Proteomic insights into an expanded cellular role for cytoplasmic lipid droplets^[S]

PROTEOMIC STUDIES

The application of proteomic analyses to CLDs within the last decade have revealed a great deal about the proteins associated with CLDs in a variety of mammals, insects, and fungi. The following sections summarize the major findings in each study and are organized according to cell/tissue origin.

SACCHAROMYCES CEREVISIAE

Saccharomyces cerevisiae was used for two proteomic studies and the results are summarized in supplementary Table I. CLDs from wild-type and deletion yeast strains were analyzed by 1D gel/MS analysis by Athenstaedt et al. (18) and proteins identified were those involved in sterol formation, lipid synthesis and lipid metabolism, enzymes, proteins associated with the ER, and several proteins with unknown functions.

A second study showed a relationship between yeast CLDs and peroxisomes (19). Proteins identified using proteomics were localized using electron microscopy (EM), immunoEM, and fluorescence microscopy in intact S. cerevisiae cultured in either control glucose (2%) or in oleic acid. In cells cultured with oleic acid, there were increased numbers of CLDs and peroxisomes. Although only observed in a small number of cases (3.2% of the cell sections where both CLDs and peroxisomes were visible), the peroxisomes were found to extend “pexopodia” into adjacent lipid droplets. Formation of pexopodia was correlated to the nutritional state of the cells, with pexopodia formation being promoted when cells were forced to scavenge fatty acids from CLDs. CLDs from these oleic acid cultures were fractionated and a shotgun approach was employed for proteomic analysis. Proteins identified were classified into three categories: 1) proteins that had previously been identified as LD proteins by Athenstaedt et al. (18), 2) mitochondrial, peroxisomal and/or ER-associated proteins, and 3) cytosolic proteins. The peroxisomal proteins identified were all matrix components that are involved in the β-oxidation of oleic acid. Many mitochondrial proteins were identified and these were further classified as either matrix proteins, matrix proteins localized to nucleoids, or inner/outer membrane or nucleoid proteins. Numerous resident proteins from the ER as well as membrane trafficking proteins, nuclear proteins, chaperones, enzymes, plasma membrane-associated proteins, and two histones, and numerous cytosolic proteins were observed.

DROSOPHILA

Two recent studies were carried out in Drosophila and their results are summarized in supplementary Table II. Ceremelli et al. (15) characterized CLDs isolated from the embryos of Drosophila followed by shotgun proteomic analysis to identify 127 proteins that appeared in more than three separate studies. [Note: 453 additional proteins were identified in only one study (397 proteins) or two studies (56 proteins)]. Proteins were classified into several groups. Lipid metabolism proteins, membrane trafficking proteins (Rab 8, and Rab 11), heat shock proteins, and other organellar proteins were observed. Surprisingly, several histone proteins (H2A, H2B, and H2Av) were identified repeatedly and at high abundances within these CLD preparations. Further orthogonal studies demonstrated that during the first few hours of embryonic development, the protein levels of H2A and H2B increased but not in direct proportion to the number of nuclei, indicating that the embryos have enough H2A and H2B to package chromatin for thousands of nuclei at the time of fertilization. Importantly, Ceremelli et al. (15) demonstrated that the large deposits of maternal histones can be stored on CLDs and rapidly and efficiently transferred to chromatin as they are needed, thereby providing a new embryo with a source of preexpressed histones for rapid embryonic development. Furthermore, by storing the proteins in an inactive form on CLDs, retardation of embryonic growth that might result from the unregulated presence of large quantities of free histones is also avoided.

The second proteomic study of Drosophila using 1D gel/MS analysis, by Beller et al. (16), focuses on lipid droplets from Drosophila fat body tissue to examine the lipid droplet subproteome from Drosophila predisposed to obesity (adp⁶⁰ and induced Lsd-2:EGFP) or leanness (Lsd-2⁵¹). This group identified 248 proteins, some that were labeled using GFP to localize the proteins within intact cells. EM studies of the cells and Western blot analyses were performed. Of the total 248 proteins identified, 60 proteins were identified in all four genotypes studied, 127 were found in wild-type larvae, 137 proteins were found in ⁶⁰ larvae, 153 were found in induced Lsd-2:EGFP, and 159 were found in Lsd-2⁵¹ mutant larvae. Proteins identified were separated into two classes: class A, composed of proteins that were identified reproducibly in separate lipid droplet preparations, and class B, which was composed of proteins identified only once. In class A, 60 proteins were identified as being common to all four genotypes with smaller numbers of proteins identified that overlapped just one or two genotypes, with a very small number of proteins identified that were genotype specific. The lack of large qualitative differences between these genotypes indicates that genotype characteristics producing leanness or obesity of adult Drosophila may not be reflected in the lipid droplet subproteome of the Drosophila embryos at this early stage of their development. One exception to this finding was the observation of the Regucalcin protein in adp⁶⁰ (obesity predisposed genotype), a homolog of the vertebrate protein senescence marker protein-30 (SMP30) that has been shown in mice to affect cellular lipid droplets, body weight, and lifespan. Other proteins identified repeatedly in different CLD preparations are proteins involved in fatty acid/lipid metabolism, enzymes modifying short- and long-chain fatty acids, membrane trafficking proteins (Rabs 2, 5, and 6), chaperones, and ER-resident proteins.

MAMMALIAN CELLS AND TISSUES

SUMMARY OF PROTEOMIC STUDIES

Over two hundred mammalian proteins have been identified across a number of proteomic analyses of enriched CLDs. [A comprehensive list of all identified CLD proteins in yeast (110 proteins), Drosophila (296 proteins), and mammals (229 proteins) organized according to the function/cellular localization can be found in supplementary Tables I, II, and III, respectively.] Table 1 summarizes the CLD proteins identified in at least three proteomic studies from mammalian cells/tissues. These proteins seem to fall into several distinct functional categories.

The noted “gold standard” marker for CLDs are members of the PAT family [named for the first three members perilipin, adipophilin (also known as adipocyte differentiation related protein, ADRP, or ADFP), and tail interacting protein 47 (TIP47), but the PAT-family also includes lipid storage droplet protein 5 (LSDP5, also known as OXPAT), myocardial lipid droplet protein (MLDP, also called PAT-1), and S3-12] (51). Though PAT proteins are all known to associate with CLDs, they are not all found in every cell. ADRP has been localized to lipid droplets in cultured murine 3T3-Ll adipocytes, murine MA-10 Leydig cells, CHO fibroblasts, and human HepG2 hepatoma cells (43) and TIP47 is similarly found in many cell types (52). However, perilipin has only been found in adipocytes and steroidogenic cells (53). The LSDP5 (OXPAT) protein is found in tissues that rely on β-oxidation of fatty acids for energy production such as heart muscle, brown adipose tissue, and liver (54–56), and S3-12 is found in white adipose tissue (57). Two related proteins, LSD1 and LSD2, are expressed in insects and found in the fat body, a tissue that has similarities to both liver and adipose tissue (58–61).

PAT proteins are thought to be responsible for lipid homeostasis within cells, but the distinctions and specificities of the family members are not yet fully understood. PAT proteins share a conserved 100 amino acid homologous N-terminal PAT domain (the exception is S3-12) (51) and repeating sequences of 11 amino acids thought to form amphipathic α-helices that may assist in embedding the proteins within lipid droplets (33, 34, 51). Phosphorylation controls the functions of some PAT family proteins like perilipin, and the Drosophila PAT family homologs LSD1 and LSD2 in facilitating lipolysis and CLD motility (60, 62). Bickel et al. (63) very recently published a helpful review of PAT proteins.

The role of CLDs in lipid metabolism is critical to maintaining lipid homeostasis and, not surprisingly, proteins involved in lipid metabolism are observed ubiquitously in the CLDs. These include two 17-β hydroxysteroid dehydrogenases, acetyl-CoA carboxylase α, CGI-49 protein, CGI-58 protein (lipase modulator), dehydrogenase/reductase (SDR family) member 1, lanosterol synthase, long-chain-fatty-acid-CoA ligases, NADH-cytochrome b5 reductase 3, patatin-like phospholipase domain-containing protein 2 [also called adipocyte triglyceride lipase (TTS-2.1) (iPLA2ξ)], protein disulfide-isomerase A3, squalene epoxidase, and NAD(P)-dependent steroid dehydrogenase-like protein. These proteins play a variety of roles in lipid synthesis (long-chain-fatty-acid-CoA ligases, lanosterol synthase, squalene epoxidase), lipid homeostasis (long-chain-fatty-acid-CoA ligases), and lipid degradation (patatin-like phospholipase domain-containing protein 2, CGI-58 (64).)

Aside from the PAT family proteins and those involved in lipid metabolism, several proteomic studies have also identified so-called “refugee proteins” that seem to be unrelated to known lipid droplet functions (4) that fall into several categories: membrane trafficking proteins [Arf1, Rab, and Rho proteins (21)], signaling proteins (PKC, Ras, AKAP), cytoskeletal proteins [actinin, filamin A, myosin heavy chain (21)], enzymes (alcohol dehydrogenase), chaperones [pyruvate kinase, peroxiredoxin 1 (21)], and proteins associated with cellular organelles.

Membrane trafficking proteins commonly observed in CLD preparations include proteins from several small GTPase families: Ras-related Rab proteins, ADP Ribosylation factor (ARF) proteins, as well as related proteins phospholipases and caveolins. Though the relationship between CLDs and the small GTPases is unclear, in vivo colocalization studies using immunofluorescence seem to suggest that these interactions are not due to contamination during fractionation (44, 65).

In particular, the Rab protein family of small Ras-related GTPase proteins is well represented in the proteomic studies of CLDs, whereas other members of the small GTPase families such as Rho and Arf proteins are less frequently observed. Rab proteins are known to be important for vesicular trafficking, vesicle budding, and fusion. Although twenty Rab proteins were identified in mammalian studies, Rab effector proteins were not observed in large numbers. The presence of both the small GTPase proteins and their effector proteins is needed for synthesis of a complete pathway. Effector proteins may simply not be present in CLDs or they could be present at such low abundances in CLDs prior to enrichment and purification that they are either lost during enrichment or are below the limit of detection of the analysis. Another possibility is that the effector proteins are only recruited to CLDs under specific conditions (21).

Alternatively, the presence of incomplete and fragmented protein pathways on CLDs in vivo could be the result of CLDs acting as a reservoir of hydrophobic proteins within the cell that functions to transport and assist with synthesis of protein pathways (21). Transfer of substrates and products between CLDs and organelles or other cellular structures could be facilitated by the Rab proteins and other membrane trafficking proteins.

The phospholipid monolayer surrounding CLDs contain abundant phosphatidylcholine and lysophosphatidylcholine (13) that are the substrates for phospholipase D (PLD) (reviewed in ref. 66). PLD1 is involved in many cellular processes like membrane fusion and vesicular trafficking (reviewed in ref. 66). The small protein Arf1 directly activates PLD1, and has received particular attention with respect to its interactions with lipid droplets. Using immunofluorescence, Arf1 and PLD1 have been shown to localize to CLDs (44) and Arf1 was shown to directly bind ADRP (65). A GDP-restricted form of Arf1 caused ADRP dissociation from CLDs and did not inhibit triacylglycerol formation but did prevent formation of new CLDs, which indicates a prevention of CLD budding (65). Arf1 was found in one proteomic study by Bartz et al. (21) along with the ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1, Uniprot: Q8N6T3).

Proteins associated with the ER are also commonly observed in CLD preparations. This is not surprising in light of the fact that lipid droplet biogenesis is thought to occur at the surface of the ER (reviewed in refs. 1, 2, 5, 7, 8, and 67). Numerous mechanisms for LD biosynthesis have been proposed (68–72). Currently, the most widely accepted model is that CLDs are formed by generation of lipid esters by the ER that are deposited into the membrane. As the quantity of lipid esters exceeds the molar proportion that can be assimilated within the phospholipid bilayer, the lipid ester begins to segregate between the two leaflets. This process is represented in Fig. 1. A budding process occurs whereby the lipid droplet begins to separate away from the membrane to form an independent lipid droplet in the cytoplasm (7). Micro-droplets of lipids are formed at the cisternae of the ER and aggregation of those micro-droplets forms larger CLDs (10). Organelles other than the ER are also commonly found in close proximity to CLDs and CLDs have been found to contain proteins associated with the Golgi apparatus (73), peroxisomes (11, 19), mitochondria (22), and the plasma membrane (24), and CLDs are frequently found in close physical contact with these organelles.

CONCLUSIONS AND PERSPECTIVES

The most interesting categories of proteins identified through proteomic analysis are those that do not have known functions within the classical view of the CLD. These “refugee proteins” (4) have prompted an ongoing debate questioning whether these proteins are bona fide CLD proteins. It has been suggested that CLDs may be more susceptible than other cellular structures or organelles to poor isolation due to the intrinsic hydrophobicity of the CLD structure (4, 74). This potential for in vitro contamination is intrinsic to every fractionation protocol. Therefore, in vivo localization studies (immuno-electron microscopy and/or fluorescence microscopy) are required for confirmation of bona fide CLD proteins.

Ideally, genetic studies would follow to validate functional significance (58, 61). Some of the CLD proteomic studies discussed above incorporated genetic knockout or knockdown strategies for functional validation in S. cerevisiae and Drosophila (16, 18, 19). A mouse variant knockout of perilipin has proven to be an extremely valuable tool for demonstrating that perilipin acts as a phosphorylation-controlled gatekeeper that can regulate access to the neutral lipid core of the CLD for lipases, thereby acting as a control on the lipolysis of CLDs (45, 46). Knockouts have been reported for adipose differentiation-related protein (also known as adipophilin, ADRP, or ADFP) (75) as have knockdown studies of ADRP using antisense oligonucleotides (76). Unfortunately, knockout or knockdown studies may sometimes produce ambiguous results that may not clearly define a phenotype related to the reduction in a protein, and this case has been reported for the ADRP phenotype. [(75, 76) and reviewed in (63)]

Knockouts of other CLD-related proteins have been reported for the senescence marker protein-30 (SMP30) (77), adipocyte triglyceride lipase (ATGL) (78), apoA-II (79), hepatic lipases (79), α-synuclein (80, 81), hormone sensitive lipase (82, 83), caveolin-1 (84), and vimentin (85). Knockouts of CLD-related proteins for other species have also been reported, such as the Drosophila adipose gene (adp) (86), and the Drosophila PAT protein analog LSD2 (58, 61).

Supplementary Material