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Demonstrated and inferred metabolism associated with cytosolic lipid droplets

2009; Elsevier BV; Volume: 50; Issue: 11 Linguagem: Inglês

10.1194/jlr.r001446

1539-7262

Joel Goodman,

Plant biochemistry and biosynthesis

Cytosolic lipid droplets were considered until recently to be rather inert particles of stored neutral lipid. Largely through proteomics is it now known that droplets are dynamic organelles and that they participate in several important metabolic reactions as well as trafficking and interorganellar communication. In this review, the role of droplets in metabolism in the yeast Saccharomyces cerevisiae, the fly Drosophila melanogaster, and several mammalian sources are discussed, particularly focusing on those reactions shared by these organisms. From proteomics and older work, it is clear that droplets are important for fatty acid and sterol biosynthesis, fatty acid activation, and lipolysis. However, many droplet-associated enzymes are predicted to span a membrane two or more times, which suggests either that droplet structure is more complex than the current model posits, or that there are tightly bound membranes, particularly derived from the endoplasmic reticulum, which account for the association of several of these proteins. Cytosolic lipid droplets were considered until recently to be rather inert particles of stored neutral lipid. Largely through proteomics is it now known that droplets are dynamic organelles and that they participate in several important metabolic reactions as well as trafficking and interorganellar communication. In this review, the role of droplets in metabolism in the yeast Saccharomyces cerevisiae, the fly Drosophila melanogaster, and several mammalian sources are discussed, particularly focusing on those reactions shared by these organisms. From proteomics and older work, it is clear that droplets are important for fatty acid and sterol biosynthesis, fatty acid activation, and lipolysis. However, many droplet-associated enzymes are predicted to span a membrane two or more times, which suggests either that droplet structure is more complex than the current model posits, or that there are tightly bound membranes, particularly derived from the endoplasmic reticulum, which account for the association of several of these proteins. Cytosolic lipid droplets, originally thought to be simply coalesced neutral lipids waiting for lipolysis at metabolic demand, are now known to be considerably more complicated both structurally and functionally. There is general agreement that droplets are comprised of a core of neutral lipids, principally triglycerides and steryl esters, surrounded by a leaflet of phospholipids into which are embedded a specific subset of cellular proteins, the most abundant of which are members of the PAT family (see below) in animal cells (1Martin S. Parton R.G. Lipid droplets: a unified view of a dynamic organelle.Nat. Rev. Mol. Cell Biol. 2006; 7: 373-378Crossref PubMed Scopus (884) Google Scholar). However, this model is probably too simple; there is evidence from physical probes of droplets isolated from yeast mutants unable to synthesize triglycerides or steryl esters that these two molecular families are partially segregated within the core, with thin shells of steryl esters forming concentric hollow spheres around an inner core composed principally of triglycerides (2Czabany T. Wagner A. Zweytick D. Lohner K. Leitner E. Ingolic E. Daum G. Structural and biochemical properties of lipid particles from the yeast Saccharomyces cerevisiae.J. Biol. Chem. 2008; 283: 17065-17074Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The next layer of complexity is the functional inhomogeneity of droplets. Subsets of droplets within the same cells exist with different populations of PAT proteins, differentiating among different sizes, ages, and levels of metabolic activity (3Brasaemle D.L. Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis.J. Lipid Res. 2007; 48: 2547-2559Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar, 4Wolins N.E. Brasaemle D.L. Bickel P.E. A proposed model of fat packaging by exchangeable lipid droplet proteins.FEBS Lett. 2006; 580: 5484-5491Crossref PubMed Scopus (310) Google Scholar). Perhaps most surprisingly, droplets may be comprised, at least in some cases, not of the layered core-phospholipid shell architecture at all but a knot of tightly woven endoplasmic reticulum (ER) surrounded by secreted neutral lipid, itself encased with a single leaflet. Such a model is based on electron microscopic thin sections (5Wan H.C. Melo R.C. Jin Z. Dvorak A.M. Weller P.F. Roles and origins of leukocyte lipid bodies: proteomic and ultrastructural studies.FASEB J. 2007; 21: 167-178Crossref PubMed Scopus (159) Google Scholar), freeze fracture-immunogold evidence (6Robenek H. Buers I. Hofnagel O. Robenek M.J. Troyer D. Severs N.J. Compartmentalization of proteins in lipid droplet biogenesis.Biochim. Biophys. Acta. 2009; 1791: 408-418Crossref PubMed Scopus (63) Google Scholar), immunohistochemical studies of ER luminal proteins within the droplet (7Dvorak A.M. Morgan E.S. Tzizik D.M. Weller P.F. Prostaglandin endoperoxide synthase (cyclooxygenase): ultrastructural localization to nonmembrane-bound cytoplasmic lipid bodies in human eosinophils and 3T3 fibroblasts.Int. Arch. Allergy Immunol. 1994; 105: 245-250Crossref PubMed Scopus (26) Google Scholar), and the identification of these proteins, notably ER chaperones, in several proteomic studies. Although certainly, such a complex structure must obey physical laws governing aqueous interactions with hydrophobic lipids and artifacts in processing for electron microscopy do occur, it may be best at present to keep an open mind and consider that droplets may not have the same structure among tissues and that they may take multiple physical forms in rapid order as they dynamically perform their functions. What are these functions? The most obvious one is lipid metabolism, namely the biogenesis and breakdown of the neutral lipids contained within the droplet. Although this conclusion predates proteomic studies (8Carey G.B. Mechanisms regulating adipocyte lipolysis.Adv. Exp. Med. Biol. 1998; 441: 157-170Crossref PubMed Scopus (71) Google Scholar), these recent studies have revealed the breadth and conservation of metabolic reactions that occur at or near the droplet surface, the subject of this review. Moreover, proteomics has demonstrated the surprising fact that droplets are likely to be very active in organellar communication because they are replete in rab proteins and other trafficking molecules. Our knowledge from proteomic studies of droplet trafficking and communication is discussed separately in this thematic review series. A major caveat must be kept in mind when evaluating droplet proteomics data: besides droplet trafficking through transient interactions with vesicles or target organelles such as early endosomes (9Liu P. Bartz R. Zehmer J.K. Ying Y.S. Zhu M. Serrero G. Anderson R.G. Rab-regulated interaction of early endosomes with lipid droplets.Biochim. Biophys. Acta. 2007; 1773: 784-793Crossref PubMed Scopus (134) Google Scholar), droplets make extensive, tight, and long-lasting synapses with the endoplasmic reticulum, mitochondria, and peroxisomes (10Binns D. Januszewski T. Chen Y. Hill J. Markin V.S. Zhao Y. Gilpin C. Chapman K.D. Anderson R.G. Goodman J.M. An intimate collaboration between peroxisomes and lipid bodies.J. Cell Biol. 2006; 173: 719-731Crossref PubMed Scopus (261) Google Scholar, 11Perktold A. Zechmann B. Daum G. Zellnig G. Organelle association visualized by three-dimensional ultrastructural imaging of the yeast cell.FEMS Yeast Res. 2007; 7: 629-638Crossref PubMed Scopus (47) Google Scholar). The fact that ER, mitochondrial, peroxisomal, and a few plasma membrane proteins are found with such high frequency in the droplet proteome probably reflects these tight interorganellar interactions, perhaps similar to the mitochondrially associated membranes (MAMs) that link mitochondria with ER (12Hayashi T. Rizzuto R. Hajnoczky G. Su T.P. MAM: more than just a housekeeper.Trends Cell Biol. 2009; 19: 81-88Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar). The molecular basis for droplet-mediated synapses are not yet known. Besides the frequent occurrence of specific nondroplet organelle proteins in the droplet proteome, adventitious contamination of droplets is unlikely in view of the unique density of droplets that allow their flotation to the top of aqueous buffers and density gradients after centrifugation while all other cell components sink (which also permits several washes with high recovery), and the nonrandom coisolation of subsets of proteins from other organelles, such as the β-oxidation peroxisomal enzymes (10Binns D. Januszewski T. Chen Y. Hill J. Markin V.S. Zhao Y. Gilpin C. Chapman K.D. Anderson R.G. Goodman J.M. An intimate collaboration between peroxisomes and lipid bodies.J. Cell Biol. 2006; 173: 719-731Crossref PubMed Scopus (261) Google Scholar), which suggests specialized regions for metabolically-productive droplet interactions at the synapses. Droplet-ER interactions are a special case; it is the rule rather than the exception that enzymes of lipid metabolism that are found in the droplet proteome are also found to varying extents in the ER. This has been well documented in yeast through genome-wide green fluorescent protein (GFP)-tagging (13Huh W.K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Global analysis of protein localization in budding yeast.Nature. 2003; 425: 686-691Crossref PubMed Scopus (3235) Google Scholar, 14Natter K. Leitner P. Faschinger A. Wolinski H. McCraith S. Fields S. Kohlwein S.D. The spatial organization of lipid synthesis in the yeast Saccharomyces cerevisiae derived from large scale green fluorescent protein tagging and high resolution microscopy.Mol. Cell. Proteomics. 2005; 4: 662-672Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Erg6p, an enzyme in the latter part of the ergosterol biosynthetic pathway, is the only droplet protein in the pathway with a near-exclusive droplet localization in yeast; Erg1p, Erg7p, and Erg 27p are dually localized, and the pattern changes depending on metabolic state. Whether this general rule is specific for yeast, in which droplets remain on the ER surface (15Szymanski K.M. Binns D. Bartz R. Grishin N.V. Li W.P. Agarwal A.K. Garg A. Anderson R.G. Goodman J.M. The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology.Proc. Natl. Acad. Sci. USA. 2007; 104: 20890-20895Crossref PubMed Scopus (425) Google Scholar), is not yet clear. However, several examples already exist in mammalian cells: cytochrome b5 reductase (DT diaphorase) and various sterol dehydrogenases (see Table 1), were classically considered ER proteins. Many enzymes of sterol metabolism that appear in droplet proteomes have multiple membrane spans and it is difficult to imagine them arranged in the single leaflet surrounding a hydrophobic core of neutral lipids. A solution to this problem, besides that of invoking internal ER cisternae within droplets, is to consider these enzymes in a specialized ER compartment that is very close to, and tightly bound with, the droplet (a "droplet synapse") that separates from the bulk ER during fractionation, copurifying with droplets. If this structure resembles that of MAMs in contact with mitochondria, it would explain the frequent coisolation of ER luminal chaperones with droplets because chaperones such as luminal HSP70 are directly involved in MAM structure (12Hayashi T. Rizzuto R. Hajnoczky G. Su T.P. MAM: more than just a housekeeper.Trends Cell Biol. 2009; 19: 81-88Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar).TABLE 1Metabolic functions of droplets as revealed by proteomicsProteinReference(s)CommentsFatty Acid SynthesisATP citrate lyase(e)Generates acetyl-CoAAcetyl-CoA carboxylase/ACC1(i) (j) (n) (o)(e)Generates malonyl CoA3-Oxoacyl(ACP) synthase(e)Drosophila; early step in FA synthesisFatty acid synthase(e)DrosophilaDiaphorase 1/Cytochrome b5 reductase(g)(h)(j) (l) (n) (o)Redox carrier in FA elongation and many othersFatty acid desaturase 2(e) (m)Many hydrophobic spans likelyFatty Acid ActivationAcyl-CoA synthetase/ACSL1(g) (n)Fatty acid-CoA ligaseAcyl-CoA synthetase/ACSL3(g)(h)(i) (j) (l) (n) (o)Fatty acid-CoA ligaseAcyl-CoA synthetase/ACSL4(g)(h) (j) (l) (n)Fatty acid-CoA ligaseAcyl-CoA synthetase/ACSL5(m)LACS2Acyl-CoA synthetases/FAA1, FAA4, FAT1(a) (d)Yeast enzymes; FAT1 is a FA transporter; may have synthetase activitySteroid SynthesisSqualene epoxidase/ERG1(a) (i) (j) (o)(d)Lanosterol synthase/ERG7(a)(g) (h) (i) (j) (m) (o)(d)NAD(P) steroid dehydrogenase like (NSDHL)/ERG26(g)(h) (i)(m) (o)Sterol synthesis3-keto reductase 17 βHSD7/ERG27(b)*(c)*(g) (j)(n) (o)(d)Sterol synthesisC24-methyltransferase/ERG6(a) (c)* (d)Specific to ergosterol synthesis in fungi17 β-HSD11 (retinal short chain dehydrogenase)(h) (i) (j) (l) (m) (n) (o) (e)Testosterone biosynthesis; steroid metabolism17 β-HSD4(l)Bile salt snthesis17 β-HSD13(m)A short-chain dehydrogenase17 β-HSD3(m)Steroid metabolismTriglyceride SynthesisAcylDHAP reductase/AYR1(d)Determined early biochemically (68Athenstaedt K. Daum G. 1-Acyldihydroxyacetone-phosphate reductase (Ayr1p) of the yeast Saccharomyces cerevisiae encoded by the open reading frame YIL124w is a major component of lipid particles.J. Biol. Chem. 2000; 275: 235-240Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar)LysoPA acyltransferase/SLC1(d)Determined earlier biochemically (69Athenstaedt K. Daum G. Biosynthesis of phosphatidic acid in lipid particles and endoplasmic reticulum of Saccharomyces cerevisiae.J. Bacteriol. 1997; 179: 7611-7616Crossref PubMed Scopus (96) Google Scholar)DAG acyltransferase/DGA1Determined biochemically in yeast (70Sorger D. Daum G. Synthesis of triacylglycerols by the acyl-coenzyme A:diacyl-glycerol acyltransferase Dga1p in lipid particles of the yeast Saccharomyces cerevisiae.J. Bacteriol. 2002; 184: 519-524Crossref PubMed Scopus (173) Google Scholar)LipolysisHormone-sensitive lipase(f)(g)Diglyceride lipase [first characterized in (71Vaughan M. Berger J.E. Steinberg D. Hormone-sensitive lipase and monoglyceride lipase activities in adipose tissue.J. Biol. Chem. 1964; 239: 401-409Abstract Full Text PDF PubMed Google Scholar)]Fat-specific gene 27(g)Lipase activityATGL(n) (o)Triglyceride lipaseMonoglyceride lipase(m)Tgl3, Tgl4, Tgl5(a)Yeast triglyceride lipases [for Tgl4 and 5 see (60Athenstaedt K. Daum G. Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae are localized to lipid particles.J. Biol. Chem. 2005; 280: 37301-37309Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar)]Tgl1p, Yeh1p(a)Yeast steryl ester lipases; Yeh1 localized in (62Koffel R. Tiwari R. Falquet L. Schneiter R. The Saccharomyces cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 genes encode a novel family of membrane-anchored lipases that are required for steryl ester hydrolysis.Mol. Cell. Biol. 2005; 25: 1655-1668Crossref PubMed Scopus (106) Google Scholar)PLC α(n)Phospholipase A1(n)Lipase ModulatorsPerilipin(g)PAT familyADRP(g)(h) (i) (k) (l) (m) (n) (o)PAT familyTIP47(g)(h) (l) (m) (o)PAT familyS3-12(g)PAT familyLSD2(e)(f)PAT family (Drosophila)CGI-58(g) (i) (n) (o) (f)Regulator of ATGL; has endogenous acyltransferase activity (72Ghosh A.K. Ramakrishnan G. Chandramohan C. Rajasekharan R. CGI-58, the causative gene for Chanarin-Dorfman syndrome, mediates acylation of lysophosphatidic acid.J. Biol. Chem. 2008; 283: 24525-24533Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar)Caveolin 1(g) (m) (n)May bridge perilipin with PKA to stimulate lipolysisOther Redox EnzymesCytochrome p450(e)Mostly in ERCytochrome b5(e)Mostly in ERAlcohol dehydrogenase 4(j) (m)(n) (e)Most in cytoplasm. Broad specificity, including retinols, aliphatic alcohols, and steroidsAldehyde dehydrogenase /ALDH3B1(g)Can oxidize medium and long chain aldehydesGlyceraldehyde phosphate dehydrogenase(a)(h) (l) (m) (n) (o) (e)Cytosolic glycolytic enzyme, but often found with dropletsXanthine oxidoreductase(k)Identified in mammary tissue onlyGulonolactone oxidase(m)Drosophila; missing in humans. Role in ascorbic acid synthesisShort-chain dehydrogenase/reductase member 1(g) (j) (n)(e)Unknown substrateOther EnzymesAcyl-CoA:ethanol o-acyltransferase /EHT1(a)(d)Generation of medium-chain ethyl estersSCCPDH (CGI49)(h)(n) (o)Degradation of lysinePI4 phosphatase/SAC1(n)Serine palmitoyltransferase subunit 1 isoform a(n)Sphingolipid synthesisSAM-dependent methyltransferase(j)Biosynthesis of phosphatidylcholinePossible ContaminationSterol carrier protein 2-related form(l) (e)May have thiolase activity. Peroxisomal contamination?Palmitoyl-protein thioesterase(j) (n)Lysosomal contamination?ER carboxyesterase(k)Mammary; used to make triglyc for lipooproteinsATPsynthase2(g)Mitochondrial contaminationCarbamoyl P Synthetase 1(m)Mitochondrial contaminationPyruvate carboxylase(g)(k)(e)Mitochondrial contamination?Fatty acid translocase/CD36(g)Plasma membrane contamination?Lipoprotein lipase (LPL)(g)Plasma membrane contamination*Non proteomics screens.(a) (29Athenstaedt K. Zweytick D. Jandrositz A. Kohlwein S.D. Daum G. Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae.J. Bacteriol. 1999; 181: 6441-6448Crossref PubMed Google Scholar).*(b) (GFP screen) (13Huh W.K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Global analysis of protein localization in budding yeast.Nature. 2003; 425: 686-691Crossref PubMed Scopus (3235) Google Scholar).*(c) (GFP screen) (14Natter K. Leitner P. Faschinger A. Wolinski H. McCraith S. Fields S. Kohlwein S.D. The spatial organization of lipid synthesis in the yeast Saccharomyces cerevisiae derived from large scale green fluorescent protein tagging and high resolution microscopy.Mol. Cell. Proteomics. 2005; 4: 662-672Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar).(d) (10Binns D. Januszewski T. Chen Y. Hill J. Markin V.S. Zhao Y. Gilpin C. Chapman K.D. Anderson R.G. Goodman J.M. An intimate collaboration between peroxisomes and lipid bodies.J. Cell Biol. 2006; 173: 719-731Crossref PubMed Scopus (261) Google Scholar).(e) (73Beller M. Riedel D. Jansch L. Dieterich G. Wehland J. Jackle H. Kuhnlein R.P. Characterization of the Drosophila lipid droplet subproteome.Mol. Cell. Proteomics. 2006; 5: 1082-1094Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar).(f) (74Cermelli S. Guo Y. Gross S.P. Welte M.A. The lipid-droplet proteome reveals that droplets are a protein-storage depot.Curr. Biol. 2006; 16: 1783-1795Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar).(g) (23Brasaemle D.L. Dolios G. Shapiro L. Wang R. Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3–L1 adipocytes.J. Biol. Chem. 2004; 279: 46835-46842Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar).(h) (75Fujimoto Y. Itabe H. Sakai J. Makita M. Noda J. Mori M. Higashi Y. Kojima S. Takano T. Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7.Biochim. Biophys. Acta. 2004; 1644: 47-59Crossref PubMed Scopus (274) Google Scholar).(i) (76Umlauf E. Csaszar E. Moertelmaier M. Schuetz G.J. Parton R.G. Prohaska R. Association of stomatin with lipid bodies.J. Biol. Chem. 2004; 279: 23699-23709Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar).(j) (24Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic.J. Biol. Chem. 2004; 279: 3787-3792Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar).(k) (77Wu C.C. Howell K.E. Neville M.C. Yates 3rd, J.R. McManaman J.L. Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells.Electrophoresis. 2000; 21: 3470-3482Crossref PubMed Scopus (186) Google Scholar).(l) (78Sato S. Fukasawa M. Yamakawa Y. Natsume T. Suzuki T. Shoji I. Aizaki H. Miyamura T. Nishijima M. Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.J. Biochem. 2006; 139: 921-930Crossref PubMed Scopus (139) Google Scholar).(m) (79Turro S. Ingelmo-Torres M. Estanyol J.M. Tebar F. Fernandez M.A. Albor C.V. Gaus K. Grewal T. Enrich C. Pol A. Identification and characterization of associated with lipid droplet protein 1: a novel membrane-associated protein that resides on hepatic lipid droplets.Traffic. 2006; 7: 1254-1269Crossref PubMed Scopus (157) Google Scholar).(n) (40Bartz R. Zehmer J.K. Zhu M. Chen Y. Serrero G. Zhao Y. Liu P. Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation.J. Proteome Res. 2007; 6: 3256-3265Crossref PubMed Scopus (244) Google Scholar).(o) (5Wan H.C. Melo R.C. Jin Z. Dvorak A.M. Weller P.F. Roles and origins of leukocyte lipid bodies: proteomic and ultrastructural studies.FASEB J. 2007; 21: 167-178Crossref PubMed Scopus (159) Google Scholar). Open table in a new tab *Non proteomics screens. (a) (29Athenstaedt K. Zweytick D. Jandrositz A. Kohlwein S.D. Daum G. Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae.J. Bacteriol. 1999; 181: 6441-6448Crossref PubMed Google Scholar). *(b) (GFP screen) (13Huh W.K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Global analysis of protein localization in budding yeast.Nature. 2003; 425: 686-691Crossref PubMed Scopus (3235) Google Scholar). *(c) (GFP screen) (14Natter K. Leitner P. Faschinger A. Wolinski H. McCraith S. Fields S. Kohlwein S.D. The spatial organization of lipid synthesis in the yeast Saccharomyces cerevisiae derived from large scale green fluorescent protein tagging and high resolution microscopy.Mol. Cell. Proteomics. 2005; 4: 662-672Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). (d) (10Binns D. Januszewski T. Chen Y. Hill J. Markin V.S. Zhao Y. Gilpin C. Chapman K.D. Anderson R.G. Goodman J.M. An intimate collaboration between peroxisomes and lipid bodies.J. Cell Biol. 2006; 173: 719-731Crossref PubMed Scopus (261) Google Scholar). (e) (73Beller M. Riedel D. Jansch L. Dieterich G. Wehland J. Jackle H. Kuhnlein R.P. Characterization of the Drosophila lipid droplet subproteome.Mol. Cell. Proteomics. 2006; 5: 1082-1094Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). (f) (74Cermelli S. Guo Y. Gross S.P. Welte M.A. The lipid-droplet proteome reveals that droplets are a protein-storage depot.Curr. Biol. 2006; 16: 1783-1795Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). (g) (23Brasaemle D.L. Dolios G. Shapiro L. Wang R. Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3–L1 adipocytes.J. Biol. Chem. 2004; 279: 46835-46842Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). (h) (75Fujimoto Y. Itabe H. Sakai J. Makita M. Noda J. Mori M. Higashi Y. Kojima S. Takano T. Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7.Biochim. Biophys. Acta. 2004; 1644: 47-59Crossref PubMed Scopus (274) Google Scholar). (i) (76Umlauf E. Csaszar E. Moertelmaier M. Schuetz G.J. Parton R.G. Prohaska R. Association of stomatin with lipid bodies.J. Biol. Chem. 2004; 279: 23699-23709Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). (j) (24Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic.J. Biol. Chem. 2004; 279: 3787-3792Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). (k) (77Wu C.C. Howell K.E. Neville M.C. Yates 3rd, J.R. McManaman J.L. Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells.Electrophoresis. 2000; 21: 3470-3482Crossref PubMed Scopus (186) Google Scholar). (l) (78Sato S. Fukasawa M. Yamakawa Y. Natsume T. Suzuki T. Shoji I. Aizaki H. Miyamura T. Nishijima M. Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.J. Biochem. 2006; 139: 921-930Crossref PubMed Scopus (139) Google Scholar). (m) (79Turro S. Ingelmo-Torres M. Estanyol J.M. Tebar F. Fernandez M.A. Albor C.V. Gaus K. Grewal T. Enrich C. Pol A. Identification and characterization of associated with lipid droplet protein 1: a novel membrane-associated protein that resides on hepatic lipid droplets.Traffic. 2006; 7: 1254-1269Crossref PubMed Scopus (157) Google Scholar). (n) (40Bartz R. Zehmer J.K. Zhu M. Chen Y. Serrero G. Zhao Y. Liu P. Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation.J. Proteome Res. 2007; 6: 3256-3265Crossref PubMed Scopus (244) Google Scholar). (o) (5Wan H.C. Melo R.C. Jin Z. Dvorak A.M. Weller P.F. Roles and origins of leukocyte lipid bodies: proteomic and ultrastructural studies.FASEB J. 2007; 21: 167-178Crossref PubMed Scopus (159) Google Scholar). The metabolic functions of droplets, as revealed or confirmed by proteomic studies, can be grouped into fatty acid synthesis and activation, sterol biosynthesis, triglyceride biosynthesis, and fatty acid mobilization from sterol esters and triglycerides. Table 1 lists the identified droplet enzymes in these pathways as found by proteomics technology in Saccharomyces cerevisiae, Drosophila melanogaster, rodents, and humans. The ones that are repeatedly found with droplets are discussed below. The first step in fatty acid synthesis is the generation of malonyl-CoA, catalyzed by acetyl-CoA carboxylase, the rate-limiting enzyme in the pathway. The multi-domain fatty acid synthase (FAS) generates palmitic acid (slightly longer chains in yeast) from acetyl-CoA and malonyl-CoA. Most of the carboxylase and FAS are cytosolic although FAS is detected in the droplet proteome in flies (Table 1). Further elongation of palmitic acid and fatty acid desaturation occurs by enzymes in the ER in mammals and probably in yeast (16Tehlivets O. Scheuringer K. Kohlwein S.D. Fatty acid synthesis and elongation in yeast.Biochim. Biophys. Acta. 2007; 1771: 255-270Crossref PubMed Scopus (321) Google Scholar). Finally, fatty acids must be activated to CoA thioesters to be oxidized in the mitochondria and peroxisomes or added to glycerol or sphinganine to form triglycerides or sphingolipids, respectively. Both acetyl-CoA carboxylase and acyl-CoA synthetases have been identified in several droplet proteomes (Table 1). Although much of acetyl-CoA carboxylase localizes in the cytosol, there is some controversy about the localization of the particulate form. Early reports suggested that a fraction of mammalian acetyl-CoA carboxylase was associated with mitochondria or peroxisomes [references contained within (17Geelen M.J. Bijleveld C. Velasco G. Wanders R.J. Guzman M. Studies on the intracellular localization of acetyl-CoA carboxylase.Biochem. Biophys. Res. Commun. 1997; 233: 253-257Crossref PubMed Scopus (21) Google Scholar)] but later work did not confirm these observations in rat liver; on the contrary, evidence involving cytoskeletal agents suggested that some of the enzyme was associated with microtubules (17Geelen M.J. Bijleveld C. Velasco G. Wanders R.J. Guzman M. Studies on the intracellular localization of acetyl-CoA carboxylase.Biochem. Biophys. Res. Commun. 1997; 233: 253-257Crossref PubMed Scopus (21) Google Scholar). In S. cerevisiae, the protein was originally localized to the surface of the ER (18Ivessa A.S. Schneiter R. Kohlwein S.D. Yeast acetyl-CoA carboxylase is associated with the cytoplasmic surface of the endoplasmic reticulum.Eur. J. Cell Biol. 1997; 74: 399-406PubMed Google Scholar). Later work from the same group indicated that in well-oxygenated growing cultures the protein is cytosolic, whereas in nutrient-limited conditions it appears close to mitochondria (16Tehlivets O. Scheuringer K. Kohlwein S.D. Fatty acid synthesis and elongation in yeast.Biochim. Biophys. Acta. 2007; 1771: 255-270Crossref PubMed Scopus (321) Google Scholar) and has been found in the yeast mitochondrial proteome (19Reinders J. Zahedi R.P. Pfanner N. Meisinger C. Sickmann A. Toward the complete yeast mitochondrial proteome: multidimensional separation techniques for mitochondrial proteomics.J. Proteome Res. 2006; 5: 1543-1554Crossref PubMed Scopus (299) Google Scholar). Caution must be brought to evaluating localization of tagged carboxylase because fusions at the carboxy terminus may not rescue the null mutant (16Tehlivets O. Scheuringer K. Kohlwein S.D. Fatty acid synthesis and elongation in yeast.Biochim. Biophys. Acta. 2007; 1771: 255-270Crossref PubMed Scopus (321) Google Scholar). The appearance of acetyl-CoA carboxylase in the droplet proteome may be the result of association of other organelles with droplets, although because the enzyme is classically negatively regulated by fatty acids, it is tempting to speculate that this association occurs on the droplet surface. A study of the effects of incubation with oleic acid (making fatty acid synthesis redundant) on droplet localization of acetyl-CoA carboxylase has not been performed. Elongation of fatty acids beyond palmitate in mammals and perhaps in yeast (16Tehlivets O. Scheuringer K. Kohlwein S.D. Fatty acid synthesis and elongation in yeast.Biochim. Biophys. Acta. 2007; 1771: 255-270Crossref PubMed Scopus (321) Google Scholar) is promoted by elongase and desaturase enzymes localize