news and views and cholesterol-rich microdomains. But exactly how they modulate Ras signalling remains to be determined. Analysis of Ras signalling in truly caveolin-deficient cells in mice and nematodes should help in this regard. h Paul W. Sternberg is in the Department of Biology, California Institute of Technology, 1201 East California Blvd, Pasadena, California 91125, USA.
Sandra L. Schmid is at the Scripps Research Institute, 10550 N. Torrey Pines, La Jolla, California 92037, USA. e-mail:
[email protected] 1. Anderson, R. G. W. Annu. Rev. Biochem. 67, 199–225 (1998). 2. Simons, K. & Ikonen, E. Nature 387, 569–572 (1997). 3. Okamoto, T., Schlegel, A., Scherer, P. E. & Lisanti, M. P. J. Biol. Chem. 273, 5419–5422 (1998). 4. Roy, S. et al. Nature Cell Biol. 1, 98–105 (1999) 5. Scheel, J., Srinivasan, J., Honnert, U., Henske, A. & Kurzchalia,
Cholesterol: stuck in traffic Sushmita Mukherjee and Frederick R. Maxfield
Niemann–Pick type C is a genetic disorder thought to be partly characterized by a defect in cholesterol storage in lysosomes. New findings show that in fact cholesterol accumulates in late endosomes, and this accumulation leads to the redistribution of membrane proteins.
t is becoming increasingly clear that the trafficking of intracellular membranes is controlled by a complex interplay of both the lipid and the protein components of the membranes. On page 113 of this issue, Kobayashi et al.1 provide fresh evidence for the function of lipids in controlling protein distribution, and suggest a surprising cellular site in which mutations in the Niemann– Pick type C (NPC) protein, NPC1, can alter lipid distributions. Membrane added to the plasma membrane by secretory vesicles is taken back into the cell, and recycled into intracellular organelles, by the process of endocytosis. The part played by endocytic organelles in the appearance of the NPC phenotype has been the focus of two recent papers, one by Kobayashi et al.1 and another by Neufeld et al.2. To understand these papers, we need to know about some key features of the endocytic trafficking pathways (Fig. 1; reviewed in ref. 3), remembering that although there has been considerable progress in describing endocytic membrane traffic, some fundamental uncertainties remain. The early sorting endosome appears to be the initial recipient of material that is internalized by clathrin-dependent and clathrin-independent endocytosis; clathrin is a ‘coat’ protein that is involved in the formation of transport vesicles from membranes. Many membrane proteins and lipids are then returned to the cell surface, mainly via a second element of the early endosomal system, the endocytic
I
recycling compartment (ERC). Some membrane proteins and lipids, however, are retained in the early sorting endosome, and, within a few minutes of membrane internalization, this organelle, along with its fluid contents, acquires the properties characteristic of a late endosome, including a reduced pH, a change in the
T. V. Nature Cell Biol. 1, 127–129 (1999). 6. Fire, A. et al. Nature 391, 806–811 (1998). 7. Campbell, S. L., Khosravi-Far, R., Rossman, K. L., Clark, G. J. & Der, C. J. Oncogene 17, 1395–1413 (1998). 8. Lui, P., Ying, Y.-S. & Anderson, R. G. W. Proc. Natl Acad. Sci. USA 94, 13666–13670 (1997). 9. Johnson, L. et al. Genes Dev. 11, 2468–2481 (1997). 10. Tang, Z. et al. J. Biol. Chem. 272, 2437–2445 (1997). 11. Church, D., Guan, K.-L. & Lambie, E. J. Development 121, 2525– 2535 (1995). 12. Song, S. K. et al. J. Biol. Chem. 271, 9690–9697 (1996).
types of Rab protein associated with the membrane, and the acquisition of various acid hydrolases3. Many of these acid hydrolases are delivered from the transGolgi network (TGN) by binding to mannose-6-phosphate receptor (MPR), which moves between the TGN and late endosomes. The presence of MPR is considered to be one of the defining characteristics of the late endosomes. Indigestible material and some membrane proteins, particularly the heavily glycosylated lysosomeassociated membrane proteins (LAMPs), are delivered from late endosomes to lysosomes, which are acidic, hydrolase-rich organelles that lack MPR and whose function is to digest extracellular material uptaken by endocytosis. However, as delivery to lysosomes is slow, most degradation of internalized proteins and lipids in cultured fibroblasts and many other cells actually occurs in the late endosomes. The endocytic traffic pathways have several branches, and bidirectional traffic occurs between many of the organelles. The
Coated pit
? Endocytic recycling compartment (pH 6.4 _ 6.5)
(cholesterol?) Early sorting endosome (pH 5.9 _ 6.0)
Tubule pinched off from sorting endosome
Trans-Golgi network (pH 6.0 _ 6.5)
Vesicle Late endosome (pH 5.0 _ 6.0) (LAMP +, MPR + )
NPC (?)
X
Golgi Vesicle (cholesterol?)
Lysosome (pH 5.0 _ 5.5) _ (LAMP +, MPR )
Endoplasmic reticulum
Figure 1 Trafficking pathways in a nonpolarized cell such as a fibroblast. Note that many of the pathways are bidirectional and that several organelles act as branch points feeding into different pathways. The membrane composition of each organelle at steady state is determined by the overall kinetics of each step in the pathway, as well as specific retention or acceleration mechanisms for specific membrane components. Also note that the definitions of some of the endocytic organelles are based on the presence or absence of membrane markers (such as mannose-6-phosphate receptor (MPR) or lysosome-associated membrane proteins (LAMPs)), so that in disease situations such as Niemann–Pick type C (NPC), in which the trafficking of those very markers is affected, it may become difficult to identify an organelle correctly. The most likely site at which the NPC1 mutation acts is indicated.
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news and views functional properties of each organelle depend upon its membrane composition, and this must be maintained despite a large flux of membrane through most organelles. For many years, membrane proteins drew most attention as determinants of membrane trafficking; numerous cytoplasmic sequence motifs have been identified and shown to be necessary for the correct targeting of membrane proteins in the endocytic pathway3. In several cases, these motifs bind to coat proteins, and this concentrates certain proteins in forming vesicle buds and allows selective targeting of membrane proteins. This targeting plays a key part in determining the protein composition of each organelle. The lipid composition also varies among organelles, but there has been little understanding of how this is achieved. Lateral differences (known as rafts or microdomains) in lipid composition have been proposed to be important in lipid sorting, perhaps by inclusion or exclusion of microdomains in various budding vesicles4,5. Earlier, Kobayashi et al.6 showed that lipid whorls inside late endosomes were enriched in an unusual lipid, lysobisphosphatidic acid (LBPA). These internal membrane whorls could be sites for retention of various lipids and membrane proteins. LBPA is an inverted-cone-shaped molecule (with much larger head group than tail cross-sectional area) that would be expected to enter highly curved membrane regions (that is, the whorls), and could retain other molecules in the whorls by specific lipid–protein and lipid–lipid interactions. Now, Kobayashi et al.1 have studied fibroblasts obtained from patients with NPC, an autosomal recessive genetic disorder that is caused by mutations in NPC1, a polytopic membrane protein. NPC is characterized by accumulation of cholesterol and other lipids in intracellular organelles, which have been proposed to be lysosomes7. Surprisingly, Kobayashi et al.1 find that lipoprotein-derived cholesterol mainly
...The redistribution of NPC1 as a consequence of changes in cholesterol concentration is reminiscent of the effects of changes in cholesterol content... on the traffic of glycosylphosphatidylinositolanchored proteins and transmembrane proteins... E38
accumulated in the LBPA-rich late endosomes, and not in lysosomes as proposed. This is consistent with late endosomes being the site of cholesterol ester hydrolysis, but it is unexpected that the excess cholesterol would remain in the late endosomes. If late endosomes are the site of cholesterol accumulation in NPC cells, one might expect to find the NPC1 protein in the same organelle. However, Neufeld et al.2 found that, in normal fibroblasts, the NPC1 is in a compartment that lacks MPR but contains LAMP2. In most cases, such a compartment would be considered a lysosome, but Neufeld et al. concluded that the compartment was not a lysosome because it did not become enriched in lowdensity-lipoprotein-derived cholesterol. With the new findings of Kobayashi et al.1 indicating that cholesterol actually accumulates in late endosomes, it seems more likely that much of the NPC1 is found in lysosomes at steady state. How could a protein that is found mainly in lysosomes affect cholesterol efflux from late endosomes? This is especially puzzling if one thinks in terms of unidirectional traffic from late endosomes to lysosomes. However, many studies, cited in Neufeld et al.2, show that retrograde traffic from lysosomes to late endosomes occurs. If NPC1 is carried back to late endosomes by this retrograde traffic, it could participate in the cholesterol efflux from the endosomes. Indeed, when cholesterol traffic is blocked pharmacologically, the NPC1 protein becomes redistributed to cholesterolrich organelles2 that are identified as late endosomes by Kobayashi et al 1. The redistribution of NPC1 as a consequence of changes in cholesterol concentration is reminiscent of the effects of changes in cholesterol content (usually reduced cholesterol levels) on the traffic of glycosylphosphatidylinositol-anchored proteins and transmembrane proteins in the plasma membrane, the TGN and the ERC4,8–10. Kobayashi et al.1 show that an increase in late-endosome cholesterol content alters the bidirectional traffic of MPR so that more receptor is found in late endosomes and less is in the TGN. The redistribution of MPR occurs after the accumulation of cholesterol, so retention of MPR is a consequence, not a cause, of altered cholesterol levels. It is not known how increased cholesterol amounts cause retention of NPC1 or MPR in late endosomes. NPC1 has a putative sterol-binding domain, but MPR is a single-transmembrane-domain protein that shows no known association with cholesterol. In fact, the effect on MPR in NPC cells, as well as the alterations seen in fluidphase endocytosis2, indicates that the mutation may cause a generalized change in endocytic membrane traffic. It remains to be seen whether such a general trafficking defect is the primary effect of the NPC
mutation or a consequence of a specific alteration in cholesterol traffic. The latter possibility is quite likely: retention of excess cholesterol in the late endosomes of NPC cells might cause an overall change in membrane elasticity, making it more difficult for cells to bud transport vesicles. One difficulty in understanding cholesterol efflux from late endosomes is that we have an inadequate understanding of the properties of this organelle. There is good evidence that clathrin-coated pits and the adaptor protein AP-1 are important in the budding of vesicles from the TGN that carry MPR to late endosomes3. We don’t have equivalent information yet about the return trip to the TGN. Unfortunately, the discovery that NPC1 may be involved in exit from late endosomes does not provide much insight into the molecular mechanism. Shifting the focus of cholesterol efflux from lysosomes to late endosomes has important consequences for understanding cholesterol metabolism (reviewed in refs 7,11). Lipoproteins carry most of their cholesterol as esters, which are hydrolysed after endocytosis. When excess free cholesterol is found in this pathway, it must be either esterified or removed from the cell to maintain cholesterol homeostasis. Both of these processes require transport to the plasma membrane. Although there are no known high-flux pathways from lysosomes to the plasma membrane, there are high-flux paths from late endosomes to the TGN and from the TGN to the plasma membrane. It is now clear that, first, lipid constituents are actively sorted in endocytic pathways; second, lipids regulate the sorting of proteins and other lipids; and third, membrane proteins are major determinants of lipid trafficking and sorting. A knowledge of these complex interactions will be essential in developing a complete understanding of endosomal membrane traffic. One hopes and expects that this, in turn, can lead to therapies for NPC and other storage disorders. h Sushmita Mukherjee and Frederick R. Maxfield are at the Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA. e-mail:
[email protected] 1. Kobayashi, T. et al. Nature Cell Biol. 1, 113–118 (1999). 2. Neufeld, E. B. et al. J. Biol.Chem. 274, 9627–9635 (1999). 3. Mukherjee, S., Ghosh, R. N. & Maxfield, F. R. Physiol. Rev. 77, 759–803 (1997). 4. Simons, K. & Ikonen, E. Nature 387, 569–572 (1997). 5. Mukherjee, S., Soe, T. T. & Maxfield, F. R. J. Cell Biol.144, 1271– 1284 (1999). 6. Kobayashi, T. et al. Nature 392, 193–197 (1998). 7. Liscum, L. & Klansek, J. J. Curr. Opin. Lipidol. 9, 131–135 (1998). 8. Mayor, S., Sabharanjak, S. & Maxfield, F. R. EMBO J. 17, 4626– 4638 (1998). 9. Rodal, S. K. et al. Mol. Biol. Cell 10, 961–974 (1999). 10. Subtil, A. et al. Proc. Natl Acad. Sci. USA (in the press). 11. Tabas, I. Curr. Opin. Lipidol. 6, 260–268 (1995).
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