is plasma membrane lipid composition defined in the exocytic or the endocytic pathway?

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14 BOURNE, H. R., SANDERS, D. A. and McCORMICK, F. (1990) Nature 348,125-132

15 ffOH, T. et al. (1993) Proc. Nail Acad. Sci. USA 90, 975-979 16 SHIBUYA, E., POLVERINO, A., CHANG, E., WIGLER, M. and

RUDERMAN, I. (1993) Proc. NatlAcad. Sci. USA 89, 9831-9835 17 FIELD, J. etal. (1988) MoL Cell. Biol. 8, 2159-2165 18 MACDONALD, S. G. etal. (1993) MoL Cell. Biol. 13, 6615-6620 19 TRAVERSE, S. etal. (1993) OncogeneS, 3175-3181 20 LEEVERS, S. I., PATERSON, H. F. and MARSHALL, C. J. (1994)

Nature 369, 411-4i4 21 MARSHALL, C. J. (1994) Curr. Opin. Genet. Dev. 4, 82-89 22 STOKOE, D., MACDONALD, S. G., CADWALLADER, K.,

SYMONS, M. and HANCOCK, I. F. (1994) Sdence 264,1465-1467 23 FREED, E., MACDONALD, S., SYMONS, M., McCORMICK, F.

and RUGGIERI, R. Science (in press) 24 IRIE, K. et al. Science (in press)

25 SOZERI, O. et al. 0992) Oncogene 7, 2259-2262 26 KOLCH, W. et aL (1993) Nature 364, 249-252 27 BOLLAG, G. and McCORMICK, F. (1991) Annu. Rev. Cell. Biol. 7,

601--632 28 KffAYAMA, H., SUGIMOTO, Y., MATSUZAKI, T., IKAWA, Y. and

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(1994) Cell 77, 841--852 33 RIDLEY, A. I. and HALL, A. (1992) Cell 70, 389-399 34 RIDLEY, A. I., PATERSON, H. F., IOHNSTON, C. L., DIEKMANN,

D. and HALL, A. (1992) Cell 70, 401-410

Is plasma membrane lipid

composition defined in the exocytic or the

endocytic pathway?

/ laL, A ~ I'A1 m a m t i a I Istilt m t im. I [ ~ / a

Compared with intracellular membranes, the plasma membrane

is rich in cholesterol and sphingomyelin. How does this distinct

composition arise? Here David Allan and Karl-losef Kallen take

a critical view of the belief that these lipids arrive at the

plasma membrane via vesicular traffic from the Golgi complex and

propose instead that they may be accreted in the endocvtic

recvcling pathway.

There is a dramatic difference between the lipid composition of the plasma membrane and that of most intracellular membranes, in particular the endoplas- mic reticulum (ER). The plasma membrane is rich in cholesterol, sphingomyelin (SM), glycolipids and phosphatidylserine and poor in phosphatidylinositol, cholesterol ester and triacylglycerol, whereas the ER (the main site of lipid biosynthesis) is rich in

phosphatidylcholine and phosphatidylinositol but contains only small amounts of those lipids that are characteristic of plasma membranes 1.

A substantial bulk flow of ER lipids to the plasma membrane must be a consequence of the vesicular transport processes that move proteins to the cell surface from their site of synthesis in the ER, but how does the cell generate and maintain the peculiar lipid composition of the plasma membrane if large quan- tities of ER llpids are continually entering it from the secretory pathway?

Despite the recent interest in lipid trafficking 2, this question has still not been answered satisfactorily. There is no obvious lipid equivalent of the sorting signals that target proteins to particular organelles, although cytosollc lipid-transfer proteins have been identified that can exchange Ilplds between different membranes s. These may play a role in glycerollpld translocatlon ~,2 but it is difficult to imagine how they could maintain lipid gradients between the ER and plasma membrane or affect the distribution of SM or glycolipids. The sphingoliplds are generally locked into lumenal membrane surfaces and do not undergo significant transbllayer movement (flip-flop) so they can move between organelles only by vesicular transport processes. Although a small proportion of sphingolipids and cholesterol may be associated with caveoli in the plasma membrane 4 and there is considerable evidence for limited lipid microdomains, in general it is assumed that most lipids diffuse freely in membranes. Thus, most liplds are not sorted between compartments of a vesicular transport pathway - i.e. lipids generally follow bulk membrane flow.

The Golgl lipid gradient Nonetheless, i t has become c o m m o n l y accepted

that the l ip id compos i t ion of t ransport vesicles changes gradual ly across the Golgi complex, so that the vesicles u l t imate ly fusing w i t h the plasma membrane are r ich in cholesterol and SM s.6. This l ip id gradient model is wide ly believed because i t is

350 © 1994 Elsevier Science Ltd 0962-8924/94/$07.00 TRENDS IN CELL BIOLOGY VOL. 4 OCTOBER 1994

FORUM

compatible with our understanding of the mechanism of vesicular transport of proteins and also because it is supported by data suggesting that Golgi and secretory granule membranes have a lipid composition intermediate between that of the ER and plasma membrane 7. Electron microscopy of filipin-labelled cells also indicates a gradient of cholesterol across the Golgi stack s.9. In addition, enzymes that synthesize SM and the simpler glycolipids are present in the cis Golgi cisternae ~°-~z and enzymes for elongation of glycolipid carbohydrate chains are present at pro- gressively more distal sites in the secretory pathway ~3, similar to the distribution of enzymes involved in glycoprotein maturation. Thus, in this model the composition of the plasma membrane is essentially defined in the exocytic pathway.

The idea of a gradual cis to trans enrichment of cholesterol and plasma membrane phospholipids through the Golgi complex carries considerable con- ceptual potency. Indeed, Bretscher and Munro recently postulated that lateral segregation of cholesterol and SM through the Golgi complex could provide a basis for protein segregation in Golgi mere- branes 6. A major purpose of this article is to point out that despite the apparent attractions of this model there is compelling evidence against the idea of a lipid gradient through the Golgi, and that an alterna- tire paradigm fits the data better.

Evidence against the Golgi lipid gradient The compositional evidence that supports the idea

of a cholesterol and SM gradient through the Golgi is unconvincing. Most of this evidence comes from liver fracttonation studies, but one of the main activi- ties of hepatocytes is secretion of llpoproteins; the Golgi complex of these cells thus contains substantial amounts of lumenal llpoprotein that is not only rich in cholesterol and SM but difficult to remove completely t4. Also, hepatocytes endocytose large amounts of plasma lipoproteins, and few attempts have been made to separate the lipoproteln.contalnlng vesicles In the exocyttc pathway from those in the endocyttc pathway ts016. Clearly, endocytlc vesicles could be another source of contamination, con. trlbutlng not only Ilpoproteins but also plasma membrane llpids to a putative Golgi fraction. To com- pound these potential problems, if the plasma membrane recycling pathway merges with the Golgi complex at the trans Golgi network (TGN) or is other- wise physically associated with the Golgi complex in the juxtanuclear region of the cell 17 it would be difficult to separate components of the two pathways. This could explain the findings with fillpin s09, which in any case represent an indirect measure of cholesterol concentration and have not been confirmed in some studies using different cell types ~s,~9.

When well-characterized Golgi membranes have been purified free of intralumenal contamination, it seems clear that there is essentially no difference in lipid composition between ER and Golgi membranes (Table 1). This is true ~ven ia pxepa~'ations enriched in trans Golgi element~ as judged by their high specific activity of galactosyltransferase ~4. Careful separation of exocytic and endocytic membrane

TABLE 1 - PROPORTIONS OF SPHINGOMYEUN AND CHOLESTEROL IN LIVER CELL MEMBRANES"

Lipid ER Golgi PM Endosomes

Sphingomyelin 5 + 2 6 + 3 17 + 1 25 + 10 Cholesterol 16 + 4 20:1:6 52 67 + 13

aData pooled and averaged from Refs 14-16, 20 and 35. Results are expressed as mole % of total phospholipid. PM, plasma membrane.

vesicles from rat liver showed that in terms of the cholesterol: phospholipid ratio exocytic vesicles resembled ER whereas endocytic vesicles resembled plasma membrane 1s,~6,2°. If, as these results suggest, there is no convincing indication of a cholesterol or SM concentration gradient between the ER and the trans Golgi cisternae, then how are cholesterol and SM concentrated in the plasma membrane and which intracellular organelles are involved in the process?

A post-Golgl site of SM synthesis? The answers to these questions probably require a

deeper knowledge of the mode of synthesis and sub- cellular localization of SM. It has been known for years that cholesterol has a higher aff inity for SM than for other phospholipids I and more recent evidence suggests that the SM content of plasma membrane exerts a remarkable influence on cholesterol localization, esterification and biosynthesis 21,22. In- deed, cholesterol might be expected to accumulate at tile site of synthesis of SM.

SM has been reported to be synthesized in the cis/medial Golgi 1°-12. However, other evidence suggests a non-Golgi location for SM synthesis 23,24, and we have obtained data suggesting that the major site of synthesis of SM for delivery to the plasma membrane is not the Golgl complex but the endocytic recycling pathway 2s,26. First, BHK cells exposed to monensin (which blocks transport between the medial and trans Golgi cisternae) fail to convert ceramide to plasma membrane SM, suggesting that ceramide must be transported to a site distal to the medial Golgi complex to be converted into plasma membrane SM 2s. Second, a major site of SM synthesis is revealed when ceramide formed by enzymic degra- dation of cell surface SM is converted back into SM that is again found at the cell surface 26. The rate of resynthesis, which is about fivefold greater than normal SM synthesis in actively growing cells, suggests .................................. that it represents the activity of a major intracellular site involved in the normal biosynthesis of SM. Resynthesis is unaffected by monensin or brefeldin A (BFA), which block vesicular transport through the Golgi complex, but is inhibited under conditions that interfere with endocytosis, for example in mitotic or energy-depleted cells. The dependence on endocytosis makes it unlikely that synthesis of SM occurs at the plasma membrane. Similar results have been reported for hepatocytes by Shiao and Vance 27 and support the concept of a Golgi-independent site of plasma membrane SM biosynthesis. This site could be part of the plasma membrane recycling pathway,

David Allan is at the Dept of Physiology, University College London, London, UK WC1E 6JJ; and Karl-loser Kallen is at the Medizinische Klinik I, Iohannes- Gutenberg Universitat, 55101 Mainz, fRG.

TRENDS IN CELL BIOLOGY VOL. 4 OCTOBER 1994 351

Golgi

ER ~

FIGURE 1

A model for the synthesis of SM and the associated accretion of cholesterol in an endosomal compartment. This model shows the movement of ceramide

(filled circles) by vesicular transport from its site of synthesis in the ER, through the Golgi complex, to the plasma membrane (PM) and thence by endocytosis to the site of SM synthesis (S) in a plasma membrane recycling compartment.

The phosphocholine headgroup of SM is shown as a cross. It is conceivable that the TGN is a constituent of the recycling pathway, so that ceramide could move

directly into endosomal compartments without passing through the plasma membrane. Cholesterol (rectangles) is envisaged to pass via a cytosolic carrier

protein irom '~:, ~lte of synthesis in the ER to the endosomes, where it is accreted uy association with SM before delivery to the plasma membrane.

which may Include the TGN (Fig. 1). There is evi. dence that BFA blocks exit from the TGN but has no effect on recycling 2s, Indicating that the TGN is not involved in the recycling pathway. However, other work suggests at least a small role for the TGN in plasma membrane recycling in BHK cells 2~.

Consequences of SM resynthesls In the endocytlc pathway

If plasma membrane SM is synthesized in the endo. cytic recycling pathway, then this is the first indication of a biosynthetic activity associated with endosomes. However, it is not clear how ceramide endogenously synthesized in the ER can reach a putative endosomal site of SM synthesis. Vesicular transport of ceramide through the Golgi complex seems to be involved rather than protein.mediated dif- fusion through the cytosoi, since monensin blocks conversion of newly synthesized ceramide into plasma, membrane SM 2s. Ceramide could therefore enter the plasma membrane recycling pathway from the TGN or it could even reach the plasma membrane and then be endocytosed, as shown in Fig 1. The longer pathway via the plasma membrane would be consistent with the observation that glucosyl- ceramlde (synthesized from ceramide in the cis Golgi complex) reaches the plasma membrane more quickly than SM ~o.

Importantly, this model implies that plasma membrane lipid composition could be determined largely by compositional modifications that occur in the recycling pathway rather than in the Golgi complex.

FORUM

In BHK cells, bulk surface membrane is endocytically recycled with a half-time of about 40 min whereas newly synthesized lipid appears at the surface with a half-time of 6 h (based on cell doubling time), so recycling brings more lipid to the surface than de hove synthesis in a given time z6. Furthermore, most endocytosed membrane lipid is recycled and does not 'leak' into the early part of the exocytic pathway ~7.2°, so lipid modifications that occur in the recycling pathway would be retained in the plasma membrane and would help to preserve its special identity. The recycling pathway may also act to repair plasma membrane sphingolipids that have been degraded, as it seems to do for SM broken down by an extra- cellular shingomyelinase 26. It might also regulate the levels of ceramide and its derivatives that are involved in signal transduction processes 3t.

One of the most interesting aspects of the model proposed here is its potential to explain the incorporation of cholesterol into the plasma membrane. Although plasma membranes contain considerably more cholesterol than SM (Table 1), the distribution of SM appears to determine the intracellular localization of cholesterol 22. Thus synthesis of SM in the endocytic recycling pathway might govern the incorporation of cholesterol into this compartment and thence into the plasma membrane. The most likely way in which cholesterol could reach this compartment from its site of synthesis in the ER is via a cytosolic carrier protein 3. This model can explain why transport of cholesteroP 2,33 and SM 2s-27 to the plasma membrane is not affected by monensln or BFA (and therefore does not require an intact Golgi complex) but is sensitive to reduced temperature and energy depletion (which inhibit vesicula, t,,msp~rt processes including endocytosis) a2,:~:~, it would also explain why cholesterol reaches the plasma membrane more quickly than the bulk of Golgi lipids :~2, because it would traverse a relatively short vesicular transport path to the cell surface. The recycling compartment where we propose cholesterol is incorporated could correspond to a known specific class of vesicles rich in newly synthesized cholesteroF u,-~4, although the ability of these vesicles to synthesize SM has not been tested.

The concept that recycling endosomes can synthesize plasma membrane SM and that this deter- mines plasma membrane cholesterol content represents a new way of looking at plasma membrane lipid biosynthesis. It suggests that the plasma membrane achieves and retains its distinctive lipid composition because its major lipids are incorporated in the recycling pathway, which has little vesicular commerce with the exocytic pathway '7. The model proposed here avoids the difficulties associated with the concept of a gradient of lipids through the exocytic pathway t2, and explains present experimental data on SM and cholesterol incorporation into the plasma membrane more effectively than the concept of a Goigi lipid gradient. Furthermore, predictions of the model, in terms of the localization of SM synthesis and cholesterol accretion in endosomal recycling vesicles, can be tested experimentally. Ultimately, the enigmas of intracellular lipid

352 TRENDS IN CELL BICLOGY VOL. 4 OCTOBER 1994

FORUM

trafficking will be resolved only when we puri fy the enz~nes involved in sphingolipid synthesis and make antibodies to study localization by immuno- histochemical methods. This approach is likely to improve dramatically our understanding not just of plasma membrane lipid biosynthesis but of membrane trafficking in general.

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Acknowledgements

We thank Paul Quinn for stimulating discussion of some of the ideas presented here. We also acknowledge the financial support of The Wellcome Trust, The Medical Research Council and the German Academic Exchange Service (DAAD).

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is plasma membrane lipid composition defined in the exocytic or the endocytic pathway?

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