Caveolin, cholesterol, and lipid bodies

Seminars in Cell & Developmental Biology 16 (2005) 163–174

Review

Caveolin, cholesterol, and lipid bodies

Sally Martin, Robert G. Parton∗

Institute for Molecular Bioscience and Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, Qld 4072, Australia

Available online 16 February 2005

Abstract

In mammalian cells a complex interplay regulates the distribution of cholesterol between intracellular membrane compartments. Oneimportant aspect of cholesterol regulation is intracellular cholesterol storage in neutral lipid storage organelles called lipid droplets or lipidbodies (LBs). Recent work has thrust the LB into the limelight as a complex and dynamic cellular organelle. LBs play a crucial role inmaintaining the cellular levels of cholesterol by regulating the interplay between lipid storage, hydrolysis and trafficking. Studies of caveolins,caveolar membrane proteins linked to lipid regulation, are providing new insights into the role of LBs in regulating cholesterol balance.© 2005 Elsevier Ltd. All rights reserved.

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Keywords: Caveolin; Cholesterol; Lipid body; Targeting; Regulation

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.1. A word about nomenclature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. New insights into LBs as dynamic organelles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3. LBs have multiple and diverse functions in different cell types. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14. Biogenesis of LBs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5. A role for LBs in pathogenesis?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6. The protein composition of LBs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7. Caveolin, LBs and the regulation of cholesterol levels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.1. Caveolins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2. Caveolin and cholesterol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.3. Linking caveolin, cholesterol, and signal transduction; functional studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.4. LB targeting of caveolin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

169169169

70170

171171171

7.5. Structural considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8. Functional effects of caveolin on the LB. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8.1. Studies of caveolin mutants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.2. Regulation of lipolysis in adipocytes; studies of caveolin-1 null mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3. Caveolin and atherosclerosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations:Cav, caveolin; ER, endoplasmic reticulum; FC, free cholesterol; Hep C, hepatitis C; HSL, hormone-sensitive lipase; LB, lipid body; PAT,perilipin/ADRP/TIP47; PKA, cAMP-dependent protein kinase/protein kinase A; PM, plasma membrane

∗ Corresponding author. Tel.: +61 7 3346 2032; fax: +61 7 3365 4422.E-mail address:[email protected] (R.G. Parton).

1084-9521/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.semcdb.2005.01.007

164 S. Martin, R.G. Parton / Seminars in Cell & Developmental Biology 16 (2005) 163–174

1. Introduction

1.1. A word about nomenclature

The presence of lipid-filled storage organelles in the cyto-plasm of cells has been known for a long time. These struc-tures are evolutionarily conserved organelles, present in alleukaryotes fromSaccharomyces cerevisiaeto mammals andplants. Initially thought of as simple cytoplasmic lipid inclu-sions the functional importance of these organelles in mam-malian cells has been long overlooked. It is increasingly clearthat the LB is a highly dynamic, motile organelle, which in-teracts with numerous organelles. The contents of the LB arerapidly turned over, and the organelle can be rapidly formedor dispersed depending upon the need of the cell. Given thedynamic nature of the organelle we feel that the term lipiddroplet is a somewhat outdated description of a complex or-ganelle. While the term adiposome has been suggested as analternative name for this organelle[1], this suggests a directconnection to adipocytes. As LBs appear to be ubiquitousorganelles we believe that the most apt description of thisorganelle is the lipid body.

2. New insights into LBs as dynamic organelles

ed ont thep thef on-n wass ffectso ts ofs eolinw cells eo nousc andt ds[ rolh ewi -m iblel

3d

dedb eins( ti-fi ts toi lec mpo-s or-

Fig. 1. Annotated electron micrograph illustrating the morphological fea-tures of a LB in a mammalian cultured cell, and cartoons of some key com-ponents. The figure shows a micrograph of a primary mouse fibroblast witha LB in close proximity to the RER, a mitochondrion, and a multivesicu-lar (late) endosome, organelles with which the LB must communicate. TheLB contains triglycerides and cholesterol esters, which form the neutral lipidcore, and is surrounded by a phospholipid monolayer. PAT proteins cover thecytoplasmic surface of the monolayer. The proposed association of a cave-olin molecule with the LB is shown schematically with its intramembranedomain contacting the lipid core.

ganisms and cell types. In mammals, LBs can be present inthe cytosol[11,12], secreted as lipoproteins[13,14], or un-dergo apocrine secretion from mammary epithelial cells[15].In this review we will focus predominantly on the cytosolicLB. Studies of LBs have historically focused on cell typesin which the storage of neutral lipids is directly linked to thefunction of the cell type, such as adipocytes and steroidogeniccells. However, all cell types we have examined, includingboth immortalized and primary cultured cells, have the poten-tial to generate LBs under conditions of elevated fatty acids,suggesting that all mammalian cells are capable of LB bio-genesis.

The primary function of LBs is the storage and provisionof fatty acids and cholesterol. The neutral lipid core of LBscontains diacylglycerols, triacylglycerols, cholesteryl esters,retinyl esters and free cholesterol in varying proportions de-pending upon cell type. Unlike most cell types, the LBs ofadipocytes contain high levels of free cholesterol. In 3T3-F442A adipocytes the proportion of free cholesterol presentin the LB is up to 30% of total cellular cholesterol and isreadily released following depletion of cell surface choles-terol, suggesting a role for adipocytes as a cholesterol ‘sink’for whole body cholesterol levels[16]. In contrast, LBs inother cell types are typically low in free cholesterol and highin cholesteryl esters. Steroidogenic cells of the adrenal cor-tex, ovary and testis contain abundant LBs that are enrichediI hichs lunge s for

In recent years increased attention has been focushe LB as an important lipid regulatory organelle due tootential link between the regulation of LB function and

unctional maintenance of the cell surface. A functional cection between these two intracellular compartmentsuggested by an analysis of the targeting and cellular ef mutants of caveolins, integral membrane componenurface pits termed caveolae. A truncation mutant of cavas found to have profound effects on the function ofurface lipid raft domains[2], while targeting to the surfacf the LB [3]. Recent studies have shown that endogeaveolins can also associate with the LB in a regulatedransient manner[1,4,5]. As caveolin binds both fatty aci6] and cholesterol[7] and has been implicated in cholesteomeostasis[8–10], these studies are starting to provide n

nsights into lipid regulation. In this review we will first sumarize the cell biology of LBs before considering the poss

inks between cholesterol, caveolin, and LBs.

. LBs have multiple and diverse functions inifferent cell types

The LB is composed of a core of neutral lipids surrouny a monolayer of phospholipids, cholesterol and protreviewed in[11]; seeFig. 1). While LBs have been idened in all eukaryotic organisms from mammals and plannsects,Dictyostelium, andS. cerevisiaethere has been littomparative analysis of these organelles. The size, coition and regulation of LBs vary considerably between

n cholesteryl esters, the precursors of steroid synthesis[17].n addition, there are a number of other cell types in wtorage of neutral lipids is a physiological necessity. Inpithelial cells LBs act as the repositories of substrate

S. Martin, R.G. Parton / Seminars in Cell & Developmental Biology 16 (2005) 163–174 165

surfactant production[18]. In liver stellate cells LBs are themajor storage sites of retinyl esters, the precursors of Vita-min A [19], while in retinal pigment epithelia the precursorsof 11-cis-retinal, a chromophore of rhodopsin, are stored asretinyl esters in retinosomes, a type of LB[20]. The produc-tion and secretion of milk fat globules from mammary epithe-lium [15], and the storage of arachidonic acid in eosinophilsand other leukocytes involved in the inflammatory response[21] are other examples of the specific role LBs can play incells with very different physiological functions.

4. Biogenesis of LBs

LBs can be formed and catabolized rapidly in response tochanging lipid and cholesterol levels[4]. Most cells growingin tissue culture contain LBs under normal conditions andcan be stimulated to synthesize more by elevating fatty acidlevels in the media. Serum starvation or removal of fatty acidsresults in the catabolism of stored lipids and loss of the or-ganelle. In the current model of LB formation neutral lipidsare synthesized from fatty acids and cholesterol by enzymespresent at the endoplasmic reticulum (ER) and deposited inthe hydrophobic domain between the phospholipid leafletsof the ER membrane[11,22,23]. This mechanism of forma-tion gives rise to a unique cytosolic organelle surrounded by as las-m l off theE cturem m-b phos-pe ng afl inggi em on-c e[ s tot r, int budsf micl

LBp llL uda is oft n ith malf om-p n ofL e. Int of theE teinc stent

with this, LBs identified using Cav3DGV form at discrete sitesin the ER[4]. Ample free cholesterol and lysophosphatidyl-choline (lyso-PC) were also identified in the LB membrane,distinct from the cholesterol-poor ER membrane[25]. Ascholesterol and lyso-PC form a tight stoichiometric complexin vitro [27], the presence of high levels of lyso-PC in the LBmembrane could act to stabilize the cholesterol component.Whether this is important in defining the site of LB formationor for the function of the LB is not yet known.

It should also be noted that the LB in mammalian cells isoften enwrapped by additional membranous structures (seeFig. 2). This is best documented for the wrapping of ER cis-ternae around the LB[28]. The bilayer of the ER appears toshow a specific, and presumably regulated, interaction withthe cytoplasmic surface (monolayer) of the LB. This complexorganization of multiple layers of membranes is a fairly fre-quent observation in cultured cells and should be consideredwhen interpreting light microscopic localization studies orbiochemical analysis of LBs. In addition to the formation ofLBs from the ER, a close interaction with the ER may facil-itate lipid exchange between these two membrane systems.

5. A role for LBs in pathogenesis?

por-t betes,a rly,w por-t lipidr ra-p pli-c whichL tor.F lipids 8 LBp sis,d ds inm

lsob cal-ip arkopt ithflt thef

theL HepC osis[ ro-p e

ingle monolayer of phospholipids derived from the cytopic leaflet of the ER membrane. In support of this mode

ormation, continuity between the cytoplasmic leaflet ofR and the surface of LBs has been shown by freeze-fraicroscopy[24] and in more recent studies the LB merane has been confirmed as a hemi-membrane (i.e.holipid monolayer) by electron microscopy[25]. Furthervidence for the ER origin of the LB has been shown usiuorescent-tagged truncation mutant of caveolin. Followrowth of cells in serum-free media Cav3DGV-YFP is local-

zed exclusively to the ER[4]. Addition of fatty acids to thedia results in the rapid formation of LBs with the c

omitant incorporation of Cav3DGV into the LB membran4]. The biogenesis of cytosolic LBs has many similaritiehe biogenesis of lipoprotein particles in the ER. Howevehe case of lipoprotein, the nascent lipoprotein particlerom the lumenal leaflet of the ER rather than the cytoplaseaflet and is subsequently secreted[13,14].

Although ER resident proteins have been identified inreparations[1,5,26] it is not currently known whether aBs remain in continuity with the ER after formation, or bway to form a mature, independent organelle. Analyshe phospholipids of the LB hemi-membrane has showas a distinct composition from that of the bulk microso

raction[25] suggesting that either the hemi-membrane cosition is altered as the LB matures or that the formatioBs occurs at a specific subdomain of the ER membran

he latter situation, a specialized membrane subdomainR is likely to be enriched in the enzymes and other proomponents necessary for the formation of LBs. Consi

Some of the most widespread and economically imant human diseases, including atherosclerosis and diare associated with malfunctions in lipid regulation. Cleahile there are many causes for these conditions it is im

ant to understand the normal mechanisms underlyingegulation including LB function, in order to identify theeutic targets. While a role of LBs has not been directly imated in these conditions, there are human diseases inB malfunction is known to be a direct contributory facor example, Chanarin–Dorfman syndrome, a neutraltorage disease, is caused by mutations in the CGI-5rotein [29,30], and results in hepatic steatosis, ichthyoevelopmental defects and the deposition of neutral lipiany cell types[31,32].A number of pathologically important proteins have a

een associated with LBs, although the role of this lozation in the pathogenesis is not known. The�-synucleinrotein is a major component of Lewy Bodies, the hallmf Parkinson’s disease[33]. Point mutants of�-synuclein areartially localized to LBs in cultured cells[34]. The wild

ype protein is targeted to the LB following treatment watty acids and prevents the hydrolysis of triglycerides[34]eading to the suggestion that localization of�-synuclein tohe membrane of the LB could act as a focal point forormation of fibrillary tangles.

One of the most intriguing proteins to localize toB membrane is the hepatitis C (Hep C) core protein.

infection is associated with liver cirrhosis and steat35]. The Hep C core protein is cleaved from the viral protein by signal peptide peptidase[36], a presenilin-typ


Fig. 2. Caveolae and lipid bodies. Electron micrograph of caveolae (upper panels) and lipid bodies (LB) in cultured fibroblasts. Note the ER profiles (arrowheads)wrapping around the lipid bodies and the mitochondrion (Mito) in the lower panels (right panel is higher magnification of central region of left panel).

intramembrane-cleaving aspartic peptidase present in the ER[37]. Following release from the ER the core protein trafficsto the LB membrane[38]. The reason for this translocationis not currently known. The development of steatosis in HepC infection has been shown to be a viral effect, suggestinga possible connection to the targeting of the core protein tothis compartment. Intriguingly, expression of the viral coreprotein alone in transgenic mice causes steatosis[39]. Un-derstanding this process is fundamentally important for un-derstanding the pathophysiology underlying the disease.

6. The protein composition of LBs

The unique nature of the LB phospholipid monolayermakes it thermodynamically unlikely that transmembraneproteins can associate with this organelle. To date, no trans-membrane proteins have been identified as LB components.In mammalian cells the best characterized LB proteins arethe PAT domain family of proteins (reviewed[40]; Fig. 1).There are three mammalian PAT domain proteins: perilipin,adipophilin/ADRP and TIP47, and a fourth related but struc-turally divergent protein, S3-12. PAT proteins are evolution-arily conserved. InDrosophila two PAT domain proteins,Lsdp1 and Lsd2, are expressed in the fat body, an organsimilar to adipose tissue. Loss of Lsd2 function results in

‘lean’ flies (reduced TAG content) whereas over-expressionresults in obesity (fat flies)[41,42]suggesting a direct role inLB function. Perilipin is expressed exclusively in adipocytesand steroidogenic cells. Perilipin knock-out mice are leanand resistant to diet-induced diabetes[43] again indicatingan important role in LB formation. In contrast to perilipin,ADRP and TIP47 are ubiquitously expressed and their regu-lation and function is less well understood. ADRP is thoughtto be involved in the conversion of small LBs to large LBs[40,44] and is known to bind fatty acids[45]. TIP47 wasfirst identified as an adapter for the mannose 6-phosphatereceptor/IGF-IIR and Rab9 present on late endosomes[46].The localization of TIP47 to LBs was a controversial issuefor some time, due in large part to the high degree of similar-ity between the PAT family members and cross-reactivity be-tween antibodies raised against these proteins. Recent studiesusing GFP-tagged TIP47 proteins[47] and proteomic analy-sis[5,26,48]have provided additional evidence for the pres-ence of TIP47 on LBs. Nevertheless, the functional associa-tion with late endosomes has been confirmed by recent datashowing that down-regulation of TIP47 affects the stabilityand expression of late endosomal Rab9[49]. Any additionalrole(s) that TIP47 plays in the function of the LB remains tobe elucidated.

The complete protein composition of mammalian LBs isnot currently clear. Apart from the PAT domain proteins, four


separate proteomic analyses of LB proteins in 2004 identifiedonly one protein, lanosterol synthase, consistently associatedwith LBs [1,5,26,48]. The diverse nature of the remainingprotein compositions is likely to reflect differences betweencell types, differences in the metabolic state of the cell atthe time the LBs were isolated (i.e. neutral lipid formationversus lipolysis), and varying degrees of contaminating com-ponents. However, it has been possible to draw some compar-isons. In the yeastS. cerevisiae, proteins associated with LBsare predominantly involved in the synthesis and activationof fatty acids and sterols[50]. This is a generally consistentfinding in mammalian studies, where a large number of LBproteins are also involved in lipid metabolism or transport.In addition, ER proteins such as BiP were also frequentlyidentified in LBs confirming the ER origin of this organelle[1,5,26]. However, groups of proteins involved in membranetrafficking and signaling have also been identified as LB com-ponents. Some of these, including members of the Rab fam-ily of small GTPases, particularly Rab5c, Rab7 and Rab18have been identified in multiple analyses[1,5,26,48], sug-gesting the potentially exciting prospect of the LB as a targetfor membrane trafficking with the endosomal system and theER, or from organelles such as peroxisomes or mitochondria.

In a recent study the protein composition of LBs isolatedfrom 3T3-L1 adipocytes under basal conditions was com-pared to that of LBs from lipolytically active cells[5]. In-t theL st-i ina

7l

7

facec st in-2( ex-iC rfacew lipidr ur-f iser nc rma-t olaef inc pressC av3c cells,r forc ero-o ar-

acterized members of the family, both form homo-oligomericcomplexes and are palmitoylated on multiple sites[66,67].While Cav2 does not appear to form homo-oligomers it ispalmitoylated in the absence of Cav1 or Cav3 (S. Martin,R.G. Parton, unpublished data). Generally, caveolins behaveas integral membrane proteins and the conserved 33 aminoacid-hydrophobic domain in the center of the polypeptidechain is believed to adopt a hairpin loop embedded in themembrane ([68,69]; Fig. 1).

Caveolae and caveolins have been implicated in numerouscellular functions[63], most of which lie outside the scopeof this article. However, of interest to the focus of this revieware the increasing number of studies implicating caveolae andcaveolins in the regulation of lipid balance, and in particular,cholesterol homeostasis.

7.2. Caveolin and cholesterol

Early morphological studies showed that the membraneassociated with surface caveolae has a distinct sterol com-position[70,71]. Although controversial at the time[72,73]later studies showed that the caveolar structure is particularlysensitive to cholesterol perturbation[74]. Cav1 was subse-quently shown to directly bind free cholesterol (FC) with anapproximate 1:1 stoichiometry and this was shown to be atight interaction even resisting SDS treatment[7]. A strikingi withF n ofC tablefth veolino nd1 nding[

arec e inc encei osedt con-c thisc aseopc ech-a ar atp of thep cave-o veo-l ur-f siono FCi np e-o oft rol

erestingly, one of the proteins found to associate withB specifically under lipolytic conditions is Cav1, sugge

ng a direct role for caveolins in the regulation of lipolysisdipocytes.

. Caveolin, LBs and the regulation of cholesterolevels

.1. Caveolins

The caveolin proteins are major proteins of cell suraveolae (reviewed in[51–53]; Fig. 2). Mammals expreshree isoforms of caveolin; caveolin-1 (Cav1), caveolCav2) and caveolin-3 (Cav3), with both Cav1 and Cav2sting as two splice variants of unknown function[51,54,55].av1 and Cav3 are predominantly present at the cell suhere they localize to caveolae, a specialized form of

aft domain[53]. In contrast, Cav2 targets to the cell sace only when oligomerized with Cav1, and is otherwestricted to the Golgi complex[56]. Expression of Cav1 iells that normally lack caveolin causes the de novo foion of caveolae showing that Cav1 is sufficient for caveormation in such a system[57]. Cav3 plays a similar roleaveolae formation in striated muscle, which does not exav1[58,59]. Genetic ablation of expression of Cav1 or Causes the loss of caveolae in non-muscle and muscleespectively[60–63]. Cav2 does not appear to be requiredaveolae formation but interacts with Cav1 to form hetligomeric complexes[64,65]. Cav1 and Cav3, the best ch

ndependent demonstration of the interaction of caveolinC at the whole cell level was shown by the identificatioav1 as the major protein cross-linked to a photoactiva

orm of cholesterol in whole MDCK cells[75]. Palmitoyla-ion is not required for caveolin targeting to caveolae[67] butas been suggested to increase the stabilization of caligomers[76]. Palmitoylation of Cav1 at positions 143 a56 has been suggested to be required for cholesterol bi

77].These findings demonstrate that FC and caveolin

losely linked but the exact role of caveolin and caveolaholesterol homeostasis still remains unclear and evids largely circumstantial. For example, it has been prophat cholesterol on the surface of mammalian cells isentrated within caveolae. However, the evidence forlaim mainly comes from treatment with cholesterol oxidr is based on subcellular fractionation[78–80]. Both ap-roaches may have limited specificity for caveolae[81,82]. Ifholesterol is highly concentrated within caveolae the mnisms underlying this concentration also remain uncleresent. Cholesterol is such an abundant componentlasma membrane (PM) that a 1:1 interaction betweenlin and FC would not cause a great enrichment in ca

ae, which only comprise a minor fraction of the cell sace in most cell types. Interestingly, however, expresf Cav1 in a caveolae-null cell causes enrichment of

n a low density ‘lipid raft’ fraction to higher levels tharedicted for a 1:1 ratio[83] suggesting that although cavlin only binds with molar stoichiometry the architecture

he caveola might facilitate high packing/flux of choleste


through caveolae. Specific epitopes throughout the caveolinprotein are masked by cholesterol upon caveolin reachingthe PM and can only be revealed by cholesterol removal (A.Pol, S. Martin, R.G. Parton, manuscript in preparation). Likemany aspects of cholesterol trafficking, the direct involve-ment of caveolin has been difficult to show definitively, evenwith the availability of caveolin-negative cells[10]. How-ever, many different avenues of research—including studiesof the regulation of caveolin expression, functional studieswith caveolin mutants, and examination of the effect of cave-olin expression—now point to a role for caveolin in lipidregulation even if the exact mechanisms remain elusive.

7.3. Linking caveolin, cholesterol, and signaltransduction; functional studies

Cav1 is regulated at the transcriptional level by FC lev-els in the ER through sterol-responsive promoter elements[9,84,85]. High FC levels cause a decrease in caveolin tran-scription and this is accompanied by changes in caveolaedensity on the PM[85,86]. The Cav2 promoter also containsSRE-like sequences that are proposed to link Cav2 transcrip-tion to intracellular cholesterol levels, although a strong linkbetween cellular sterols and Cav2 expression has not beenproven[86]. Cav1 protein levels[87], and Cav1 traffickingthrough the Golgi complex (A. Pol, S. Martin and R.G. Par-t se toc cle-s (P.F.M t re-c argeto ta-s of ac ande wl ipidr ights

lsos cialt spe-c ormo ve,t ss tivel spe-c y-a g-n no ver-e theC tralls aticpb but

these studies point to a role for caveolin in regulating choles-terol balance which influences signaling processes occurringthrough putative lipid raft domains. The work of Fielding andFielding[90] has provided further links between the choles-terol regulating properties of caveolin and signal transduc-tion. These studies suggest that phosphorylation of a con-served Ser in Cav1 (Ser80) changes FC binding[91]. Consis-tent with this model, PDGF stimulation caused Ser80 phos-phorylation and increased FC efflux suggesting reciprocalregulation of signaling and cholesterol regulation. Other stud-ies have associated changes in Ser80 with trafficking defects[92,93]. Cav1(S80E), which mimics constitutively phospho-rylated Cav1, is targeted to the ER[93].

In view of the above studies, caveolin-deficient cells mightbe expected to show severe disruption of cholesterol regula-tion. This is clearly not the case—to date the analysis of FCbalance in cells from KO mice lacking Cav1 (and with greatlydecreased Cav2 levels)[61,94]or from cell lines which lackcaveolins[10] show efficient delivery of FC to the PM. Yetthere is evidence for a facilitatory role of caveolin in choles-terol transport in several different cell systems upon caveolin(re)expression. A clear consensus is difficult; some studieshave shown an effect of caveolin expression on newly syn-thesized FC efflux to serum acceptors or purified apoA1 butnot to cyclodextrin[82] while other studies showed no effecton the release of newly synthesized FC efflux but stimulationov larf howt rt ine ath-w cifics

oryr ula-t helpm eolini fairlyi in-tca sionw angew ex-p linsa ave-o sur-f ento cili-t ortedt -ia -n forc olicp eolin

on, manuscript in preparation) also change in responhanges in FC levels. No similar regulation of the muspecific Cav3 isoform by cholesterol has been observedery, S.J. Nixon, R.G. Parton, unpublished results) bu

ent studies have shown that the CAV3 gene is a direct tf ROR�, a key transcriptional regulator of lipid homeosis in skeletal muscle which programs the expressionascade of genes involved in lipid uptake, catabolism,nergy expenditure[88]. At present, this is one of the fe

inks between the muscle-specific form of caveolin and legulation but suggests that all three caveolin isoforms mhare similar roles in lipid metabolism.

A general role for caveolins in lipid regulation is aupported by studies of caveolin mutants, including artifiruncation mutants and point mutants corresponding toific amino acid changes occurring in patients with a ff Limb Girdle Muscular Dystrophy. As described abo

he truncation mutant Cav3DGV or its Cav1 equivalent, wahown to affect surface FC levels and to inhibit putaipid raft-dependent signaling pathways mediated by aific palmitoylated Ras isoform[2]. The muscular dystrophssociated mutant, Cav3C71W, also specifically disrupted sialing in the same system[89]. In both cases the inhibitiof signaling was rescued by cholesterol addition or by oxpression of wild type Cav1 or Cav3. However, whileav3DGV mutant accumulated on LBs and affected neu

ipid balance (see the following), the Cav3C71W mutant washown to associate with caveolae at the PM with no dramerturbation of cellular lipid balance[89]. The mechanismy which the mutant affects cholesterol is still unclear

f the efflux of pre-existing FC to serum acceptors[10]. Iniew of the universal importance of FC transport in celluunction, it may not be surprising that caveolins, which sight tissue specificity, are not essential for FC transpovery cell type, but may provide a specialized transport pay or a higher level of regulation in response to speignals.

Accepting that caveolin could play either a facilitatole in FC transport or could provide a higher level of region of FC transport, by what mechanism could caveolinaintain cholesterol balance? At steady state most cav

s present in surface caveolae. Caveolae are generallymmobile[95,96]but a small number of caveolae can beernalized and this process can be stimulated[96,97]. Theaveolin protein is very immobile in PM caveolae[95,96]nd even after internalization of entire caveolae and fuith endosomal compartments caveolin does not exchith other caveolin pools as shown by photobleachingeriments[96]. It therefore appears unlikely that caveore rapidly equilibrating between different domains. Clin expression would then cause the generation of more

ace caveolae, which could act as a ‘sink’ for recruitmf cholesterol from intracellular membranes and/or fa

ate release of cholesterol. Cholesterol can be transphrough the cell by vesicle-independent[98] and even energyndependent mechanisms[99] with trafficking dictated byffinity for distinct membrane domains[23]. This mechaism would be quite distinct from a direct transport roleaveolins moving FC through the cell. However, a cytosool of caveolin has been described and this pool of cav


was shown to be complexed with chaperones and carryingnewly synthesized cholesterol[77]. Complex formation de-pends on palmitoylation[77], which is intriguing in view ofdata showing that caveolin is cotranslationally inserted intothe ER[69], and then passes to post-Golgi compartments be-fore being irreversibly palmitoylated[100]. Thus, caveolinwould have to be extracted from this post-Golgi membranesystem and released into the cytosol to generate the choles-terol transport complex. Further experiments with a specificinhibitor of this complex formation, Cyclosporin A[101], aswell as the application of novel fluorescent cholesterol deriva-tives (such as dihydroergosterol[102]) to follow cholesteroltransport should provide more insights into the regulation ofcholesterol transport in diverse cell types.

7.4. LB targeting of caveolin

In recent years several studies have identified the LB asa possible target organelle of the caveolins[1,3,5,103,104].The identification of LBs as a target was first describedfor the Cav3DGV mutant, which serendipitously inhibits thecatabolism of LBs resulting in an accumulation of neutrallipids in the cell[4]. In some systems localization of cave-olin to the ER appears to be a factor in its targeting to LBs.The attachment of an ER retrieval motif to the C-terminus ofcaveolin is sufficient to cause its localization to LBs[104].I sest s thel r-v olini ERaH Bsh usingd ivos toL howntm thef lipid[ asso-c s arec tionsa oteinc lvesa iatedc o-f t toL heC dert 1o . An Fore undt ys hys-

iological role. These findings came from an interesting invivo system, the regenerating liver[3]. After partial hepate-ctomy, in which two thirds of the liver is removed, there isa hormone-dependent mobilization of fat from adipose tis-sue causing plasma free fatty acid levels to rise dramatically.This causes a massive accumulation of LBs in the hepato-cytes of the regenerating liver. During this process both Cav1and Cav2 redistribute from the PM to accumulate on LBs[3].Interestingly, both caveolins redistribute from LBs before theLBs are catabolized providing further evidence for regulatedassociation with LBs. This represents a powerful system tostudy the role of caveolin-LB association in vivo.

7.5. Structural considerations

Despite extensive studies of LB-associated proteins, todate there is no clear consensus sequence that would allowthe prediction of LB association. LB proteins fall into two cat-egories, those synthesized in the ER that associate with theLB during biogenesis, such as the HepC core protein and theplant oleosins[106], and those synthesized on free polysomesthat associate with LBs after formation such as perilipin andADRP. It is not certain how and when caveolin associateswith the LB or whether the ER is an obligatory step in theassociation with the LB. Structurally oleosins contain a longhydrophobic central domain that inserts into the phospholipidm bya ,P ughm kedb inh -g toL rtantf tm do-m ch asa mainh s ord fb es inb ces-s ated.O n-c hortm ionals

8

8

olin,c ev-i nly

n addition, treatment of cells with brefeldin A, which cauhe Golgi complex to be absorbed into the ER, increaseocalization of caveolin to the LBs[103,104]. These obseations led to the suggestion that LB targeting of caves restricted to newly synthesized protein transiting thend is not a physiological destination of caveolins[22,104].owever, this view of artefactual targeting of caveolin to Las been refuted by a number of independent studiesifferent techniques to study LBs in cultured cells and in vystems[1,4,5]. It now appears that targeting of caveolinsBs is a regulated process. Cav1 and Cav2 have been s

o traffic to LBs upon addition of fatty acids[1,4]. Further-ore, the retrieval of Cav2 from LBs following removal of

atty acid from the medium precedes the loss of neutral4], suggesting an active regulation rather than passiveiation. The pathways involved in this regulated procesompletely unclear at present and raise intriguing quesbout the mechanisms by which an integral membrane pran be transported to and from LBs. Whether this invocytosolic intermediate, as postulated for caveolin-medholesterol transport[77] remains unknown. Different isorms of caveolin may also vary in their ability to targeBs; the Cav2� isoform was readily recruited to LBs, but tav2� isoform remained largely in the Golgi complex un

he same conditions in this system[103]. In this study Cavnly localized to LBs when cells were treated with BFAumber of other studies have identified caveolin in LBs.xample, LBs formed using an in vitro assay were also foo contain both Cav1 and Cav2[105]. Finally, a recent studuggests that LB association of caveolins may have a p

onolayer and neutral lipid core of the oil body, flankedmphipathic N- and C-terminal regions[22,106]. In contrastAT domain proteins appear to associate with the LB throultiple, partially redundant hydrophobic domains flany amphipathic regions[107]. A similar structure to oleosas been identified in the HepC core protein[108] and sugested for Cav1[109]. Studies of the targeting of caveolinsBs have shown that the intramembrane domain is impo

or LB association, but not sufficient[103,109]. It appears thaaintaining the structural integrity of the intramembraneain is more important than the sequence itself, inasmulanine-scanning mutagenesis of the intramembrane doas no effect on LB targeting, whereas certain insertioneletions cannot be tolerated[109]. Structural integrity ooth the intramembrane domain and flanking sequencoth the N- and C-terminal domains of the protein are neary. The precise targeting domains remain to be elucidne difficulty in the analysis of LB targeting previously e

ountered for the PAT proteins is the observation that sotifs are less important than the overall three dimens

tructure[110,111].

. Functional effects of caveolin on the LB

.1. Studies of caveolin mutants

Despite the growing evidence for a link between caveholesterol regulation, and LBs, there is currently littledence to link them directly. At the cellular level, the o


evidence for a functional role of caveolin in LB regulationhas been suggested by analysis of the artificial truncation mu-tant, Cav3DGV. Expression of the LB-targeted Cav3DGV hasprofound effects on lipid and cholesterol homeostasis[3,4].Cav3DGV induces a decrease in cell surface cholesterol levelsand a concomitant increase in late endosomal cholesterol thatimpacts on the functioning of cell surface lipid raft domains[2,89]. There is also an accumulation of neutral lipids in LBsdue to their slowed catabolism[4], presumably due to inhibi-tion of the hydrolysis and catabolism of stored triglyceridesand cholesteryl esters.

LBs have been shown to undergo microtubule-based bidi-rectional and saltatory motility. This has been most elegantlydescribed in the developingDrosophilaembryo where theco-ordinated movement of LBs during early embryogenesisis tightly controlled. InDrosophila the gene product of theKlarsicht mutant is involved in organizing motors on the sur-face of the LB[112,113]. In mammalian cells, motility ofLBs has been described using the lipophilic fluorescent dyeNile red[4,114]or GFP-ADRP[107]. Using GFP-ADRP itwas show that most LBs oscillate within a given area andseem to be in close apposition to the ER membrane, but afew undergo rapid translocation. While the motility of LBsis dependent upon microtubules[4] and the dynamitin com-plex [114], the formation of LBs is not[109]. Expression ofCav3DGV completely blocks the microtubule-based motilityo nt-i ofg dentL

8c

tesi cav1[ sioni tiono acidl arer op ex-p fort tes isa allya e-t ina pase( P-dA A,w sur-f ca-t -p ory-l n

shown to interact directly with, and negatively regulate, PKA[120]. Consistent with this, PKA activity is markedly up reg-ulated in adipocytes from Cav1−/− mice [116]. However,intriguingly adipocytes from Cav1−/− mice show a bluntedresponse to lipolytic stimulation, suggesting PKA activity issomehow uncoupled from HSL activity. In adipocytes bothPKA and Cav1 were found to interact with phosphorylatedperilipin, and the interaction between PKA and perilipin wasinhibited in Cav1−/− adipocytes, leading to the suggestionthat Cav1 mediates the interaction between PKA and per-ilipin. While there appears to be a functional link betweenCav1, perilipin and PKA in adipocytes[116], it is unclearwhether a single protein complex containing these proteins,or the interplay between several protein complexes that to-gether coordinate the stimulated lipolysis of triglycerides,mediates these effects. As Cav1 binds only to phosphorylatedperilipin, this suggests that the interaction between cav1 andperilipin must be down-stream of the interaction betweenperilipin and PKA[116]. While there is much to be doneto completely define the interactions between cav1, perilipinand PKA in adipocytes there is clearly a functional link be-tween these proteins. It will be of great importance to de-termine whether these proteins form a single multifunctionalcomplex, and to define the intracellular localisation of thevarious components under conditions of lipid deposition andlipolysis.

8

ntlyo , areo ons.M sclec ingf roticp esi witha no tp is in-d scle-r dis-e netica orticC int de-v emia[ -v cel-l de-sb typest con-n cularf

f LBs [4]. Whether the loss of motility is a factor in preveng the catabolism of LBs is not yet known and it will bereat interest to elucidate the role of microtubule-depenB motility in LB function.

.2. Regulation of lipolysis in adipocytes; studies ofaveolin-1 null mice

The differentiation of 3T3-L1 fibroblasts into adipocys associated with a large increase in the expression of115]. In mice fasting induces an increase in Cav1 expresn adipose tissue concomitant with the increased mobilizaf triacylglycerides and an elevation of serum free fatty

evels[116]. Cav1−/− mice show reduced adiposity andesistant to diet induced obesity[94] in a similar manner terilipin−/− mice [43] suggesting a link between cav1ression and lipid regulation. The only evidence to date

he presence of caveolin at the surface of LBs in adipocyrecent proteomic analysis of LBs isolated from lipolyticctive 3T3-L1 adipocytes[5]. This study suggests a link b

ween caveolin localization to LBs and lipolysis. Lipolysisdipocytes is mediated largely by hormone sensitive liHSL), under complex regulation by perilipin and cAMependent protein kinase/protein kinase A (PKA)[117,118].gonist stimulation of�-adrenergic receptors activates PKhich subsequently phosphorylates both perilipin at the

ace of the LB and cytosolic HSL, resulting in the transloion of HSL from the cytosol to the LB surface[117]. Phoshorylated perilipin is believed to allow access of phosph

ated HSL to its triglyceride substrates[119]. Cav1 has bee

.3. Caveolin and atherosclerosis

Fatty streaks in the arterial wall, consisting predominaf cholesterol loaded macrophage-derived foam cellsne of the earliest visible signs of atherosclerotic lesiacrophages, together with proliferating smooth mu

ells, accumulate cholesterol and lipids eventually formoam cells, resulting in the generation of the atherosclelaque[121]. Lesional instability of atherosclerotic plaqu

s linked to macrophage apoptosis, which is associatedn increase in Cav1 expression[122] and an accumulatiof cholesterol in the ER[123]. While it is not known aresent whether these two events are related thereirect evidence that the expression of Cav1 in atherootic sites could be important in the progression of thease. Atherosclerosis-prone mice, developed by the geblation of apolipoprotein E (ApoE), have increased aav1 levels[124]. Subsequent genetic ablation of Cav1

he ApoE−/− background confers protection against theelopment of atherosclerosis, despite hypercholesterola126]. Furthermore, treatment of ApoE−/− mice with rosuastatin, an HMG-CoA reductase inhibitor that lowersular cholesterol levels, returns Cav1 levels to normal,pite persistent elevated plasma cholesterol[124]. A num-er of statins have been shown to have differential, cellpecific effects on caveolin expression[85,124,125]. Fur-her work is required to determine the nature of theection between caveolin regulation and normal vas

unction.


9. Conclusions

Research into the formation, composition, structure, dy-namics, and function of LBs is entering an exciting new era.For an organelle so crucial to regulation of cellular lipid bal-ance, the present state of understanding of this organelleis surprisingly limited. This perhaps reflects the particularchallenges associated with studying lipid regulation and traf-ficking in general. In its role as a regulator of lipid balancein the cell, the LB must communicate indirectly with manyother membrane systems such as the endosomes, mitochon-dria, endoplasmic reticulum, peroxisomes, and PM. The pro-posed association of many different Rab proteins with theLB membrane raises new questions about more direct inter-actions with cellular organelles. It is also now clear that theLB is a dynamic motile organelle, which can be generated orcatabolised very rapidly in response to cellular demands.

Caveolins have emerged as unexpected associates of LBs.The perturbation of LB function by mutant caveolin pro-teins and the associated change in specific signaling pathwayssuggests that lipid imbalance can have very specific effectson PM processes. This highlights the importance of under-standing changes in cellular lipid regulation in human diseasestates such as obesity and atherosclerosis, two disease statesin which caveolins have been implicated. The regulated andreversible association of caveolins with the LB is a fascinatingp icals n butt onall nullm o be-i ionsb e.

A

rantf nala f theA senta n andc

R

.be

hem

ngbyCell

G.bod-

ies and induces intracellular cholesterol imbalance. J Cell Biol2001;152:1057–70.

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[5] Brasaemle DL, Dolios G, Shapiro L, Wang R. Proteomic analy-sis of proteins associated with lipid droplets of basal and lipolyti-cally stimulated 3T3-L1 adipocytes. J Biol Chem 2004;279:46835–42.

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[8] Fielding CJ, Fielding PE. Caveolae and intracellular trafficking ofcholesterol. Adv Drug Deliv Rev 2001;49:251–64.

[9] Ikonen E, Parton RG. Caveolins and cellular cholesterol balance.Traffic 2000;1:212–7.

[10] Ikonen E, Heino S, Lusa S. Caveolins and membrane cholesterol.Biochem Soc Trans 2004;32:121–3.

[11] Murphy DJ. The biogenesis and functions of lipid bodies in ani-mals, plants and microorganisms. Prog Lipid Res 2001;40:325–438.

[12] Murphy DJ, Vance J. Mechanisms of lipid-body formation. TrendsBiochem Sci 1999;24:109–15.

[13] Hussain MM. A proposed model for the assembly of chylomicrons.Atherosclerosis 2000;148:1–15.

[14] Olofsson SO, Asp L, Boren J. The assembly and secretionof apolipoprotein B-containing lipoproteins. Curr Opin Lipidol

ilk.

ttlereri-ndedSci

ultzwithenic

cytethe-iol

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on-tures

Im-

urr

Biol

edaacees

heique

rocess, which is not yet fully understood. The physiologignificance of this association also remains to be showhere is accumulating circumstantial evidence for functiinks between caveolin and LBs from studies of caveolin-

ice and of liver regeneration. With surface caveolae alsng linked to cholesterol regulation, more direct connectetween caveolae and LBs may be revealed in the futur

cknowledgments

The work of the authors is supported by a Program Grom the NHMRC of Australia. The Centre for Functiond Applied Genomics is a Special Research Centre oustralian Research Council. We would like to thank prend past members of the Parton laboratory for discussioomments on the manuscript.

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Caveolin, cholesterol, and lipid bodies