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Originally published In Press as doi:10.1074/jbc.M403920200 on May 10, 2004

J. Biol. Chem., Vol. 279, Issue 29, 30490-30497, July 16, 2004
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CGI-58 Interacts with Perilipin and Is Localized to Lipid Droplets

POSSIBLE INVOLVEMENT OF CGI-58 MISLOCALIZATION IN CHANARIN-DORFMAN SYNDROME*

Tomohiro Yamaguchi, Naoto Omatsu, Shuhei Matsushita, and Takashi Osumi{ddagger}

From the Graduate School of Life Science, Himeji Institute of Technology, University of Hyogo, 3-2-1 Koto, Kamigori, Hyogo 678-1297, Japan

Received for publication, April 8, 2004 , and in revised form, May 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipid droplets (LDs) are a class of ubiquitous cellular organelles that are involved in lipid storage and metabolism. Although the mechanisms of the biogenesis of LDs are still unclear, a set of proteins called the PAT domain family have been characterized as factors associating with LDs. Perilipin, a member of this family, is expressed exclusively in the adipose tissue and regulates the breakdown of triacylglycerol in LDs via its phosphorylation. In this study, we used a yeast two-hybrid system to examine the potential function of perilipin. We found direct interaction between perilipin and CGI-58, a deficiency of which correlated with the pathogenesis of Chanarin-Dorfman syndrome (CDS). Endogenous CGI-58 was distributed predominantly on the surface of LDs in differentiated 3T3-L1 cells, and its expression increased during adipocyte differentiation. Overexpressed CGI-58 tagged with GFP gathered at the surface of LDs and colocalized with perilipin. This interaction seems physiologically important because CGI-58 mutants carrying an amino acid substitution identical to that found in CDS lost the ability to be recruited to LDs. These mutations significantly weakened the binding of CGI-58 with perilipin, indicating that the loss of this interaction is involved in the etiology of CDS. Furthermore, we identified CGI-58 as a binding partner of ADRP, another PAT domain protein expressed ubiquitously, by yeast two-hybrid assay. GFP-CGI-58 expressed in non-differentiated 3T3-L1 or CHO-K1 cells was colocalized with ADRP, and the CGI-58 mutants were not recruited to LDs carrying ADRP, indicating that CGI-58 may also cooperate with ADRP.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipid droplets (LDs)1 are subcellular structures, containing triacylglycerol or cholesterol ester. They are surrounded by a phospholipid monolayer, and function as energy depots (1–3). Moreover, LDs seem to have important roles in lipid trafficking and disorders such as obesity, inflammation, and atherosclerosis (4). Recently, LDs have been characterized as ubiquitous organelles existing in most types of cells and tissues of higher eukaryotes (1). However, the mechanism of the biogenesis of LDs is not entirely clear. It is expected that, similar to other organelles, the protein components associated with the membrane surface would regulate the dynamics of LDs. Indeed, a specific set of proteins called the PAT domain family consisting of four members (perilipin, ADRP, TIP47, and S3–12) that share a conserved region at the N terminus (except for S3–12 carrying the region at the C terminus) have been identified as factors associated with the surface of LDs (5). Perilipin is a phosphoprotein that is selectively expressed in adipocytes and steroidogenic cells, involved in cAMP-dependent lipolysis (6–8). ADRP is ubiquitously expressed in mammalian cells, being involved in lipid uptake, LD formation, milk lipid secretion, and surfactant biosynthesis, and has been used as a marker for diseases involving fat-accumulating cells (9–14). S3–12 coats the surface of LDs that are distinct from those surrounded by perilipin in adipocytes (15). TIP47 is predicted to exist in LDs or the trans-Golgi network and may regulate intracellular membrane trafficking (16–18). Despite these functions, the PAT proteins seem to lack any catalytic domains. Hence, it is expected that partners of PAT proteins in the cells help with the functions. Hormone-sensitive lipase (HSL) is a well-characterized partner of perilipin. Perilipin basically blocks the access of HSL to LDs in quiescent adipocytes and thus restricts the lipolytic activity. On the other hand, under conditions of lipolysis, perilipin is multiphosphorylated by cAMP-dependent protein kinase (PKA), and facilitates the access of HSL to LDs, thereby promoting lipolysis (8, 19). It was reported that perilipin-null mice exhibited a markedly reduced fat cell mass and increased basal lipolysis (20, 21). In contrast, HSL-null mice displayed relatively mild phenotypes, not being obese, but retaining about half of the wild-type level of triacylglycerol lipase activity (22, 23). Moreover, an in vitro study using cell lines lacking endogenous HSL showed that the knockout lines still had a significant level of lipolytic activity (24, 25). These studies indicate that perilipin is a major regulator of lipolysis, and several other cofactors including HSL participate in this event. To elucidate the regulatory mechanism of lipolysis, it is necessary to identify the proteins cooperating with perilipin. In this study, we found that CGI-58 (comparative gene identification-58), which is known as a causal gene of Chanarin-Dorfman syndrome (CDS), interacts with perilipin as well as ADRP, on the surface of LDs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Two-hybrid Screening—A Zucker rat adipose tissue cDNA library (DupLEX-A, OriGene), constructed in a pJG4–5 plasmid encoding the B42 activation domain was subjected to yeast two-hybrid screening for an interactor. cDNAs of perilipin and ADRP were obtained from mouse adipocyte total RNA by reverse transcription (RT)-PCR, and subcloned into a bait vector pEG202-NLS encoding the LexA DNA-binding domain. Part of the sequence of perilipin (corresponding to amino acid residues 1–250) was subcloned into pEG202-NLS. The plasmid was introduced into the yeast strain EGY48, and screening was carried out according to the manufacturer's protocol. From 4.7 x 106 transformants, Leu+ colonies were selected through growth on agar plates lacking leucine, and verified by galactose-dependent lacZ expression. Plasmids were recovered from the yeast cells and sequenced. Several positive clones were isolated, one of them containing a fulllength cDNA sequence similar to Riken 1300003D03, which was judged to be a rat homolog of human CGI-58. HSP86 was isolated as another positive clone. For the screening of an interactor for ADRP, the fulllength sequence of ADRP was subcloned into pEG202-NLS and subjected to screening as described above. From 3.3 x 107 transformants, several positive clones were isolated. Some of the clones were isolated multiple times independently; four clones encoded the C-terminal region of IRG-47, four other clones were similar to Riken 1300003D03.

Cell Culture—3T3-L1 cells were maintained as described previously (26). For differentiation, confluent cells (day 0) were treated with a hormone mixture containing 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 5 µg/ml insulin in Dulbecco's modified Eagle's medium (high glucose)/10% fetal calf serum. After 48 h (day 2), the mixture was removed, and cells were maintained in the same medium supplemented with 5 µg/ml of insulin. CHO-K1 cells were maintained in F12/10% fetal calf serum.

Plasmids—Because some of the yeast two-hybrid clones contained a full-length CGI-58 cDNA, the entire open reading frame of rat CGI-58 was amplified by PCR, and subcloned into pBluescript (Stratagene). The cDNA was transferred to a GFP expression vector, phGFP (105), encoding a GFP mutant that has an increased brightness (27), using appropriate restriction sites. A series of truncation or point mutations of CGI-58 were generated by PCR or employing appropriate restriction sites, and subcloned into phGFP (105). Mutations were verified by DNA sequencing. For the bacterial expression of GST- and T7-tagged proteins, cDNA fragments were inserted into pGex-KG (28) and pET21 (Novagen)

Antibodies—An antibody was raised in a rabbit against GST-fused full-length rat CGI-58 expressed in Esherichia coli. The antibody was affinity-purified with Hi-Trap NHS-activated columns (Amersham Biosciences) coupled with GST and GST-CGI-58. Guinea pig polyclonal anti-perilipin, anti-ADRP and mouse monoclonal anti-ADRP antibodies were purchased from Progen. Rabbit anti-lactose dehydrogenase (LDH) was a gift from Dr. Usuda. A monoclonal antibody to the T7 tag was obtained from Invitrogen.

Immunodetection of CGI-58—3T3-L1 cells were washed with phosphate-buffered saline (PBS) and dissolved in the heated SDS-PAGE sample buffer. Rat adipose tissue was homogenized in 0.25 M sucrose containing 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride. Aliquots of the extracts were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, probed with the affinity-purified CGI-58 antibody, and detected by enhanced chemiluminescence (ECL) (Amersham Biosciences). Amounts of proteins were estimated using LDH as an internal control.

DNA Transfection and Immunofluorescence Microscopy—3T3-L1 cells grown on coverslips were transfected with the expression vectors encoding the GFP-fused full-length or mutants of CGI-58. Transfection was carried out using LipofectAMINE 2000 (Invitrogen), according to the manufacturer's directions. After transfection for 24 h, cells were fixed with cold methanol for 2 min for ADRP staining, or PBS containing 4% paraformaldehyde for 20 min at room temperature for perilipin staining. Fixed cells were permeabilized in 0.2% Triton X-100/PBS for 20 min and blocked with 2% bovine serum albumin/PBS. Cells were then incubated with primary polyclonal antibodies against ADRP and perilipin for 1 h, washed with PBS, and incubated with Cy3-conjugated secondary antibody (Jackson ImmunoResearch) for 1 h. After washing with PBS, cells were mounted and observed under a confocal microscope (LSM510, Carl Zeiss). For the detection of endogenous CGI-58, differentiated 3T3-L1 cells (day 3) were double immunostained with antiperilipin and affinity-purified anti-CGI-58, followed by appropriate Cy3- or fluorescein isothiocyanate-labeled secondary antibodies (Jackson ImmunoResearch). CHO-K1 cells were transfected by a calcium phosphate method as described previously (29). Cells were fixed with cold methanol for 2 min and then treated as described above. A monoclonal antibody for ADRP (Progen) was used for the immunodetection.

RT-PCR—The procedures were as described previously (26). Total RNA was isolated from 3T3-L1 cells using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Total RNA (1 µg) was mixed with a downstream primer mixture (10 pmol each) in a total volume of 20 µl, and RT was carried out for 1 h at 43 °C with Moloney murine leukemia virus reverse transcriptase (Invitrogen). After RT, PCR was performed with 2 µl of the RT product as a template, 10 pmol each of the upstream and downstream primers and recombinant TaqDNA polymerase (TaKaRa). PCR products were analyzed with a 2% agarose gel, and determined in a fluorescence imaging analyzer, FLA3000 (Fuji).

GST Pull-down Experiment—Recombinant GST-fused proteins were expressed in E. coli BL21(DE3) and affinity-purified on glutathione-agarose (28). The amounts of the recombinant proteins were standardized on Coomassie Blue-stained SDS-polyacrylamide gels. T7-tagged mouse perilipin and ADRP were produced in E. coli BL21(DE3) using an expression vector, pET21. Cells were harvested and lysed by sonication in ice-cold PBS containing 1 mM EDTA, 1 mM dithiothreitol, and proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml of leupeptin, 10 µg/ml of aprotinin, and 1 µg/ml of pepstatin A). After the addition of Triton X-100 at 0.5%, lysates were centrifuged at 10,000 x g for 30 min at 4 °C. The resulting supernatant containing a T7-tagged protein was mixed with glutathione-agarose beads containing 15 µg of different GST-fused proteins. After incubation overnight at 4 °C, the beads were washed four times with a binding buffer (PBS containing 1 mM EDTA, 1 mM dithiothreitol, 0.5% Triton X-100), and resuspended in 100 µl of the SDS-PAGE loading buffer. The samples (20 µl each) were separated by SDS-PAGE and analyzed by Western blotting with an anti-T7 tag monoclonal antibody. Immunoblots were developed using ECL. For the binding experiment with native perilipin and the GST-CGI-58 fusion protein, the total 3T3-L1 cell homogenate was used as the source of perilipin. Differentiated 3T3-L1 cells (day 5) were washed with PBS and harvested in binding buffer containing 1 mM phenylmethylsulfonyl fluoride and proteinase inhibitor mixture (Sigma). Any insoluble material was removed by centrifugation. The supernatant was used as a sample for the GST pull-down experiment.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of CGI-58 as a Binding Partner of Perilipin and ADRP—Using the yeast two-hybrid assay, we screened a rat adipose tissue cDNA library with mouse perilipin as bait. The part of perilipin (residues 1–250) used was common to isoforms A, B, and C, and contained the entire PAT domain and subsequent {beta}-sheet region (30, 31). Several clones, which were strongly positive for both growth on agar plates without leucine and galactose-dependent lacZ expression, were isolated, and the cDNAs sequenced. One of the clones was 86.3% identical to human CGI-58 (GenBankTM AL606838 [GenBank] ), and 96.0% identical to mouse RIKEN cDNA 1300003D03 (BC037063 [GenBank] ) assumed to be a CGI-58 homolog. Thus, we judged this clone to be the rat CGI-58. It encoded the entire putative rat CGI-58, a protein of 351 amino acid residues. Furthermore, we also performed a yeast two-hybrid screening with the full-length mouse ADRP as bait, using the same library. CGI-58 was again isolated among the positive clones. Two of four CGI-58 clones obtained in this screening encoded the entire protein sequence. The two other clones lacked the region corresponding to the N-terminal 6 and 12 amino acids but encoded the remaining region up to the C terminus. CGI-58 has been reported as a causal gene for CDS [MIM 275630 [OMIM] ]) (32). CDS is a rare autosomal recessive form of nonbullous congenital ichtyosiform erythroderma (NICE) and is characterized by the intracellular accumulation of LDs in many types of tissues (33–35). The CGI-58 protein sequence is largely conserved from rodents to humans (Fig. 1A). It belongs to a large protein family defined by a {alpha}/{beta} hydrolase fold (36), and contains sequences highly conserved among members of the esterase/lipase/thioesterase subfamily (36). However, as compared with the conserved sequence motif of lipases (LLGHSLGG), the serine residue that is part of the catalytic triad is replaced by asparagine in the corresponding region of CGI-58 (LLGHNLGG; underlined in Fig. 1A).



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  		FIG. 1.
Identification of CGI-58 as a protein interacting with perilipin and ADRP. A, amino acid sequence alignment of CGI-58 from human, mouse, and rat. Residues highlighted indicate the point mutations found in CDS patients. Asterisks show the residues that are diverged among species. A line below the sequence indicates a predicted lipase motif. B, GST pull-down assay of the binding of perilipin and ADRP to CGI-58. A GST fusion protein containing the full-length CGI-58 was used in the pull-down experiment. T7-tagged full-length perilipin-(1–517), truncated perilipin-(1–416), full-length ADRP-(1–425), and truncated ADRP-(1–260) are the inputs. GST alone was analyzed as a control. Input and bound proteins were analyzed by immunoblotting with an anti-T7 antibody and detected by ECL. Numbers on the left indicate the positions of molecular mass standards.

 
In Vitro Binding of CGI-58 to Perilipin and ADRP—To confirm the interactions found in yeast two-hybrid screening, we performed GST pull-down analyses. Total lysate of bacteria expressing recombinant epitope-tagged perilipin or ADRP, the full-length or C-terminally truncated form was applied to glutathione-agarose beads containing GST alone or a GST-fused version of the full-length rat CGI-58. Both the full-length and truncated perilipin and ADRP bound to GST-CGI-58 but not GST alone (Fig. 1B). These findings in vitro support the interaction of CGI-58 with perilipin and ADRP and suggest that the C-terminal regions of perilipin and ADRP are not critical for the binding.

Endogenous CGI-58 Is Principally Located on the Surface of LDs in Differentiated 3T3-L1 Cells—In order to characterize the endogenous CGI-58, an antibody was raised against the recombinant fusion protein expressed in bacteria. The antibody generated was affinity-purified and used in an immunoblot analysis of total protein from rat adipose tissue and 3T3-L1 cells. We expected the antibody to react with both the rat and mouse CGI-58, based on the sequence conservation (only two amino acids differ). The antibody recognized a single protein in the adipose tissue lysate at a position of ~45 kDa (Fig. 2A), which was somewhat larger than the estimated size of rat CGI-58 (39.1 kDa). However, the CGI-58 expressed from a mammalian expression vector was also detected with this antibody, as a band of apparently larger size, close to 45 kDa (data not shown). Thus, we consider that this band indeed represented CGI-58, and the slower electrophoretic mobility would be a characteristic of the protein. On the blot of 3T3-L1 lysate, interestingly, we observed a dramatic change in the protein expression upon adipocyte differentiation. For the lysates prepared from the cells on day 0 (before differentiation), we did not detect any band with the anti-CGI-58 antibody. In contrast, for the cell lysates on day 5 (about 60% of cells differentiated), the antibody detected several bands (Fig. 2B). The major band (indicated by an arrow in Fig. 2B) corresponded to the one detected for the rat adipose tissue lysate (confirmed by another blot where lysates were electrophoresed side by side; data not shown). Other bands were much fainter than the major one. Although they are possibly nonspecific, another explanation would be the presence of alternative splicing variants of CGI-58. Indeed, based on a data base analysis in AceView of NCBI, several variants of human CGI-58 are predicted. These results indicate that protein expression of CGI-58 is induced upon adipocyte differentiation.



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  		FIG. 2.
Detection of endogenous CGI-58 by Western blotting (A and B) and immunofluorescence (C) analyses. A, total protein was prepared from rat adipose tissue and 20 µg were separated by SDS-PAGE. The blotted membrane was probed with an anti-CGI-58 antibody. Numbers on the left indicate the positions of molecular mass standards. B, whole cell lysates were prepared from non-differentiated (-) and differentiated (+) 3T3-L1 cells. Endogenous CGI-58 was analyzed as in A. To compare the protein amounts, LDH was used as a control. C, differentiated 3T3-L1 cells were fixed and double-immunostained with anti-CGI-58 and anti-perilipin antibodies. The cells were observed with a confocal microscope. Bar, 10 µm.

 
We next determined the subcellular localization of CGI-58 in differentiated 3T3-L1 cells by the indirect immunofluorescence method. Differentiated 3T3-L1 cells were fixed and double immunostained with the anti-CGI-58 and anti-perilipin antibodies (Fig. 2C). Endogenous CGI-58 mainly displayed a cytoplasmic ring-shaped distribution, similar to that of perilipin, suggesting that CGI-58 is colocalized with perilipin on the surface of LDs. CGI-58 was also expressed with a diffuse pattern in the cytosol (Fig. 2C), where perilipin was absent. When non-differentiated 3T3-L1 cells were immunostained for CGI-58, no fluorescence was detected (data not shown).

CGI-58 mRNA Level Increased in the Early Stage of Adipocyte Differentiation—We next followed the change in the mRNA level of CGI-58 during the differentiation of 3T3-L1 adipocytes, in comparison with that of PAT proteins and adipose protein/fatty acid-binding protein (aP2), a marker for adipogenesis (37). A ribosomal protein, 36B4, was used as an internal control, unaffected by the differentiation. RT-PCR was performed using total RNA prepared from cells on the indicated days, 0 to 8. Before differentiation (day 0), CGI-58 mRNA was expressed at a low level, whereas after induction, the mRNA level increased immediately (day 1), and remained constant during the course of the differentiation (Fig. 3). This result was consistent with that for the CGI-58 protein expression (see Fig. 2B). On the other hand, the results for perilipin, ADRP, and aP2 were all consistent with those of a previous report (10, 37): mRNAs of perilipin and aP2 were not detectable until day 3 and then increased, consistent with the observation that these genes are regulated by the same transcription factor, peroxisome proliferator-activated receptor (PPAR) {gamma}2 (37–39). ADRP showed an initial decrease in mRNA level, followed by a large increase from a relatively early stage of differentiation (10). The mRNA level of S3–12 increased from day 3 and continued to rise, nearly corresponding to the increase in protein described before (15). These results indicate that CGI-58 mRNA increases at an initial stage of differentiation, prior to the increases in PAT protein mRNAs. Thus, CGI-58 is possibly regulated by a mechanism distinct from that of PAT proteins. In addition, we noticed that TIP47 (putative mouse sequence, AK004970 [GenBank] ) was the only PAT domain protein unaltered in its mRNA expression during the differentiation, and so likely to be functionally unrelated to adipogenesis.



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  		FIG. 3.
CGI-58 mRNA expression is up-regulated in the early stages of adipocyte differentiation. 3T3-L1 cells were maintained and induced to differentiate as described under "Experimental Procedures." Total RNA was prepared from the cells on the days indicated. mRNA expression was estimated by RT-PCR using appropriate primers for the mouse sequences. 36B4 unaffected by differentiation was used as a control. Numbers of PCR cycles were 20 for 36B4 and 23 for the rest, set in the exponential amplification phase. Note that the expression level of CGI-58 was elevated from day 0 to day 1.

 
Ectopically Expressed CGI-58 Is Recruited to LDs in 3T3-L1 Cells—To elucidate the mechanisms behind the localization of CGI-58 to LDs, we first examined whether ectopic CGI-58 was correctly recruited to LDs similar to the endogenous protein. We introduced an expression vector with GFP fused to the full-length CGI-58 into differentiated 3T3-L1 cells and compared the distribution of the fusion protein and perilipin using immunofluorescence microscopy. GFP-CGI-58 showed a ringshaped subcellular distribution, overlapping with perilipin detected by the specific antibody (Fig. 4A). Moreover, when GFP-CGI-58 was expressed in the non-differentiated cells, a punctate distribution was observed over the cytoplasmic fluorescence, being colocalized with ADRP (Fig. 4B). In contrast, GFP per se distributed only in the cytosol and exhibited no particulate distribution under either set of conditions (Fig. 4). These results indicate that ectopically expressed CGI-58 was recruited to LDs and colocalized with perilipin and ADRP.



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  		FIG. 4.
CGI-58 fused to GFP was colocalized with endogenous perilipin or ADRP in the cells. A, differentiated 3T3-L1 cells were transiently transfected with an expression vector for GFP-CGI-58 or GFP. Cells were immunolabeled with antibodies to perilipin, followed by Cy3-coupled secondary antibody, and the fluorescence of the antibody and GFP were observed with a confocal microscope. B, non-differentiated 3T3-L1 cells were transfected and processed as in A, except that an anti-ADRP antibody was used for immunolabeling. Bar, 10 µm.

 
We next investigated the motif of CGI-58 that are necessary for its recruitment to LDs. For this purpose, a series of truncated mutants of CGI-58 were generated as GFP-fused proteins (Fig. 5). After transfection, we examined whether the CGI-58 mutants were colocalized with perilipin and ADRP, in differentiated and non-differentiated 3T3-L1 cells, respectively. For both perilipin and ADRP, removal of the first 18 amino acids from the N terminus of CGI-58 (residues 19–351) affected its ability to be recruited to the surface of LDs, but its localization on the LDs was apparent in some cells (marked ±). All other mutant CGI-58s failed to be recruited to LDs. Even when a short fragment was removed from the C terminus (residues 1–317), the targeting was completely impaired. These observations indicate that nearly the entire region of CGI-58 is necessary for the localization at the surface of LDs.



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  		FIG. 5.
Analysis of CGI-58 truncation mutants for recruitment to LDs. The full-length and truncated CGI-58s were tagged with GFP at the N terminus. Numbers indicate residues of CGI-58. The constructs were expressed in the differentiated and non-differentiated 3T3-L1 cells, and analyzed for their localization at LDs. Cells were immunostained, and the fluorescence of the antibody and GFP were observed with a confocal microscope. The full-length construct exhibited colocalization with perilipin or ADRP and is marked positive (+). The construct containing residues 19–351 exhibited colocalization in some cells, and a diffuse cytosolic pattern in others, and is marked (+/-). With regard to all other mutants, only a diffuse cytosolic distribution was observed, which is marked negative (-).

 
Point Mutations, Causal for CDS, Abrogated the Recruitment of CGI-58 to LDs—CDS is a rare autosomal recessive disease of lipid metabolism characterized by the presence of abnormally large amounts of lipid droplets in several organs. Patients with CDS develop an abnormal intracellular accumulation of LDs containing triacylglycerol (TAG) in many tissues. Recently, CGI-58 mutations were identified as the cause of CDS (32). Nine distinct mutations were found in the CGI-58 gene of CDS patients (32, 40). They were: (a) three point mutations (Q130P, E7K, and E260K: numbers mean amino acid positions of human CGI-58); (b) two nonsense mutations (S33X and R184X); (c) two splice site mutations, leading to aberrant splicing in exons 3 and 6; (d) a deletion in exon 1; and (e) an insertion in exon 4, both causing a frameshift and leading to a premature termination of translation. In the mutations of b–e, substantial portions of the C-terminal region of CGI-58 would be eliminated. On the other hand, the three point mutations in a result in single amino acid substitutions at residues conserved throughout the species. Hence, these residues probably have important roles in the function of CGI-58. To investigate the significance of these residues, we introduced the corresponding mutations (E9K, Q132P, E262K) into the rat CGI-58 fused to GFP, and examined the distribution of the mutant CGI-58 proteins in 3T3-L1 cells (Fig. 6). The Q132P and E262K mutants, in contrast to the wild type, exhibited a diffuse cytoplasmic distribution without colocalization with perilipin or ADRP. Thus, these two point mutations caused mistargeting of CGI-58 in vivo. On the other hand, the E9K mutant was localized to the surface of LDs, similar to the wild-type, together with perilipin and ADRP in differentiated and non-differentiated cells, respectively. An E8K + E9K double mutant also exhibited a localization to LDs similar to the wild-type and E9K (data not shown). The results for E9K and E8K + E9K agree with the observation that removal of a short stretch of the N terminus still allowed the protein to be localized to LDs in some cells (Fig. 5).



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  		FIG. 6.
Substitutions of single amino acids corresponding to the point mutations found in CDS cause mistargeting of CGI-58 to the cytosol. GFP-CGI-58 containing point mutations, E9K, Q132P, and E262K, was expressed in differentiated (A) and non-differentiated (B) 3T3-L1 cells. Cells were immunostained with anti-perilipin (A) and ADRP (B) antibodies, respectively, and observed by confocal microscopy. Note the loss of ability to be localized to LDs of Q132P and E262K mutants, but not the E9K mutant. Bar, 10 µm.

 
We next examined the targeting of CGI-58 mutants in other cell lines. Wild-type or mutant CGI-58s fused to GFP were introduced into CHO-K1 cells, which contained ADRP but not perilipin, and the subcellular distributions were examined. Similar to results with 3T3-L1 cells, wild-type and E9K mutants were recruited to LDs and colocalized with ADRP, but Q132P and E262K mutants failed to be localized correctly (Fig. 7). Similar results were obtained with HeLa cells (data not shown). These results suggest that the effects of point mutations of CGI-58 were not restricted to the adipocytes or preadipocytes, consistent with the observation that the symptoms of CDS involve multiple organs and systems (33–35).



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  		FIG. 7.
Wild-type CGI-58 but not the mutant fused to GFP is recruited to LDs and colocalized with ADRP in CHO-K1 cells. Wild-type and mutant GFP-CGI-58s were expressed in CHO-K1 cells. GFP alone was used as a control (top panel). Cells were immunostained with anti-ADRP antibody and observed by confocal microscopy. Left panels indicate the fluorescence of GFP, and right panels show ADRP staining. Bar, 10 µm.

 
Point Mutations of CGI-58 Weaken the Ability to Interact with Native Perilipin—To examine whether the mutations found in the CDS patients affect the direct interaction of CGI-58 with perilipin, we next performed a GST pull-down assay using the GST-fused proteins of the wild-type and the mutants of CGI-58, Q132P, and E262K, defective in the localization to LDs. The fusion proteins were mixed with the total cell extract from the differentiated 3T3-L1 cells containing native perilipin, and recovered with affinity resin. Fig. 8 shows that the wild-type CGI-58 bound to perilipin, but the Q132P and E262K mutants were severely impaired in the binding. Hence, the CGI-58 mutants failed to be recruited to LDs due to the diminished affinity for perilipin, which in turn led to the functional defect. We also examined the binding of native ADRP contained in 3T3-L1 preadipocyte lysates with GST-CGI-58, but failed to detect the binding (data not shown). This is inconsistent with the results of the yeast two-hybrid assay and GST pull-down assay using recombinant proteins (Fig. 1B). A possible explanation for this would be that other factors are required for the interaction in vivo, and they are released, inactivated, or disrupted during the preparation of samples from the cells.



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  		FIG. 8.
Binding of GST-CGI-58 with native perilipin is impaired by the CDS point mutations. GST pull-down assay of perilipin using the GST-fused forms of the wild-type and mutant (A, Q132P; B, E262K) CGI-58s. The extract from differentiated 3T3-L1 cells was used as a source of native perilipin. Input and bound proteins were analyzed by immunoblotting with an anti-perilipin antibody visualized by ECL (upper panel), and Coomassie Blue staining (lower panel) to ensure equal loading of wild-type and mutant CGI-58. GST alone was used as a control. Numbers on the left indicate the positions of molecular mass standards. The arrow and asterisk indicates perilipin and GST proteins, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we identified CGI-58 as a binding partner of perilipin and ADRP using a yeast two-hybrid system. Specific interaction between CGI-58 and perilipin was confirmed in a GST-pull-down assay and supported by an immunofluorescence analysis. We further demonstrated that CGI-58 is principally located at the surface of LDs in 3T3-L1 cells, together with perilipin, and its expression is up-regulated upon stimulation for adipocyte differentiation.

The human CGI-58 gene was originally identified using a comparative proteomic approach, with the Caenorhabditis elegans proteome as an alignment template and the human EST data bases (41). The amino acid sequence of CGI-58 protein is highly conserved from humans to rodents throughout its entire length, and contains a region homologous to the {alpha}/{beta} hydrolase fold found in a large family of proteins. It is classified as a member of the esterase/lipase/thioesterase subfamily, based on the sequence of its catalytic triad (36). Importantly, CGI-58 was identified as a causal gene of CDS, a disorder characterized by the accumulation of abnormally large amounts of lipid droplets in several organs (32).

Previous studies identified nine mutations of CGI-58 in CDS patients (32, 40). Six of them would cause a premature termination of translation. The remainder were missense mutations, of which two (Q132P and E262K) disrupted recruitment to LDs, indicating that the targeting of CGI-58 is physiologically important, and its loss may trigger CDS. The other mutant (E9K) was targeted correctly like the wild type, and thus probably causes the disorder via a mechanism other than mistargeting. This mutation may impair a catalytic function of CGI-58, or its interaction with another unknown protein factor. It is also possible that this mutant cannot support the physiological function of perilipin, despite the successful physical association.

LDs are coated with a specific class of protein, the major one being perilipin in adipocytes and steroidogenic cells, and ADRP in most other cell types (6, 7, 10). They are loosely grouped as a small protein family sharing a common N-terminal motif, called the PAT domain (5). The function of the PAT domain is not clear. The domain does not seem to be necessary for recruitment to LDs in either perilipin or ADRP (42–45). Although the N-terminal region of perilipin A was shown to take part in facilitating TAG storage in LDs, a region containing the {beta}-strand region following the PAT domain rather than the PAT domain itself, was essential (42). In the present study, we used the first 250 N-terminal residues of perilipin, containing the PAT domain and the {beta}-strand region, as the bait for yeast two-hybrid screening. The corresponding region of ADRP also exhibited interaction with CGI-58. Thus, the PAT domain and the {beta}-strand region consist of a protein-protein interaction domain, and CGI-58 is an important partner of this domain.

The levels of mRNA and protein of CGI-58 were significantly increased upon the adipogenic differentiation of 3T3-L1 cells, indicating that the expression of the CGI-58 gene is dependent on physiological conditions. Many adipocyte-specific genes, including the perilipin gene (38, 39, 46, 47), are regulated by PPAR{gamma}, a master regulator of adipogenesis (37). Accordingly, we were interested in whether the CGI-58 gene is also regulated by PPAR{gamma}. The increase in CGI-58 mRNA, however, was observed at an earlier stage of differentiation, prior to that of perilipin or aP2, another PPAR{gamma} target, as well as ADRP. Hence, the expression of the CGI-58 gene is regulated by a mechanism distinct from that of regulating major adipogenic genes. The physiological meaning of why the expression of CGI-58 precedes that of its partners is also of interest.

We found CGI-58 to be a binding partner of both perilipin and ADRP, with the yeast two-hybrid screening. Moreover, overexpressed GFP-CGI-58 was colocalized with each of these proteins in differentiated and non-differentiated 3T3-L1 cells, respectively. However, it is not yet clear whether CGI-58 physiologically functions with both proteins in vivo. We would propose that CGI-58 cooperates at least with perilipin, which is supported by the following results: First, the expression of CGI-58 is induced upon differentiation of 3T3-L1 cells. Second, indirect immunofluorescence analysis showed that endogenous CGI-58 was located at the surface of LDs, together with perilipin. Third, native perilipin in the 3T3-L1 lysate bound to GST-CGI-58, and the mutant CGI-58s containing the CDS missense mutations exhibited less ability for the binding. On the other hand, at present, we have no evidence that ADRP binds to CGI-58 in vivo, though the interaction was supported by the GST pull-down experiment using the recombinant proteins. Although overexpressed GFP-CGI-58 was colocalized with endogenous ADRP in 3T3-L1 and CHO-K1 cells, there is still a possibility that CGI-58 is localized at the surface of LDs without interacting with ADRP. During the course of this study, Liu et al. (48) reported the results of a proteomic analysis on LDs isolated from CHO K2 cells. They showed that CGI-58 appeared in the LD fraction, when cells were grown with oleic acid. Given that ADRP is ubiquitously expressed, but perilipin expression is highly restricted, CGI-58 possibly cooperates with ADRP only in cell lines lacking perilipin, like CHO cells. To clarify this, it is necessary to investigate the relationship between CGI-58 and ADRP or other PAT proteins in non-adipose cells.

The most important issue remaining to be resolved is the physiological function of CGI-58 on the surface of LDs. Since CGI-58 has sequences similar to the motifs that characterize the esterase/lipase/thioesterase subfamily, one possibility is that it is itself a lipase catalyzing the breakdown of TAG or cholesterol ester. However, the active site serine in the catalytic triad common to this subfamily is replaced by asparagine in CGI-58. In the case of HSL, another member of the subfamily, substitution of this serine with glycine, alanine, cysteine, or threonine by site-directed mutagenesis completely abolished both the lipase and esterase activities (49), indicating an essential role for this residue in the enzymatic activities. Since perilipin has a critical role in the mobilization of TAG stored in the adipocytes (42), CGI-58, a binding partner of perilipin, may also contribute to the lipolytic events occurring on the surface of LDs, in addition to HSL. Further analysis is required to elucidate whether CGI-58 has lipase/esterase/thioesterase activities.

A case of CDS caused by a CGI-58 mutation was recently reported in a patient from a non-Mediterranean region. In the epidermis of this patient, the accumulation of abnormal lamellar granules and lipid vacuoles were observed, suggesting that CGI-58 participates in the lipid metabolism of lamellar granules and is thus involved in the pathogenesis of ichthyosis (40). Elucidation of the physiological functions of CGI-58 will lead to an understanding of the regulatory mechanism of lipid mobilization in LDs, and hence the cause of diseases involving abnormal lipid metabolism, including CDS.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY550934 [GenBank] .

* This work was supported by grants from Uehara Memorial Foundation (to T. Y.), a grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science (to T. O.), and the 21st Century Center of Excellence Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 81-791-58-0194; Fax: 81-791-58-0193; E-mail: osumi{at}sci.u-hyogo.ac.jp.

1 The abbreviations used are: LDs, lipid droplets; CDS, ChanarinDorfman syndrome; ADRP, adipose differentiation-related protein; PAT, perilipin, ADRP, and TIP47; LDH, lactose dehydrogenase; TAG, triacylglyceride; GFP, green fluorescent protein; CHO, Chinese hamster ovary; HSL, hormone-sensitive lipase; PBS, phosphate-buffered saline; CGI, comparative gene identification; GST, glutathione S-transferase; RT, reverse transcriptase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. T. C. Südhof for the plasmid and A. Omukae for help in the initial stages of this study.



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Perilipin A Mediates the Reversible Binding of CGI-58 to Lipid Droplets in 3T3-L1 Adipocytes
J. Biol. Chem., October 1, 2004; 279(40): 42062 - 42071.
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