, if not all, wild-type yeast strains because they do not allow the insertion of detectable amounts of probe into the plasma membrane. The end4-1 strain was suitable for these experiments based on the fact that the exoplasmic surface of the plasma membrane could be labeled. These 181
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subsequent demonstration that Drs2p is localized to the Golgi,15 casts further doubt on its identification as the transporter responsible for NBD-PS flip in S. cerevisiae.
NBD-PC and NBD-PE are actively flopped across the plasma membrane Outward-directed transport across the plasma membrane, commonly referred to as flop, has only been investigated for NBD-PC and NBD-PE. Following internalization by standard methods, both of these probes are rapidly flopped to the exoplasmic leaflet of the plasma membrane where they are degraded to non-fluorescent water soluble products by periplasmic enzymes.6 Degradation was shown to occur subsequent to flop to the exoplasmic surface by the almost complete inhibition of degradation and the retrieval of intact probes from phospholipid vesicles present in the bathing medium. Similar results were obtained when the above experiment was performed with the sec6-4 strain at its non-permissive temperature. Since the transport of secretory vesicles to the plasma membrane is blocked in this strain,16 secretion as the mechanism of efflux is ruled out. Because periplasmic degradation of NBD-PC and NBD-PE occurs faster than flop across the plasma membrane, the flop rate can be inferred from the loss of fluorescence and measured by flow cytometry. Using this approach, the flop of NBD-PC and NBD-PE was found to be almost completely blocked by ATP-depletion and by incubation at 2 ◦ C.11 However, flop was not affected by collapse of the proton electrochemical gradient. Two members of the ABC transporter family, Pdr5p and Yor1p, have been demonstrated to contribute to the flop of NBD-PE.17
Figure 3. NBD-PC flip is inhibited by CCCP. Early log phase cells were incubated with and without the protonophore CCCP (carbonyl cyanide m-chlorophenylhdrazone), chilled on ice and labeled with NBD-PC or NBD-PE solubilized in DMSO. At the indicated times, aliquots were removed and analyzed by flow cytometry. NBD-PC control (closed circles); NBD-PE control (open circles); NBD-PC with CCCP (closed squares); NBD-PE with CCCP (open squares). Used with permission of The Journal of Biological Chemistry; Hanson and Nichols (2001) JBC 276, 9861–9867.
differences imply an alteration in the plasma membrane organization and structure that may account for the observed differences in ATP-dependence and NEM-sensitivity of NBD-PC and NBD-PS flip. NBD-PA flip measured by fluorescence microscopy and extraction and purification is not ATP-dependent.10 This may reflect the ability of its protonated species to spontaneously flip,12 however its sensitivity to collapse of the proton-electrochemical gradient has yet to be investigated. No consensus has emerged regarding the identity of the putative transporter/s responsible for NBD-PC, NBD-PS and NBD-PE flip. A member of the P-type ATPase family has been proposed to account for essentially all of the flip activity observed for NBD-PS in yeast.13 This conclusion was based on the loss of NBD-PS flip measured by ‘back exchange’ at low temperature in ATP-depleted strains in which the DRS2 gene encoding for the P-type ATPase was deleted. However, no apparent inhibition of NBD-PS flip was observed in a drs2 null strain when examined by fluorescence microscopy,14 and no quantitative differences were observed between NBD-PC and NBD-PS flip in the drs2 null strain when analyzed by flow cytometry.14 Furthermore deletion of the DRS2 gene in an end4-1 strain failed to abolish NBD-PS flip.9 The
Net internalization of NBD-phospholipids is controlled by regulating flip and flop A screen of mutagenized yeast identified gain-offunction mutations in two transcription factors, PDR1 and PDR3 that resulted in the loss of net NBD-PE accumulation.6 PDR1 and PDR3 regulate the expression of a wide range of targets in response to stress.18 Among these downstream targets are several drug efflux pumps that are members of the ABC transporter family.18 This class of transporter has been shown to confer drug resistance by effluxing a wide range of drugs across the plasma membrane suggesting 182
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that the observed decrease in net internalization of NBD-PC and NBD-PE in the PDR1 and PDR3 mutants resulted from their increased flop relative to flip. This hypothesis was confirmed by flow cytometry by the demonstration that NBD-PC and NBD-PE flop rates (step 3) were accelerated in the mutant PDR3 strain (pdr3-11).11 Since flop is essentially blocked at 2 ◦ C, measurement of the total influx at this temperature allows the rate of flip to be measured in the absence of flop. Surprisingly, the 2 ◦ C flip of both NBD-PC and NBD-PE (step 3) is dramatically reduced in the gain-of-function mutant strains (PDR1-11 and pdr3-11) and increased in strains in which PDR1 and PDR3 are deleted.11 It is thus apparent that the transcription factors PDR1 and PDR3 control the net accumulation of NBD-PC and NBD-PE by regulating both their rate of flip and flop across the plasma membrane. The net accumulation of NBD-PC and NBD-PE is also almost completely inhibited during the late log phase of growth by a decrease in the rate of flip without affecting the rate of flop.11 The physiological significance of down-regulating NBD-PC and NBD-PE flip in response to nutrient starvation remains to be determined, but suggests a role in the adaptation to growth arrest.
One might predict that passive transport would result in equal labeling of all of the membranes of intracellular organelles, however this does not appear to be the case. Neither NBD-PC nor NBD-PE is detected in the vacuolar or plasma membrane. Perhaps these membranes have an inherently lower affinity (partition coefficient) for the NBD-phospholipids or perhaps their distribution is specifically directed by the activity of transfer proteins. At this time, we cannot distinguish between these and other plausible alternatives.
Intracellular sorting and metabolism of NBD-PC and NBD-PA NBD-PC is internalized predominantly by flip across the plasma membrane and subsequently transported from the cytosolic membrane compartment to the lumen of the vacuole (step 6) at 30 ◦ C. The latter step is demonstrated by first labeling wild-type cells with NBD-PC at 2 ◦ C followed by a 30 ◦ C chase. During the chase, NBD-PC fluorescence is lost from the mitochondria, nuclear envelope/ER and appears in the vacuole as diffuse fluorescence characteristic of water soluble products20 . This transport to the vacuole is blocked by energy depletion. These results demonstrate that NBD-PC is selectively transported to the vacuole from intracellular organelles by an energy-dependent process. Elimination of NBD-PC transport to the vacuole in strains deleted in VPS4 and VPS28 genes required for vesicular traffic from the prevacuolar compartment (PVC) to the vacuole20 implies that NBD-PC is internalized into the PVC (step 5) by either invagination or transport across the membrane prior to its transport to the vacuole. The intracellular metabolism of NBD-PA to NBDphosphatidylinositol and NBD-PC is also dependent on normal membrane recycling to and from the plasma membrane. Although its degradation to NBDdiacylglycerol is unaffected in a sec6-4 strain, which is blocked in the Golgi to plasma membrane transport step, the synthesis of NBD-PC and NBD-phosphatidylinositol are significantly inhibited at the non-permissive temperature in this strain background.10 This suggests that the perturbation of membrane recycling in the sec6-4 strain blocks the transport of NBD-diacylglycerol to the intracellular sites of synthesis of NBD-phosphatidylinositol and NBD-PC. Transport of NBD-PC to the vacuole and the metabolism of NBD-PA to NBD-PC and NBD-phosphatidylinositol are specific intracellular lipid trafficking and
NBD-PC and NBD-PE are passively transported to the membranes of intracellular organelles If NBD-PC and NBD-PE are internalized by flip across the plasma membrane at 2 ◦ C, they must be distributed from the cytoplasmic leaflet of the plasma membrane to that of the mitochondria, nuclear envelope/ER and other intracellular organelles (step 4) to produce the pattern of localization shown in Figure 2. Since this transport occurs at 2 ◦ C, it is unlikely to depend on vesicular transport. The simplest explanation is that the short acyl chain in the sn-2 position of both NBD-PC and NBD-PE permits their passive, spontaneous redistribution to intra-organellar membranes. Although the partition coefficient for NBD-PC is 3 × 106 ,2 this high partition coefficient is several orders of magnitude lower than that for endogenous phospholipids (∼109 )19 and results in an increased half-time of spontaneous transfer through the aqueous phase from days to a few seconds.2 Although the physical properties of the probes are adequate to explain their rapid distribution, we cannot exclude a possible role of phospholipid transfer proteins. 183
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8. Wendland B, McCaffery JM, Xiao Q, Emr SD (1996) A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15. J Cell Biol 135:1485–1500 9. Marx U, Polakowski T, Pomorski T, Lang C, Nelson H, Nelson N, Herrmann A (1999) Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein. Eur J Biochem 263:254–263 10. Trotter PJ (2000) A novel pathway for transport and metabolism of a fluorescent phosphatidic acid analog in yeast. Traffic 1:425– 434 11. Hanson PK, Nichols JW (2001) Energy-dependent flip of fluorescence-labeled phospholipids is regulated by nutrient starvation and transcription factors, PDR1 and PDR3. J Biol Chem 276:9861–9867 12. Eastman SJ, Hope MJ, Cullis PR (1991) Transbilayer transport of phosphatidic acid in response to transmembrane pH gradients. Biochemistry 30:1740–1745 13. Tang X, Halleck MS, Schlegel RA, Williamson P (1996) A subfamily of P-type ATPases with aminophospholipid transporting activity. Science 272:1495–1497 14. Siegmund A, Grant A, Angeletti C, Malone L, Nichols JW, Rudolph HK (1998) Loss of Drs2p does not abolish NBD-labeled phospholipid transfer across the plasma membrane of Saccharomyces cerevisiae. J Biol Chem 273:34399–34405 15. Chen CY, Ingram MF, Rosal PH, Graham TR (1999) Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function. J Cell Biol 147:1223– 1236 16. Walworth NC, Novick PJ (1987) Purification and characterization of constitutive secretory vesicles from yeast. J Cell Biol 105:163–174 17. Decottignies A, Grant AM, Nichols JW, de Wet H, McIntosh DB, Goffeau A (1998) ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p. J Biol Chem 273:12612–12622 18. Balzi E, Goffeau A (1994) Genetics and biochemistry of yeast multidrug resistance. Biochim Biophys Acta 1187:152–162 19. Smith R, Tanford C (1972) The critical micelle concentration of l-dipalmitoylphosphatidylcholine in water and water–methanol solutions. J Mol Biol 67:75–83 20. Hanson PK, Grant AM, Nichols JW (2002) NBD-labeled phosphatidylcholine enters the yeast vacuole via the pre-vacuolar compartment. J Cell Sci, in press.
metabolic steps that are perhaps amenable to exploration by further screening existing mutant strains in relevant trafficking pathways and to the development of genetic screens that may identify new components of these poorly understood processes.
Acknowledgements The author acknowledges the intellectual and experimental contributions of C. Angeletti, A.M. Grant, P.K. Hanson, L.S. Kean, and L. Malone. This work was supported in part by National Institutes of Health Grant GM52410 and a grant from the University Research Committee of Emory University.
References 1. van Meer G (1993) Transport and sorting of membrane lipids. Curr Opin Cell Biol 5:661–673 2. Nichols JW (1985) Thermodynamics and kinetics of phospholipid monomer–vesicle interaction. Biochemistry 24:6390–6398 3. Nichols JW (1987) Binding of fluorescent-labeled phosphatidylcholine to rat liver non-specific lipid transfer protein. J Biol Chem 262:14172–14177 4. Grant AM, Hanson PK, Malone L, Nichols JW (2001) NBDlabeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane. Traffic 2:37–50 5. Kean LS, Fuller RS, Nichols JW (1993) Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane. J Cell Biol 123:1403–1419 6. Kean LS, Grant AM, Angeletti C, Mahe Y, Kuchler K, Fuller RS, Nichols JW (1997) Plasma membrane translocation of fluorescent-labeled phosphatidylethanolamine is controlled by transcription regulators, PDR1 and PDR3. J Cell Biol 138:255– 270 7. Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128:779–792
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Internalization and trafficking of fluorescent-labeled phospholipids in yeast J. Wylie Nichols
establish and maintain this non-random distribution of lipids among and across cellular membranes are largely unknown. Thus, the questions of why and how phospholipids are sorted and transported between and across cellular membranes are among the most fundamental, unanswered questions in cell biology. Numerous approaches have been used to investigate the mechanisms of phospholipid trafficking and sorting in eukaryotic cells. These include the biochemical purification of soluble and membrane proteins that transport phospholipids between and across membranes, the development of in vitro and in situ reconstitution assays to investigate particular sorting or trafficking steps, and the use of short-chain reporter phospholipids to visualize and study sorting and trafficking in living cells. Each of these approaches has provided new insights into this difficult problem; however, future advances in this field are likely to depend on the insightful integration of information from all of the above mentioned approaches in addition to the development of novel techniques. This brief review will focus on insights gained by combining biochemical and cell biological approaches using fluorescent-labeled phospholipids with the genetic approaches available with the yeast, Saccharomyces cerevisiae.
Phospholipid reporter molecules, containing a fluorescent group attached to a short, acyl chain, spontaneously insert into the plasma membrane of yeast cells allowing retrograde trafficking to intracellular organelles as well as their metabolic fates to be monitored. This approach provides the framework for determining the dependence of particular phospholipid trafficking and metabolic steps on a wide range of genes known to be required for related membrane transport functions as well as for developing genetic screens to identify novel genes required for these processes. This review presents an overview of insights gained into phospholipid trafficking and metabolism using this approach. Key words: Saccharomyces cerevisiae / plasma membrane / NBD / flip–flop / vacuole © 2002 Elsevier Science Ltd. All rights reserved.
Introduction The organellar membranes in eukaryotic cells maintain unique compositions of lipids that include as many as 50 of the approximately 500 different cellular membrane lipids.1 Specific functions have been identified for many of these lipids. For example, membrane lipids serve as receptors, second messengers or precursors to second messengers, and anchors for membrane-associated proteins. Although a rationale for the complex lipid compositions of membranes has begun to emerge, the full significance for maintaining different lipid compositions between the membranes of cellular organelles as well as between both leaflets of the same membrane has not been elucidated. Furthermore, the molecular mechanisms that
Advantages and disadvantages of NBD-labeled phospholipids Of the numerous choices of fluorescence groups that are suitable for attachment to phospholipids, for studies in yeast, the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) moiety has been used almost exclusively. The molecule most commonly used is acylated with either a myristoyl or palmitoyl group in the sn-1 position of the glycerol backbone and with N-NBD aminocaproyl in the sn-2 position. Placing the label in the acyl chain allows for the synthesis of labeled phospholipids with each of the commonly occurring head groups. These
From the Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1084–9521 / 02 / $– see front matter
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molecules are commercially available and relatively inexpensive—a significant advantage when large amounts are required for genetic screens. The short acyl chain in the sn-2 position increases the water solubility sufficiently to allow rapid spontaneous equilibration between membranes while maintaining a strong preference for hydrophobic membranes.2 The NBD group has spectral properties that allow it to be visualized in a fluorescence microscope with a standard fluorescein filter set. The high environmental sensitivity of its fluorescence yield—the fluorescence of water soluble products is only ∼1% of the hydrophobic parent compound3 —can be exploited to monitor degradation rates in situ. One of the major disadvantages of the NBD fluorophore is its propensity to photobleach although this can be overcome by the use of a high sensitivity video camera and short exposure times. These properties allow one to introduce NBD-labeled phospholipids to the plasma membrane of living yeast cells and to monitor their fates during internalization in order to gain insight into the mechanisms of sorting and trafficking of phospholipids in yeast. Although NBD-labeled phospholipids and reporter phospholipids in general provide useful tools for the investigation of phospholipid trafficking and for the identification of phospholipid translocators, one must use caution in extrapolating their behavior to that of endogenous phospholipids. Demonstration of short-chain reporter phospholipid transport does not constitute proof of endogenous phospholipid transport in the absence of supporting evidence. However, this disadvantage is balanced by the lack of alternative approaches to address these problems and the fact, that when appropriate supporting evidence is available, the behavior of reporter phospholipids has been shown in many cases to reflect the behavior of their endogenous counterparts.4
Figure 1. Schematic of NBD-phospholipid traffic in yeast.
vesicle or DMSO suspension. Their internalization, distribution and metabolism were monitored and quantified by several different methods including fluorescence microscopy, flow cytometry, extraction and purification. Step 1 refers to internalization by inward-directed transmembrane transfer across the plasma membrane, commonly referred to as flip. Studies of the four NBD-phospholipids indicate that each is internalized predominantly by flip as opposed to endocytosis although the mechanistic details differ. NBD-PC, NBD-PS and NBD-PE are internalized into wild-type yeast cells from donor vesicles at 30 ◦ C, however their fates are distinctly different. NBD-PC is internalized predominantly to the vacuole (Figure 2) and degraded whereas a minor component is localized in the mitochondria and nuclear envelope/ER.5 On the other hand, NBD-PE and NBD-PS (not shown) are internalized to the nuclear envelope/ER and mitochondria (Figure 2) and are not transported to the vacuole. NBD-PE is not degraded intracellularly,6 however the metabolic fate of NBD-PS has not been determined. The exclusive transport and degradation of NBD-PC in the vacuole was initially interpreted to reflect that a significant fraction of that internalized was endocytosed from the plasma membrane and transported through the late endocytic pathway to the vacuole.5 However, subsequent investigations have led to the conclusion that NBD-PC, like NBD-PE and NBD-PS, is transported primarily across the plasma membrane by flip and that endocytosis is a minor contributor, if any. NBD-PC is transported to the vacuole (step 6) subsequent to its flip across the plasma
Mechanism of internalization across the plasma membrane A generalized scheme of the trafficking steps investigated with NBD-phospholipids is illustrated in Figure 1. The numbered mechanistic steps will be discussed below in relation to four NBD-labeled phospholipids (NBD-phosphatidylcholine, NBD-PC; NBD-phosphatidylserine, NBD-PS; NBD-phosphatidylethanolamine, NBD-PE; and NBD-phosphatidate, NBD-PA) studied to date. In all cases the NBD-phospholipids were spontaneously introduced to the exoplasmic surface of the plasma membrane from a 180
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Figure 2. Localization of NBD-PC and NBD-PE in yeast at 30 ◦ C. A diploid strain of S. cerevisiae was grown to mid-log phase at 30 ◦ C and incubated with vesicles containing either NBD-PC or NBD-PE. Panels A and C show cells incubated with NBD-PE and panels B and D show cells incubated with NBD-PC. Used with permission of The Journal of Cell Biology; Kean et al. (1997) JCB 138, 255–270.
membrane. These conclusions are based on several pieces of evidence. First, at 2 ◦ C, both NBD-PC, NBD-PE and NBD-PS are internalized similarly to the mitochondria and nuclear envelope/ER whereas an established morphological tracer of the endocytic pathway in yeast, FM 4-64,7, 8 remains at the plasma membrane and is not internalized. Thus, endocytosis is not required for their internalization at low temperature. At higher temperatures that block the early stages of endocytosis in the end4-1 strain, FM4-64 internalization is significantly inhibited while the internalization of NBD-PC and NBD-PE is unaffected. If the early stages of endocytosis requiring End4p contributed significantly to the total internalization of either NBD-PC or NBD-PE, a measurable decrease in the amount of internalized fluorescent phospholipid would be expected. A similar result was observed using the ‘back exchange’ assay. The end4-1 strain was labeled with NBD-PC or NBD-PS, washed, and the amount of internalization inferred from the fraction not removed with an albumin wash. Both NBD-PC and NBD-PS were internalized in the endocytosis deficient mutant.9 NBD-PA was also shown to be internalized predominantly by flip across the plasma membrane followed by its metabolism to numerous products including diacylglycerol, phosphatidylinositol, phos-
phatidylcholine, and phosphatidylethanolamine in decreasing order of magnitude.10 Although the four NBD-phospholipids appear to be flipped across the plasma membrane, the mechanisms of transport appear to differ. When measured by fluorescence microscopy and flow cytometry, flip of NBD-PC, NBD-PS, and NBD-PE are dramatically inhibited by ATP-depletion,4 NEM treatment,4 and collapse of the plasma membrane proton-electrochemical potential11 (Figure 3). These results support the conclusion that their flip occurs via a proton gradient coupled transporter susceptible to inhibition by sulfhydryl modification. However, results obtained in the end4-1 strain using the ‘back exchange’ assay demonstrated no ATP-dependence or NEM-sensitivity to the flip of NBD-PC and NBD-PS.9 This discrepancy may be the result of an interesting property of the end4-1 strain. ‘Back exchange’ experiments, in which only the NBD-phospholipid inserted into the exoplasmic surface of the plasma membrane is allowed to flip inward, are precluded in most, if not all, wild-type yeast strains because they do not allow the insertion of detectable amounts of probe into the plasma membrane. The end4-1 strain was suitable for these experiments based on the fact that the exoplasmic surface of the plasma membrane could be labeled. These 181
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subsequent demonstration that Drs2p is localized to the Golgi,15 casts further doubt on its identification as the transporter responsible for NBD-PS flip in S. cerevisiae.
NBD-PC and NBD-PE are actively flopped across the plasma membrane Outward-directed transport across the plasma membrane, commonly referred to as flop, has only been investigated for NBD-PC and NBD-PE. Following internalization by standard methods, both of these probes are rapidly flopped to the exoplasmic leaflet of the plasma membrane where they are degraded to non-fluorescent water soluble products by periplasmic enzymes.6 Degradation was shown to occur subsequent to flop to the exoplasmic surface by the almost complete inhibition of degradation and the retrieval of intact probes from phospholipid vesicles present in the bathing medium. Similar results were obtained when the above experiment was performed with the sec6-4 strain at its non-permissive temperature. Since the transport of secretory vesicles to the plasma membrane is blocked in this strain,16 secretion as the mechanism of efflux is ruled out. Because periplasmic degradation of NBD-PC and NBD-PE occurs faster than flop across the plasma membrane, the flop rate can be inferred from the loss of fluorescence and measured by flow cytometry. Using this approach, the flop of NBD-PC and NBD-PE was found to be almost completely blocked by ATP-depletion and by incubation at 2 ◦ C.11 However, flop was not affected by collapse of the proton electrochemical gradient. Two members of the ABC transporter family, Pdr5p and Yor1p, have been demonstrated to contribute to the flop of NBD-PE.17
Figure 3. NBD-PC flip is inhibited by CCCP. Early log phase cells were incubated with and without the protonophore CCCP (carbonyl cyanide m-chlorophenylhdrazone), chilled on ice and labeled with NBD-PC or NBD-PE solubilized in DMSO. At the indicated times, aliquots were removed and analyzed by flow cytometry. NBD-PC control (closed circles); NBD-PE control (open circles); NBD-PC with CCCP (closed squares); NBD-PE with CCCP (open squares). Used with permission of The Journal of Biological Chemistry; Hanson and Nichols (2001) JBC 276, 9861–9867.
differences imply an alteration in the plasma membrane organization and structure that may account for the observed differences in ATP-dependence and NEM-sensitivity of NBD-PC and NBD-PS flip. NBD-PA flip measured by fluorescence microscopy and extraction and purification is not ATP-dependent.10 This may reflect the ability of its protonated species to spontaneously flip,12 however its sensitivity to collapse of the proton-electrochemical gradient has yet to be investigated. No consensus has emerged regarding the identity of the putative transporter/s responsible for NBD-PC, NBD-PS and NBD-PE flip. A member of the P-type ATPase family has been proposed to account for essentially all of the flip activity observed for NBD-PS in yeast.13 This conclusion was based on the loss of NBD-PS flip measured by ‘back exchange’ at low temperature in ATP-depleted strains in which the DRS2 gene encoding for the P-type ATPase was deleted. However, no apparent inhibition of NBD-PS flip was observed in a drs2 null strain when examined by fluorescence microscopy,14 and no quantitative differences were observed between NBD-PC and NBD-PS flip in the drs2 null strain when analyzed by flow cytometry.14 Furthermore deletion of the DRS2 gene in an end4-1 strain failed to abolish NBD-PS flip.9 The
Net internalization of NBD-phospholipids is controlled by regulating flip and flop A screen of mutagenized yeast identified gain-offunction mutations in two transcription factors, PDR1 and PDR3 that resulted in the loss of net NBD-PE accumulation.6 PDR1 and PDR3 regulate the expression of a wide range of targets in response to stress.18 Among these downstream targets are several drug efflux pumps that are members of the ABC transporter family.18 This class of transporter has been shown to confer drug resistance by effluxing a wide range of drugs across the plasma membrane suggesting 182
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that the observed decrease in net internalization of NBD-PC and NBD-PE in the PDR1 and PDR3 mutants resulted from their increased flop relative to flip. This hypothesis was confirmed by flow cytometry by the demonstration that NBD-PC and NBD-PE flop rates (step 3) were accelerated in the mutant PDR3 strain (pdr3-11).11 Since flop is essentially blocked at 2 ◦ C, measurement of the total influx at this temperature allows the rate of flip to be measured in the absence of flop. Surprisingly, the 2 ◦ C flip of both NBD-PC and NBD-PE (step 3) is dramatically reduced in the gain-of-function mutant strains (PDR1-11 and pdr3-11) and increased in strains in which PDR1 and PDR3 are deleted.11 It is thus apparent that the transcription factors PDR1 and PDR3 control the net accumulation of NBD-PC and NBD-PE by regulating both their rate of flip and flop across the plasma membrane. The net accumulation of NBD-PC and NBD-PE is also almost completely inhibited during the late log phase of growth by a decrease in the rate of flip without affecting the rate of flop.11 The physiological significance of down-regulating NBD-PC and NBD-PE flip in response to nutrient starvation remains to be determined, but suggests a role in the adaptation to growth arrest.
One might predict that passive transport would result in equal labeling of all of the membranes of intracellular organelles, however this does not appear to be the case. Neither NBD-PC nor NBD-PE is detected in the vacuolar or plasma membrane. Perhaps these membranes have an inherently lower affinity (partition coefficient) for the NBD-phospholipids or perhaps their distribution is specifically directed by the activity of transfer proteins. At this time, we cannot distinguish between these and other plausible alternatives.
Intracellular sorting and metabolism of NBD-PC and NBD-PA NBD-PC is internalized predominantly by flip across the plasma membrane and subsequently transported from the cytosolic membrane compartment to the lumen of the vacuole (step 6) at 30 ◦ C. The latter step is demonstrated by first labeling wild-type cells with NBD-PC at 2 ◦ C followed by a 30 ◦ C chase. During the chase, NBD-PC fluorescence is lost from the mitochondria, nuclear envelope/ER and appears in the vacuole as diffuse fluorescence characteristic of water soluble products20 . This transport to the vacuole is blocked by energy depletion. These results demonstrate that NBD-PC is selectively transported to the vacuole from intracellular organelles by an energy-dependent process. Elimination of NBD-PC transport to the vacuole in strains deleted in VPS4 and VPS28 genes required for vesicular traffic from the prevacuolar compartment (PVC) to the vacuole20 implies that NBD-PC is internalized into the PVC (step 5) by either invagination or transport across the membrane prior to its transport to the vacuole. The intracellular metabolism of NBD-PA to NBDphosphatidylinositol and NBD-PC is also dependent on normal membrane recycling to and from the plasma membrane. Although its degradation to NBDdiacylglycerol is unaffected in a sec6-4 strain, which is blocked in the Golgi to plasma membrane transport step, the synthesis of NBD-PC and NBD-phosphatidylinositol are significantly inhibited at the non-permissive temperature in this strain background.10 This suggests that the perturbation of membrane recycling in the sec6-4 strain blocks the transport of NBD-diacylglycerol to the intracellular sites of synthesis of NBD-phosphatidylinositol and NBD-PC. Transport of NBD-PC to the vacuole and the metabolism of NBD-PA to NBD-PC and NBD-phosphatidylinositol are specific intracellular lipid trafficking and
NBD-PC and NBD-PE are passively transported to the membranes of intracellular organelles If NBD-PC and NBD-PE are internalized by flip across the plasma membrane at 2 ◦ C, they must be distributed from the cytoplasmic leaflet of the plasma membrane to that of the mitochondria, nuclear envelope/ER and other intracellular organelles (step 4) to produce the pattern of localization shown in Figure 2. Since this transport occurs at 2 ◦ C, it is unlikely to depend on vesicular transport. The simplest explanation is that the short acyl chain in the sn-2 position of both NBD-PC and NBD-PE permits their passive, spontaneous redistribution to intra-organellar membranes. Although the partition coefficient for NBD-PC is 3 × 106 ,2 this high partition coefficient is several orders of magnitude lower than that for endogenous phospholipids (∼109 )19 and results in an increased half-time of spontaneous transfer through the aqueous phase from days to a few seconds.2 Although the physical properties of the probes are adequate to explain their rapid distribution, we cannot exclude a possible role of phospholipid transfer proteins. 183
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8. Wendland B, McCaffery JM, Xiao Q, Emr SD (1996) A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15. J Cell Biol 135:1485–1500 9. Marx U, Polakowski T, Pomorski T, Lang C, Nelson H, Nelson N, Herrmann A (1999) Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein. Eur J Biochem 263:254–263 10. Trotter PJ (2000) A novel pathway for transport and metabolism of a fluorescent phosphatidic acid analog in yeast. Traffic 1:425– 434 11. Hanson PK, Nichols JW (2001) Energy-dependent flip of fluorescence-labeled phospholipids is regulated by nutrient starvation and transcription factors, PDR1 and PDR3. J Biol Chem 276:9861–9867 12. Eastman SJ, Hope MJ, Cullis PR (1991) Transbilayer transport of phosphatidic acid in response to transmembrane pH gradients. Biochemistry 30:1740–1745 13. Tang X, Halleck MS, Schlegel RA, Williamson P (1996) A subfamily of P-type ATPases with aminophospholipid transporting activity. Science 272:1495–1497 14. Siegmund A, Grant A, Angeletti C, Malone L, Nichols JW, Rudolph HK (1998) Loss of Drs2p does not abolish NBD-labeled phospholipid transfer across the plasma membrane of Saccharomyces cerevisiae. J Biol Chem 273:34399–34405 15. Chen CY, Ingram MF, Rosal PH, Graham TR (1999) Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function. J Cell Biol 147:1223– 1236 16. Walworth NC, Novick PJ (1987) Purification and characterization of constitutive secretory vesicles from yeast. J Cell Biol 105:163–174 17. Decottignies A, Grant AM, Nichols JW, de Wet H, McIntosh DB, Goffeau A (1998) ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p. J Biol Chem 273:12612–12622 18. Balzi E, Goffeau A (1994) Genetics and biochemistry of yeast multidrug resistance. Biochim Biophys Acta 1187:152–162 19. Smith R, Tanford C (1972) The critical micelle concentration of l-dipalmitoylphosphatidylcholine in water and water–methanol solutions. J Mol Biol 67:75–83 20. Hanson PK, Grant AM, Nichols JW (2002) NBD-labeled phosphatidylcholine enters the yeast vacuole via the pre-vacuolar compartment. J Cell Sci, in press.
metabolic steps that are perhaps amenable to exploration by further screening existing mutant strains in relevant trafficking pathways and to the development of genetic screens that may identify new components of these poorly understood processes.
Acknowledgements The author acknowledges the intellectual and experimental contributions of C. Angeletti, A.M. Grant, P.K. Hanson, L.S. Kean, and L. Malone. This work was supported in part by National Institutes of Health Grant GM52410 and a grant from the University Research Committee of Emory University.
References 1. van Meer G (1993) Transport and sorting of membrane lipids. Curr Opin Cell Biol 5:661–673 2. Nichols JW (1985) Thermodynamics and kinetics of phospholipid monomer–vesicle interaction. Biochemistry 24:6390–6398 3. Nichols JW (1987) Binding of fluorescent-labeled phosphatidylcholine to rat liver non-specific lipid transfer protein. J Biol Chem 262:14172–14177 4. Grant AM, Hanson PK, Malone L, Nichols JW (2001) NBDlabeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane. Traffic 2:37–50 5. Kean LS, Fuller RS, Nichols JW (1993) Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane. J Cell Biol 123:1403–1419 6. Kean LS, Grant AM, Angeletti C, Mahe Y, Kuchler K, Fuller RS, Nichols JW (1997) Plasma membrane translocation of fluorescent-labeled phosphatidylethanolamine is controlled by transcription regulators, PDR1 and PDR3. J Cell Biol 138:255– 270 7. Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128:779–792
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