The authors have declared that no competing interests exist.
Conceived and designed the experiments: TW RT WW. Performed the experiments: RT WW SW ZW LS W. He RF YZ XX. Analyzed the data: TW RT SW ZW. Contributed reagents/materials/analysis tools: SW ZW. Wrote the paper: TW W. Hong.
The subcellular location and cell biological function of small GTPase Rab40c in mammalian cells have not been investigated in detail. In this study, we demonstrated that the exogenously expressed GFP-Rab40c associates with lipid droplets marked by neutral lipid specific dye Oil red or Nile red, but not with the Golgi or endosomal markers. Further examination demonstrated that Rab40c is also associated with ERGIC-53 containing structures, especially under the serum starvation condition. Rab40c is increasingly recruited to the surface of lipid droplets during lipid droplets formation and maturation in HepG2 cells. Rab40c knockdown moderately decreases the size of lipid droplets, suggesting that Rab40c is involved in the biogenesis of lipid droplets. Stimulation for adipocyte differentiation increases the expression of Rab40c in 3T3-L1 cells. Rab40c interacts with TIP47, and is appositionally associated with TIP47-labeled lipid droplets. In addition, over-expression of Rab40c causes the clustering of lipid droplets independent of its GTPase activity, but completely dependent of the intact SOCS box domain of Rab40c. In addition, Rab40c displayed self-interaction as well as interaction with TIP47 and the SOCS box is essential for its ability to induce clustering of lipid droplets. Our results suggest that Rab40c is a novel Rab protein associated with lipid droplets, and is likely involved in modulating the biogenesis of lipid droplets.
Lipid droplets (LDs, also referred to as adiposome) are the unique organelle for the storage of neutral lipids, mostly triacylglycerols (TG) and cholesterol esters. Lipid droplet has a lipid core surrounded by a monolayer of phospholipids membrane, and is produced by many types of cells in animals, plants and even microorganisms (comprehensively reviewed in
It is widely accepted that LDs are primarily derived from endoplasmic reticulum (ER) membrane
The best characterized LDs surface proteins are the PAT proteins [perilipin, ADRP (adipocyte differentiation related protein or adipophilin) and TIP47 (tail-interacting protein 47 KDa)], which are classified by harboring the perilipin amino-terminal (PAT) domain within the N-terminal portion of these proteins. PAT proteins are crucial for LDs function and biogenesis
Proteomics studies demonstrated that many Rab proteins associate with lipid droplets
Rab40c is a homolog of xRab40 in Xenopus, which was shown to interact with culin5 and elongin B to form a complex, serving as E3 ligase and regulating noncanonical wnt pathway
The rabbit polyclonal antibody against Rab40c was generated by Genemed Synthesis (San Antonio, TX) using the peptide sequence (LMRHGMEKIWRPNRVFS) as the antigen. The monoclonal antibodies (mAbs) against GM130 and EEA1 were from BD (BD Biosciences, PaloAlto, CA), mAbs against rat Lamp2 was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa (Department of Biological Science, Iowa City, IA). mAb against Myc-tag (9E10) and mAb against Flag tag were obtained from American Type Culture Collection (ATCC, Manassas, VA). mAb against lysobisphosphatidicacid (LBPA) was a generous gift from Dr Jean Gruenberg(University of Geneva, Switzerland). mAb against KDEL receptor was from BD (BD Biosciences, PaloAlto, CA). Rabbit polyclonal antibody against ADRP was from SANTA CRUZ (Delaware Avenue, CA). Rabbit polyclonal anti-TIP47 antibody was purchased from AnaSpec (Fremont, CA). Rabbit mAb against Perilin was from Cell Signaling (Danvers, MA). mAb against GFP was from Clontech (Palo Alto, CA,USA). mAb against β–tubulin was from Sigma (St. Louis, MO). HRP-conjugated secondary antibodies were purchased from Pierce (Rockford, IL). Texas red-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA).
The coding regions of human Rab40a, Rab40b and Rab40c were retrieved from the cDNA library of Human fetal brain (BD Clontech) by PCR with primer1(
Human multiple tissues cDNA panels (BD Biosciences, Palo Alto, CA,USA) were used for PCR-based analysis to assess the expression levels of the transcripts for Rab40a, Rab40b and Rab40c using the primers described above. Primer 8 (
NRK cells, HepG2 cells, Hela cells and MCF7 cells were from ATCC and grown in DMEM media supplemented with 10% fetal bovine serum (Gibco, Ann Arbor, MI) in a 5% CO2 incubator at 37°C. 3T3-L1 cells were grown in DMEM medium (4.5 g/liter) supplemented with 10% newborn calf serum. Transient transfection of plasmids was conducted using either Lipofectamine 2000 reagents (Invitrogen,Gaithersburg, MD, USA) or TurboFect in vitro transfection reagent (Thermo, Massachusetts, USA) according to the manufacturer’s protocol. When inducing lipid droplets formation in HepG2 cells, 100 µg/ml oleic acid (pre-complexed with 0.1% fatty-free BSA in 0.1 M Tris·Cl, pH8.0) was added to the media. For serum starvation, cells were grown in DMEM medium (4.5 g/liter) without FBS.
Adipocyte differentiation was induced as described previously
Immunofluorescence microscopy experiments were performed as described
[]Lipid droplets fractionation was performed as described
GST-pulldown assay was performed as described previously
For western-blotting experiments, tissue lysates or the samples from GST-pull-down assay were resolved by SDS-PAGE and transferred to nitrocellulose filter. The filter was blocked with 5% milk in PBS and then incubated with primary antibody for 1 h at room temperature, followed by the incubation with HRP-conjugated secondary antibody. The blots were detected using ECL system (Pierce, Rockford, IL, USA).
pSuper vector mediated shRNA was carried out by constructing Rab40c targeting sequence (
Mean size of lipid droplets, from 50 cells of transfected cells, was estimated using the software ImageJ 1.32 (NIH) under confocal microscope. The size differences between groups were examined with
XRab40 regulates
A. sequence alignment showed that Rab40 family proteins possess conserve phosphate/Mg2+ (PM) binding motifs and SOCS box (the key residues for proper function of SOCS box are underlined), and hRab40c is likely the homolog of xRab40. B. PCR based examination of the transcript levelsof 3 Rab40 members. C. Rab40c is expressed in different tissues as examined by western-blot using Rab40c antibody. D. NRK cells were transfected with GFP-Rab40a, GFP-Rab40b or GFP-Rab40c, respectively. Immuno-fluorescence microscopy analysis showed different localization of Rab40 family proteins. Bar, 20 µm.
PCR based examination of the transcript levels of Rab40 members demonstrated that they are expressed ubiquitously in different human tissues (
In order to study the cell biological function, we generated GFP-tagged Rab40a, Rab40b and Rab40c, and examined their sub-cellular location using immuno-fluorescence microscopy. Surprisingly, these 3 members have different subcellular locations. GFP-Rab40a and GFP-Rab40b are generally distributed in the cytoplasmnucleus. Interestingly, GFP-Rab40c is mostly associated with vesiclar structures (
To investigate the properties of the GFP-Rab40c-associated vesicles, we performed co-localization experiments with markers for different organelles using immuno-fluorescence microscopy. As NRK cells give a better vesicular structure of Rab40c in our experiments, we examined the sub-cellular localization of Rab40c in NRK cells. It was observed that GFP-Rab40c did not co-localize with Golgi marker GM130, late endosomal/lysomomal markers LBPA and Lamp2 in NRK cells (
A. NRK cells were transfected with GFP-Rab40c, and then immuno-labeled with the indicated antibodies. Immuno-fluorescence microscopy showed that GFP-Rab40c is not co-localized with Golgi marker GM130, late endosomal/lysomomal markers LBPA and Lamp2 in NRK cells. B. GFP-Rab40c is not co-localized with early endosomal marker EEA1 in Hela cells. Bar, 20 µm.
As GFP-Rab40c is not associated with most of the membrane trafficking compartments, further examinations were conducted to focus on other organelles, such as mitochondria and LDs. Interestingly, GFP-Rab40c was seen to be present on the surface of LDs marked by Oil-red or Nile red in NRK cells (
A. NRK cells were transfected with GFP-Rab40c and labeled with Oil red or Nile red, then processed for immuno-fluorescence microscopy. The results showed that GFP-Rab40c is associated with LDs. B. NRK cells were co-transfected with GFP-Rab40c and Flag-Rab18, and processed for immuno-fluorescence microscopy, Rab40c is revealed by GFP and Rab18 is revealed by labeling with antibody against Flag tag. The results showed that GFP-Rab40c co-localizes partially with flag-Rab18. C. NRK cells were transfected with pCIneo-Rab40c and labeled with Oil red, and immuno-stained by antibody against Rab40c, showing that non-tagged Rab40c is also associated with LDs. D. Western blotting analysis of sucrose density-gradient fractions from HepG2. An equal volume from each fraction was loaded. Both TIP47 and Rab40c were enriched in the top floating LDs-containing fraction (lane 1). The ER-Golgi recycling protein KDEL receptor was detected in the bottom fractions. E. NRK cells were co-transfected with pCIneo-Rab40c and GFP-ERGIC-53, and immuno-stained by antibody against Rab40c. Rab40c is also associated with structures marked by ERGIC-53. Bar, 20 µm.
To further confirm Rab40c association with LDs, we purified the LDs fraction from HepG2 cells induced with oleic acid, western-blot analysis of subcellular fractions demonstrated that Rab40c is highly enriched in the top floating fraction, which contains the LDs fractions marked by TIP47 (
Although Rab40c does not colocalize with most of trafficking compartments, some Rab40c was observed to associate with ER-Golgi intermediate compartments. when closely examined by co-expressing pCIneo-Rab40c with GFP-ERGIC-53 (
Since Rab40c associates with LDs, we examined whether Rab40c is involved in LDs biogenesis. Because it is difficult to transfect GFP-Rab40c into 3T3-L1 adipocytes, we investigated the dynamic membrane association of GFP-Rab40c during LDs formation in HepG2 cells induced with oleic acids, mimicking the the formation of LDs during adipocyte differentiation. It was observed through immuno-fluorescence microscopy that GFP-Rab40c is recruited to the surface of LDs upon stimulation for the formation of LDs in a time course dependent manner. As shown in
A. LDs formation was induced by Oleic acid in HepG2 transfected with GFP-Rab40c for 0, 12, 24 and 48 h, respectively, and LDs were revealed with Oil red. immuno-fluorescence microscopy showed that GFP-Rab40c is recruited to LDs upon stimulation in a time course dependent manner. B. HepG2 cells were co-transfected with pCIneo-Rab40c and GFP-ERGIC-53, then starved for 24 h (upper panels) or stimulated with Oleic acid for 24 h afterward(lower panels), the expressed Rab40c was revealed by immuno-staining with antibody against Rab40c. The data showed that most of the expressed Rab40c co-localized with ERGIC-53 under starvation condition. However, when stimulated with oleic acids, the expressed Rab40c segregated from ERGIC-53, and become associated with the enlarged vesicles characteristic of LDs (indicated by arrow heads). Bar, 20 µm.
As the biogenesis of LDs is closely related to the ER, we examined whether Rab40c is also associated with the ER by co-expressing pCIneo-Rab40c and mCherry-Sec61, and found Rab40c did not co-localize with Sec61 (data not shown). However, a pool of Rab40c was seen to associate with ERGIC-53 containing compartments (
To further investigate the role of Rab40c in LDs biogenesis, we performed RNAi experiments to see whether the depletion of Rab40c affects the formation of LDs. HepG2 cells were transfected with pSuper-shRNARab40c, then stimulated with oleic acids, and the formation of LDs was analysed with confocal microscopy. The knockdown efficiency was monitored by western-blot using antibody against hRab40c (
A. HepG2 cells were transfected with shRNA-Rab40c or shRNA-control to achieve about 70% transfection efficiency. Western-blot showed that the protein level of Rab40c can be specifically knocked down by shRNA-Rab40c in HepG2 cells. Quantitative analysis was done by densitometry, and the data represent the mean value from 3 independent experiments. B. HepG2 cells were transfected with pSuper.GFP-shRNA-control, then stimulated with oleic acid for the indicated time, and LDs were revealed by Oil red, the data showed that shRNA-control has little effects on LDs. C. HepG2 cells transfected with pSuper.GFP-shRNA-Rab40c, and processed for immuno-fluorescence microscopy analysis as mentioned above, the results revealed that shRNA-Rab40c moderately decreased the size of LDs. D. Quantitative analysis of the mean size of LDs from 50 transfected cells, showing depletion of Rab40c significantly decreases the size of LD. *p<0.05. The error bars represent the deviation of three independent experiments. Bar, 20 µm.
The above data demonstrated that Rab40c is likely involved in the biogenesis of LDs, so we examined the expression of Rab40c during adipocyte differentiation to see whether Rab40c is involved in adipocyte differentiation. 3T3-L1 cells were induced to differentiate into adipocytes, and the expression of Rab40c was examined using peptide antibody against Rab40c. The results showed that the level of Rab40c protein increases following with the adipocyte differentiation (
A. 3T3-L1 cells were induced to differentiate into adipocytes for the indicated times, then the cells were lysed and subjected for western-blot to detect β-tubulin, Rab40c, Perilipin, ADRP or TIP47. The results demonstrated that the level of Rab40c increased during adipocyte differentiation. B. 3-T3-L1 cell lysates were incubated with GST, GST-Rab40cWT or GST-Rab40c(SOCS) coupled to GST-Sepharose 4B resin. Proteins retained on the beads were processed for western-blot using antibodies against TIP47, Perilipin or ADRP. The data demonstrated that Rab40c can interact with TIP47 but not ADRP and Perilipin. Mutations of SOCS box in Rab40c disrupted the interaction between Rab40c and TIP47. C. Immuno-fluorescence microscopy revealed that GFP-Rab40c exhibites close apposition with TIP47 upon oleic acids stimulation in HepG2 cells. Bar, 20 µm.
As PAT family proteins play key roles in the biogenesis of lipid droplets, we detected the interaction between Rab40c and ADRP, Perilipin or TIP47 through GST-pulldown assay. Interestingly, we found that Rab40c can interact with TIP47, but not ADRP and Perilipin (
Upon over-expression, Rab40c was mostly observed in the clustered LDs. Over-expression of GFP-Rab40cWT (wild type) significantly resulted in the clustering of oil red labeled LDs in NRK cells (
NRK cells were transfected with GFP-Rab40cWT (wild type), GFP-Rab40cG28N, GFP-Rab40cG28T or GFP-Rab40cQ73L, respectively. Then the cells were labeled with Oil red and processed for confocal microscopy analysis. The results demonstrated that GFP-Rab40cWT and all the mutants can significantly induce the clustering of LDs. Bar, 20 µm.
To characterize the structural sequences responsible for Rab40c in inducing the clustering of LDs, we expressed the N-terminally truncated form GFP-Rab40c (11-281) and C-terminally truncated form GFP-Rab40c (1-243) in NRK cells, and found that both the N-terminal variable region and C-terminal variable region are not essential for inducing the clustering of LDs (
A. NRK cells were transfected with the N-terminal truncated form GFP-Rab40c(11-281), C-terminal truncated form GFP-Rab40c(1-243) or GFP-Rab40c(SOCS) (mutant with mutations LPLP(212-215)AAAA in SOCS box), respectively. The cells were labeled with Oil red and processed for immuo-fulorescence microscopy. The results revealed that disruption of the SOCS box abolished the association of Rab40c with lipid droplets, and its ability to induce the clustering of LDs. B. MCF7 cell lysates expressing GFP-Rab40b or GFP-Rab40c were subjected to pull-down experiments using immobilized GST-Rab40b or GST-Rab40c. Western-blot experiments using GFP antibody demonstrated that GST-Rab40c can efficiently retain GFP-Rab40c, where as GST-Rab40b did not exhibit binding to GFP-Rab40b. C. MCF7 cell lysates expressing GFP-Rab40cWT, GFP-Rab40cQ73L or GFP-Rab40c(SOCS) were subjected to pull-down experiments using GST-Rab40c, showing that GST-Rab40c can bind to both GFP-Rab40c and GFP-Rab40cQ73L, but not GFP-Rab40c(SOCS) mutant. Bar, 20 µm.
Further investigation demonstrated that Rab40c may form homodimer or homooligomer through SOCS box. To test the self-interaction of Rab40c, GST-Rab40c coupled to GST sepharose beads was used to bind GFP-Rab40c in MCF7 cell lysates. As shown in
Proteomics studies revealed that many Rab GTPases are likely associated with LDs, but the members of the associated Rab proteins may vary in different experiments or cell types
Rab40c was shown to play roles in gastrulation of Xenopus embryo and in vesicle transport in oligodendrocytes
The well known factors regulating the biogenesis dynamics of LDs are the PAT family proteins. Perilipin is only expressed in adipocytes and steroidogenic cells, and regulates the formation and lipolysis of LDs
LDs is most likely derived from the ER or its associated membrane
Rab40c may regulate the distribution of LDs, as over-expressing Rab40c caused the clustering of LDs, this effect depends on the intact SOCS box. The SOCS (suppressors of cytokine signaling) box is a structural domain found at the C-terminus of over 70 human proteins. The SOCS box can interact with Cullin−/ElongB/C to form E3 ligase complexes, mediating cytokine receptors for proteasomal degradation