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NATURE CELL BIOLOGY | VOL 1 | JUNE 1999 | cellbio.nature.com E37

and cholesterol-rich microdomains. Butexactly how they modulate Ras signallingremains to be determined. Analysis of Rassignalling in truly caveolin-deficient cellsin mice and nematodes should help inthis 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: sternbergp@starbase1.caltech.edu

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Chem. 273, 5419–5422 (1998).

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T. V. Nature Cell Biol. 1, 127–129 (1999).

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9. Johnson, L. et al. Genes Dev. 11, 2468–2481 (1997).

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2535 (1995).

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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 thetrafficking of intracellular membranes iscontrolled by a complex interplay of both

the lipid and the protein components of themembranes. On page 113 of this issue,Kobayashi et al.1 provide fresh evidence forthe function of lipids in controlling proteindistribution, and suggest a surprising cellu-lar site in which mutations in the Niemann–Pick type C (NPC) protein, NPC1, can alterlipid distributions.

Membrane added to the plasma mem-brane by secretory vesicles is taken backinto the cell, and recycled into intracellularorganelles, by the process of endocytosis.The part played by endocytic organelles inthe appearance of the NPC phenotype hasbeen the focus of two recent papers, one byKobayashi et al.1 and another by Neufeld etal.2. To understand these papers, we needto know about some key features of theendocytic trafficking pathways (Fig. 1;reviewed in ref. 3), remembering thatalthough there has been considerableprogress in describing endocytic mem-brane traffic, some fundamental uncer-tainties remain.

The early sorting endosome appears tobe the initial recipient of material that isinternalized by clathrin-dependent andclathrin-independent endocytosis; clath-rin is a ‘coat’ protein that is involved in theformation of transport vesicles frommembranes. Many membrane proteinsand lipids are then returned to the cell sur-face, mainly via a second element of theearly endosomal system, the endocytic

recycling compartment (ERC). Somemembrane proteins and lipids, however,are retained in the early sorting endo-some, and, within a few minutes of mem-brane internalization, this organelle, alongwith its fluid contents, acquires the prop-erties characteristic of a late endosome,including a reduced pH, a change in the

I

types of Rab protein associated with themembrane, and the acquisition of variousacid hydrolases3. Many of these acidhydrolases are delivered from the trans-Golgi network (TGN) by binding to man-nose-6-phosphate receptor (MPR), whichmoves between the TGN and late endo-somes. The presence of MPR is consideredto be one of the defining characteristics ofthe late endosomes. Indigestible materialand some membrane proteins, particu-larly the heavily glycosylated lysosome-associated membrane proteins (LAMPs),are delivered from late endosomes to lyso-somes, which are acidic, hydrolase-richorganelles that lack MPR and whose func-tion is to digest extracellular materialuptaken by endocytosis. However, asdelivery to lysosomes is slow, most degra-dation of internalized proteins and lipidsin cultured fibroblasts and many othercells actually occurs in the late endosomes.

The endocytic traffic pathways have sev-eral branches, and bidirectional trafficoccurs between many of the organelles. The

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.

Coatedpit

Early sorting endosome(pH 5.9 6.0)_

(pH 5.0 6.0)_

(cholesterol?)

(cholesterol?)

Tubule pinchedoff from sorting

endosome

Lateendosome

(LAMP , MPR )+ + Vesicle

Vesicle

X

(pH 5.0 5.5)_

(pH 6.4 6.5)_

(pH 6.0 6.5)_

Lysosome

(LAMP , MPR )+ _

NPC (?)

Endocyticrecyclingcompartment

Trans-Golgi network

Golgi

Endoplasmicreticulum

?

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E38 NATURE CELL BIOLOGY | VOL 1 | JUNE 1999 | cellbio.nature.com

functional properties of each organelledepend upon its membrane composition,and this must be maintained despite a largeflux of membrane through most organelles.For many years, membrane proteins drewmost attention as determinants of mem-brane trafficking; numerous cytoplasmicsequence motifs have been identified andshown to be necessary for the correct target-ing of membrane proteins in the endocyticpathway3. In several cases, these motifsbind to coat proteins, and this concentratescertain proteins in forming vesicle buds andallows selective targeting of membrane pro-teins. This targeting plays a key part indetermining the protein composition ofeach organelle.

The lipid composition also varies amongorganelles, but there has been little under-standing of how this is achieved. Lateraldifferences (known as rafts or microdo-mains) in lipid composition have been pro-posed to be important in lipid sorting,perhaps by inclusion or exclusion of micro-domains in various budding vesicles4,5.

Earlier, Kobayashi et al.6 showed thatlipid whorls inside late endosomes wereenriched in an unusual lipid, lysobisphos-phatidic acid (LBPA). These internalmembrane whorls could be sites for reten-tion of various lipids and membrane pro-teins. LBPA is an inverted-cone-shapedmolecule (with much larger head groupthan tail cross-sectional area) that wouldbe expected to enter highly curved mem-brane regions (that is, the whorls), andcould retain other molecules in the whorlsby specific lipid–protein and lipid–lipidinteractions.

Now, Kobayashi et al.1 have studiedfibroblasts obtained from patients withNPC, an autosomal recessive genetic disor-der that is caused by mutations in NPC1, apolytopic membrane protein. NPC is char-acterized by accumulation of cholesteroland other lipids in intracellular organelles,which have been proposed to be lysosomes7.Surprisingly, Kobayashi et al.1 find thatlipoprotein-derived cholesterol mainly

accumulated in the LBPA-rich late endo-somes, and not in lysosomes as proposed.This is consistent with late endosomesbeing the site of cholesterol ester hydrolysis,but it is unexpected that the excess choles-terol would remain in the late endosomes.

If late endosomes are the site of choles-terol accumulation in NPC cells, onemight expect to find the NPC1 protein inthe same organelle. However, Neufeld etal.2 found that, in normal fibroblasts, theNPC1 is in a compartment that lacks MPRbut contains LAMP2. In most cases, sucha compartment would be considered a lys-osome, but Neufeld et al. concluded thatthe compartment was not a lysosomebecause it did not become enriched in low-density-lipoprotein-derived cholesterol.With the new findings of Kobayashi et al.1

indicating that cholesterol actually accu-mulates in late endosomes, it seems morelikely that much of the NPC1 is found inlysosomes at steady state.

How could a protein that is foundmainly in lysosomes affect cholesterol effluxfrom late endosomes? This is especiallypuzzling if one thinks in terms of unidirec-tional traffic from late endosomes to lyso-somes. However, many studies, cited inNeufeld et al.2, show that retrograde trafficfrom lysosomes to late endosomes occurs.If NPC1 is carried back to late endosomesby this retrograde traffic, it could partici-pate in the cholesterol efflux from the endo-somes. Indeed, when cholesterol traffic isblocked pharmacologically, the NPC1 pro-tein becomes redistributed to cholesterol-rich organelles2 that are identified as lateendosomes by Kobayashi et al 1.

The redistribution of NPC1 as a conse-quence of changes in cholesterol concentra-tion is reminiscent of the effects of changesin cholesterol content (usually reduced cho-lesterol levels) on the traffic of glycosylphos-phatidylinositol-anchored proteins andtransmembrane proteins in the plasmamembrane, the TGN and the ERC4,8–10.Kobayashi et al.1 show that an increase inlate-endosome cholesterol content alters thebidirectional traffic of MPR so that morereceptor is found in late endosomes and lessis in the TGN. The redistribution of MPRoccurs after the accumulation of cholesterol,so retention of MPR is a consequence, not acause, of altered cholesterol levels.

It is not known how increased choles-terol amounts cause retention of NPC1 orMPR in late endosomes. NPC1 has a puta-tive sterol-binding domain, but MPR is asingle-transmembrane-domain proteinthat shows no known association with cho-lesterol. In fact, the effect on MPR in NPCcells, as well as the alterations seen in fluid-phase endocytosis2, indicates that the muta-tion may cause a generalized change inendocytic membrane traffic. It remains tobe seen whether such a general traffickingdefect is the primary effect of the NPC

mutation or a consequence of a specificalteration in cholesterol traffic. The latterpossibility is quite likely: retention of excesscholesterol in the late endosomes of NPCcells might cause an overall change in mem-brane elasticity, making it more difficult forcells to bud transport vesicles.

One difficulty in understanding choles-terol efflux from late endosomes is that wehave an inadequate understanding of theproperties of this organelle. There is goodevidence that clathrin-coated pits and theadaptor protein AP-1 are important in thebudding of vesicles from the TGN thatcarry MPR to late endosomes3. We don’thave equivalent information yet about thereturn trip to the TGN. Unfortunately, thediscovery that NPC1 may be involved inexit from late endosomes does not providemuch insight into the molecular mecha-nism.

Shifting the focus of cholesterol effluxfrom lysosomes to late endosomes hasimportant consequences for understand-ing cholesterol metabolism (reviewed inrefs 7,11). Lipoproteins carry most oftheir cholesterol as esters, which arehydrolysed after endocytosis. Whenexcess free cholesterol is found in thispathway, it must be either esterified orremoved from the cell to maintain choles-terol homeostasis. Both of these processesrequire transport to the plasma mem-brane. Although there are no knownhigh-flux pathways from lysosomes to theplasma membrane, there are high-fluxpaths from late endosomes to the TGNand from the TGN to the plasma mem-brane.

It is now clear that, first, lipid constitu-ents are actively sorted in endocytic path-ways; second, lipids regulate the sorting ofproteins and other lipids; and third, mem-brane proteins are major determinants oflipid trafficking and sorting. A knowledgeof these complex interactions will be essen-tial in developing a complete understand-ing of endosomal membrane traffic. Onehopes and expects that this, in turn, canlead to therapies for NPC and other storagedisorders. hSushmita 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: frmaxfie@mail.med.cornell.edu

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).

...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

glycosylphosphatidylinositol-

anchored proteins and

transmembrane proteins...

© 1999 Macmillan Magazines Ltd