1 Department of Anatomy and Molecular Cell Biology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa, Nagoya 466-8550, Japan
2 Harima Institute at SPring-8, RIKEN, Mikazuki, Sayo, Hyogo 679-5148, Japan
3 Institute for Enzyme Research, The University of Tokushima, Tokushima 770-8503, Japan
Author for correspondence (e-mail: tfujimot{at}med.nagoya-u.ac.jp)
Accepted 17 March 2005
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Summary |
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Key words: Lipid droplet, Mass spectrometry, Rab18, Endoplasmic reticulum, Membrane apposition
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Introduction |
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LDs in non-adipose cells have been shown to harbor adipocyte differentiation-related protein (ADRP) and TIP47, both of which show sequence similarity to perilipin expressed in adipose cells (Londos et al., 1999). However, studies performed over the past several years have revealed that LDs contain a number of other functional molecules including major eicosanoid-forming enzymes (Bozza et al., 1997
), MAP kinase, cytosolic phospholipase A2 (Yu et al., 1998
), caveolins (Fujimoto et al., 2001
; Ostermeyer et al., 2001
; Pol et al., 2001
),
-synuclein (Cole et al., 2002
), Nir2 (Litvak et al., 2002
), NAD(P)H steroid dehydrogenase (Ohashi et al., 2003
), and
-1 receptor (Hayashi and Su, 2003
). In conjunction with the mobility and rapid transport of various lipids in and out of LDs (Frolov et al., 2000
; Prattes et al., 2000
; Pol et al., 2004
), it is becoming clear that LDs are much more dynamic and functionally active organelles than originally thought.
To elucidate the physiological functions of LDs in molecular terms, we attempted to identify molecules involved in LD function by a proteomic approach. After our preliminary report (T.F., Noriko Nakamura, S.O. and Hiroshi Kogo, The 75th Annual Meeting of the Japanese Biochemical Society, Kyoto, Japan, 2002), three groups published the results of proteomic studies of LDs from various cell types (Fujimoto et al., 2004; Liu et al., 2004
; Umlauf et al., 2004
). One of a number of proteins identified by the proteomic analysis is Rab18, which we have investigated and found to be highly concentrated in LDs, and that its expression reduced ADRP from LDs. Furthermore, Rab18 induced close apposition of LDs to an ER-derived membrane. These observations implied that Rab18 plays a crucial role in controlling the relationship between LDs and the ER, which may be important for lipid transport between the two organelles.
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Materials and Methods |
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Anti-Rab18 antibody was raised in rabbits by injecting an antigen peptide (ESENQNKGVKLSH), corresponding to amino acids 177-189 of human Rab18, bound to keyhole limpet hemocyanin, and affinity-purified by an antigen column. We verified that the antibody does not react with Rab1, Rab2, Rab3, Rab5, Rab7, Rab9 or Rab10 by immunofluorescence microscopy and western blotting using cells transfected with the respective Rab cDNA. Antibodies to lysobisphosphatidic acid (LBPA) (Kobayashi et al., 1998), and LC3 (Kabeya et al., 2000
) were kindly provided by Toshihide Kobayashi and Yasuo Uchiyama, respectively. Antibodies: ADRP (Progen, Darra, Australia), Lamp1 and Myc (Developmental Studies Hybridoma Bank, the University of Iowa, USA), EEA1 (Transduction Lab., Lexington, KY, USA), transferrin receptor (Cymbus Biotech., Flanders, NJ, USA), GFP (Molecular Probes, Eugene, OR, USA), and FLAG (Sigma-Aldrich, St Louis, MO, USA). Secondary antibodies conjugated with fluorochromes (Molecular Probes) and colloidal gold (BioCell, Cardiff, UK) were also used.
Expression vectors and siRNA
cDNAs of human Rab1, Rab9 and Rab18 were amplified from HepG2 or human fibroblast total RNA by RT-PCR, checked by sequencing and cloned in pEGFP-C (Clontech, Palo Alto, CA, USA) and pFLAG-C (Sigma-Aldrich) vectors. pEFBOS-Myc-Rab2, pCMV5-FLAG-Rab5, pEGFP-Rab7 and pEGFP-Rab10 were kindly provided by Yoshimi Takai, Toshiaki Katada, Takuya Sasaki and Mitsunori Fukuda, respectively. Small interfering RNA (siRNA) for ADRP knockdown was produced by either in vitro transcription using an siRNA construction kit (Ambion, Austin, TX, USA) or by chemical synthesis by Japan BioService (Saitama, Japan). Both plasmid expression vectors and siRNAs were transfected into cells by Lipofectamine2000 (Invitrogen, San Diego, CA, USA) according to the manufacturer's instruction. Cells were used 24-48 hours after transfection of plasmid vectors, and 48-72 hours after siRNA treatment.
Isolation of LDs by subcellular fractionation
LDs were isolated from HepG2 cells as described (Fujimoto et al., 2001). Briefly, cells were disrupted by nitrogen cavitation at 800 psi for 15 minutes at 4°C. After the nuclei were sedimented, the supernatant, adjusted to 0.54 M sucrose (3 ml), was overlaid with 0.27 M sucrose (3 ml), 0.135 M sucrose (3 ml) in disruption buffer, and buffer without sucrose (3 ml), followed by centrifugation for 60 minutes at 154,000 g in an SW41 rotor (Beckman, Fullerton, CA, USA). The LD fraction recovered at the top of the tube was used for mass analysis. For western blotting, eight fractions (1.5 ml each) were obtained from the top, mixed with 6x sample buffer, and subjected to electrophoresis and electrotransfer.
Proteomic analysis
The LD fraction purified from HepG2 cells was subjected to proteomic analysis as described previously (Kikuchi et al., 2004). Briefly, the sample electrophoresed in SDS-PAGE was stained with Coomassie Brilliant Blue, destained, and subjected to in-gel digestion with trypsin after reduction by dithiothreitol and alkylation by iodoacetamide. The resulting peptides were extracted and subjected to liquid chromatography/mass spectrometry (LC/MS) and data-dependent tandem mass (LC-MS/MS) analyses using a Q-Tof-type hybrid mass spectrometer (Micromass, Manchester, UK) interfaced on-line with a capillary HPLC (Waters-Micromass modular CapLC, Micromass). Peak lists obtained from the MS/MS spectra were used to identify proteins using the Mascot search engine (Matrixscience, London, UK).
Immunofluorescence microscopy
Cells cultured on coverslips were observed by immunofluorescence microscopy as described previously (Fujimoto et al., 2001). For most experiments, cells were fixed with 3% formaldehyde and 0.05-0.1% glutaraldehyde, permeabilized with 0.01% digitonin, and treated with 3% bovine serum albumin (BSA) before immunolabeling. LDs were visualized using BODIPY493/503 (Molecular Probes) in most experiments (Gocze and Freeman, 1994
). When triple labeling was necessary, LDs were stained with Sudan III. Although the procedure for Sudan III staining could cause some morphological changes in the LDs, correlations with antigen localization were preserved (Fukumoto and Fujimoto, 2002
). Images were acquired using a Zeiss PASCAL confocal laser scanning microscope, or a Zeiss Axiophot2 fluorescence microscope equipped with an AxioCam digital camera.
Conventional and immunoelectron microscopy
For conventional electron microscopy, cells on coverslips were fixed with 2.5% glutaraldehyde, post-fixed with 1% osmium tetroxide, stained en bloc with uranyl acetate, and embedded in Epon for thin sectioning. For some specimens, 0.7% potassium ferrocyanide was added to the osmium tetroxide solution to enhance membrane contrast. For immunoelectron microscopy, cells were fixed with 3% formaldehyde for 60 minutes, infiltrated with a mixture of sucrose and polyvinylpyrolidone, and frozen in liquid nitrogen. Ultrathin cryosections were prepared, labeled with antibodies, and embedded in methylcellulose (Liou et al., 1996). The specimens were observed using a JEOL 1200CX electron microscope operated at 100 kV.
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Results |
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A number of Rab proteins were identified by proteomic analysis, i.e. Rab1, Rab2, Rab5, Rab6, Rab7, Rab8, Rab10, Rab11, Rab14, Rab18 and Rab32. To examine the extent to which the Rab proteins exist in LDs, we transfected cells with tagged Rab cDNAs and observed their distribution in comparison with LDs stained with BODIPY493/503 or Sudan III. Six Rabs, Rab1, Rab2, Rab5, Rab7, Rab10 and Rab18, were examined. Among these Rab proteins, only Rab18 showed conspicuous and almost exclusive labeling around LDs, whereas other Rabs were generally distributed in other parts of the cell or were distributed rather diffusely in the cytoplasm, and only a small number were seen around LDs (Fig. 1A). The LD localization of Rab18 was seen when either FLAG or EGFP was used as a tag, and even when EGFP-Rab18 was observed without permeabilization. These results excluded the possibility of artifacts caused by the tag or the labeling procedure.
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We next examined whether the LD localization of Rab18 was dependent on its activation. Based on the highly conserved sequence among Rab proteins, we constructed a GTPase-deficient mutant (Q67L) and a constitutively GDP-bound mutant (S22N) of Rab18, and observed the distribution of EGFP- and FLAG-tagged molecules in HepG2 cells. Rab18(Q67L) showed localization to LDs in the same way as Rab18(WT: wild-type), whereas Rab18(S22N) was seen diffusely in the cytoplasm and did not show any concentration around LDs (Fig. 1D). These observations implied that the localization of Rab18 to LDs is regulated by its guanine nucleotide status.
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Western blotting of subcellular fractions of HepG2 showed that the anti-Rab18 antibody reacted intensely with the top floating fractions, which were highly enriched with LDs, as shown by its reactivity with anti-ADRP (Fig. 2A). When gels were loaded with an equal volume from each fraction, the immunoreactivity for Rab18 was also seen in the bottom fractions containing membrane and soluble proteins. The purity of the LD fraction was confirmed by the absence of other organelle markers; EEA1 for the early endosome, Lamp1 for the late endosome/lysosome, syntaxin 6 for the trans-Golgi network, and calnexin for the ER were only detected in the bottom fractions. In our previous study we also showed that markers for the Golgi apparatus, ER and the plasma membrane were not found in the LD fraction (Fujimoto et al., 2001).
Using immunofluorescence microscopy of HepG2 and 3T3 cells, the anti-Rab18 antibody was shown to be present in a ring around BODIPY493/503-stained LDs (Fig. 2B). Notably, a subpopulation of LDs were not labeled by anti-Rab18 antibody (arrowheads in Fig. 2B). The labeling was abolished by pre-absorption of the anti-Rab18 antibody with the antigen peptide or when it was omitted from the procedure (data not shown). These observations supported the results of LC-MS/MS and immunofluorescence microscopy of tagged Rab18, and demonstrated that endogenous Rab18 was localized to LDs.
A previous study showed that Rab18 was distributed in the endosomal vesicles of MDCK cells (Lutcke et al., 1994). To examine this possibility, double labeling for Rab18 and endosomal markers was performed. For the late endosome, we labeled for LBPA and Lamp1, and for the early endosome, we labeled for EEA1 and transferrin receptor. However, Rab18 did not overlap with any of the endosomal markers (Fig. 2C). Based on the result of immunofluorescence microscopy and western blotting, we concluded that Rab18 was localized to the LDs in the cell types examined. The disparity between the present result and those reported previously (Lutcke et al., 1994
) cannot be explained easily, but it is notable that a similar disparity with regard to the localization of TIP47 has been reported by other groups (Barbero et al., 2001
; Wolins et al., 2001
; Miura et al., 2002
). Proteins that have a propensity to localize to LDs may be affected by subtle differences in experimental conditions, and may be distributed in other locations under some circumstances.
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Effect of Rab18 overexpression on LDs
When triple labeling of endogenous Rab18, ADRP and LDs was performed in HepG2, both Rab18 and ADRP were shown to be associated with BODIPY 493/503-positive LDs as expected. However, the labeling intensity of Rab18 and ADRP showed clear reciprocity in most cases, i.e. in LDs where the Rab18 labeling was intense, ADRP labeling was relatively weak, and vice versa (Fig. 4A). These results suggested that the presence of Rab18 may decrease the amount of ADRP in LDs. Therefore, we examined the consequences of Rab18 overexpression on ADRP. When EGFP-Rab18(WT) was introduced, it was distributed around Sudan III-positive LDs in HepG2 cells, but EGFP-Rab18(WT) and ADRP hardly overlapped (Fig. 4B). The reduction of ADRP expression in the Rab18-overexpressing cell was also detected by western blotting of the total cell lysate. The cells transfected with non-tagged Rab18 cDNA expressed lower levels of ADRP than those transfected with empty vector (Fig. 4C). Similar results were obtained when cells transfected with EGFP-Rab18 cDNA were compared with those transfected with EGFP cDNA (data not shown).
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Ultrastructural analysis of Rab18-overexpressing cells
To confirm that EGFP-Rab18 was localized to the LD surface, ultrathin cryosections of transfected cells were prepared and labeled with anti-GFP. Immunogold labeling was indeed seen along the surface of LDs, although LDs were usually observed as an empty round space because of the difficulty of retaining lipid ester in ultrathin cryosections (Fig. 5A). At the same time, we often noticed thin membrane cisternae in the vicinity of EGFP-Rab18-positive LDs (arrows in Fig. 5A).
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The ER-derived membrane apposed to LDs was not likely to be the autophagic vacuole, because LC3, a marker of early autophagic vacuoles (Kabeya et al., 2000), was not detected around the EGFP-Rab18-positive LDs by immunofluorescence microscopy, and autophagic vacuoles did not appear to be increased in cells expressing EGFP-Rab18 as determined by electron microscopy (data not shown).
Down-regulation of ADRP induces membrane apposition to LDs
The above results demonstrated that overexpression of Rab18 causes a decrease in the level of ADRP in LDs as well as close membrane apposition between LDs and the ER-derived membrane. To examine whether the decrease in ADRP in LDs was the cause of the membrane apposition, we applied two different methods to decrease ADRP and examined whether similar structural changes were induced: one was knockdown of ADRP by RNA interference, and the other was brefeldin A (BFA) treatment.
For RNA interference, cells transfected with siRNA for ADRP knockdown were compared with those transfected with control scrambled siRNA. About 50% reduction of ADRP expression was verified by western blotting (Fig. 5D-1). By electron microscopy, cells treated with ADRP siRNA frequently showed LDs surrounded by the neighboring membrane cisternae (48.1%), which were very similar to the structure in Rab18-overexpressing cells. In contrast, such structures were hardly observed in cells treated with control siRNA (3.7%) (Fig. 5D-2,F).
In a previous study, we observed that the amount of ADRP in LDs was reduced significantly by treating cells with BFA (Nakamura et al., 2004). Electron microscopy of cells treated with BFA for 5 hours showed that LDs were surrounded by thin membrane cisternae in the majority of cells (82.8%). Such a disposition was seldom observed in control untreated cells (6.9%; Fig. 5E,F). Virtually all LDs in BFA-treated cells showed apposition to the ER, whereas in cells transfected with pEGFP-Rab18 cDNA or ADRP siRNA, LDs showing apposition to the ER coexisted with those without such a disposition. This difference can be explained at least partly by the penetration ratio of the procedures: BFA should affect all the cells and LDs with the same strength, but the ratio of transfected cells was about 60-70% with variable intensity as estimated by fluorescence microscopy.
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Discussion |
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Mechanism by which Rab18 causes elimination of ADRP from LD
When cells were transfected with Rab18 cDNA, LDs harboring exogenous Rab18 showed weaker ADRP labeling than the others. This observation can be explained if expression of Rab18 induces de novo formation of LDs containing little ADRP, but the following results indicated that Rab18 was recruited to pre-existing LDs and replaced ADRP. First, overexpression of Rab18 did not increase the number or the total volume of LDs observed by fluorescence microscopy (data not shown); second, even when HepG2 cells were cultured in 2% LPDS to minimize de novo LD formation, recruitment of Rab18 to remaining LDs occurred to a similar extent.
These observations raise the question of how Rab18 decreases ADRP in LDs. Interestingly, ADRP is also eliminated from LDs by expression of perilipin (Brasaemle et al., 1997) and hepatitis C virus (HCV) core protein (data cited in McLauchlan, 2000
). We also observed that the N-terminal truncation mutant of caveolin-3 that is distributed to LDs (Pol et al., 2001
) also displaced ADRP (K.T.-S. and T.F., unpublished). However, it is unlikely that these proteins compete with ADRP for common specific binding sites in LDs for several reasons. First, in the case of perilipin, despite some similarity to ADRP in the N-terminal PAT-1 domain (Londos et al., 1999
), binding to LDs is not mediated by the domain, but by the adjacent short hydrophobic segments (Garcia et al., 2003
; Garcia et al., 2004
). Second, targeting of HCV core protein and caveolins to LDs requires a long hydrophobic domain (Hope and McLauchlan, 2000; Fujimoto et al., 2001
; Ostermeyer et al., 2004
), which is not found in ADRP or perilipin. Third, Rab18 does not appear to have any structural similarity to ADRP, perilipin, or HCV core protein. These properties suggest that any proteins recruited to the LDs in large amounts could replace pre-existing ADRP sterically. Alternatively, the above proteins could recruit a common protein(s), which competes with ADRP in adherence to LDs. In the case of Rab18, effector proteins may also be involved.
Mechanism by which reduction of ADRP induces LD-ER apposition
We showed that reduction of ADRP in LDs causes close apposition of LDs and the ER-derived membrane. The detailed function of ADRP in LDs has not been determined, but its overexpression was shown to stimulate LD formation (Imamura et al., 2002; Nakamura and Fujimoto, 2003
) and its reduction was reported to result in a decrease in LDs detectable by fluorescence labeling (Nakamura et al., 2004
). These results suggested that ADRP is important for the maintenance of the LD structure, and that its reduction may compromise the stability of LDs.
Thus, the elimination of ADRP from LDs may be the only function of Rab18, i.e. reduction of ADRP may induce exactly the same results as Rab18 activation. However, it is also possible that Rab18 may activate specific effectors, which then exert downstream functions. For example, Rab18 itself or Rab18 effectors may bind to particular proteins in the ER membrane, and cause the apposition of LDs to a specific sub-compartment of the ER, whereas simple reduction of ADRP may induce nonspecific apposition to bulk ER. This kind of specificity may be important because the LD-ER apposition may be related to lipid transport as discussed in the following section.
Apposition of ER and other organelles
In hepatocytes, LDs are believed to form in an ER sub-compartment enriched with lipid ester-synthesizing enzymes; LDs are likely to be detached from the ER, and then to dock to another ER sub-compartment where lipid esters in the LDs are utilized to generate very low-density lipoproteins (VLDL) (Gibbons et al., 2000; Murphy, 2001) [for a different possible mechanism of LD formation in other cell types, see Robenek et al. (Robenek et al., 2004
)]. Thus, LDs and the ER could exist in proximity in two situations: LD formation and LD docking. If the Rab18-induced LD-ER apposition is involved in LD formation, recruitment of Rab18 should be observed concomitantly with lipid esterification. However, upon addition of oleic acid, Rab18 was detectable only at much later times than LDs or ADRP (Fig. 3). This observation suggests that the LD-ER apposition induced by Rab18 may not be related to the LD formation process.
ER has been shown to appose to other organelles (Voelker, 2003; Levine, 2004
). The specialized ER region in contact with mitochondria is referred to as the mitochondria-associated membrane, or MAM (Pickett et al., 1980
; Rusinol et al., 1994
). MAM is enriched with phosphatidylserine synthase (Vance, 1990
), and is thought to be involved in transport of phosphatidylserine from the ER to the mitochondria (Voelker, 2003
). More recently, close contact with the plasma membrane was reported in yeast, and the apposed ER region is called the plasma membrane-associated membrane, or PAM (Pichler et al., 2001
). The apposition between the ER and LDs is observed frequently in various steroidogenic cells, and has been suggested to be involved in mobilization of stored cholesterol esters for steroid synthesis (Rhodin, 1974
; Fawcett, 1981
). The present result is consistent with this hypothesis. In accordance with the nomenclature of MAM and PAM, we propose that the ER region apposed to LDs should be called the lipid droplet-associated membrane, or LAM.
Although the mechanism of phosphatidylserine transport between MAM and mitochondria is beginning to be elucidated, it is not yet known how the apposition between the ER and other organelles is formed (Voelker, 2003). The present study showed, for the first time, that a Rab protein is involved in apposition of the ER membrane. It would be of interest to determine whether other GTP-binding proteins are also involved in the formation of MAM, PAM and other membrane appositions. A number of questions remain to be answered regarding the apposition: e.g. how stable is it?; how is it regulated?; what kind of proteins and lipids are involved?, etc. Elucidation of the molecular mechanism responsible for the apposition would also lead to an understanding of its function.
Functional heterogeneity of LD
The present results showed that LDs in a cell could have ADRP and Rab18 in variable ratios, which probably reflects the diverse functional states of the LDs. As speculated above, ADRP is likely to stabilize LDs, and its reduction may lead to mobilization of the lipid content. We suppose that Rab18 may offer a physiological mechanism to regulate the amount of ADRP. ARF1 may also be involved in regulation by dissociation of ADRP from LDs (Nakamura et al., 2004). The conjecture that the amount of ADRP is correlated with LD function was supported by the observation that ER-linked LDs lacked ADRP, while independent LDs contained it (Hayashi and Su, 2003
).
It is becoming clear that the LD is not a static organelle involved only in storing excessive lipids. The contents of LDs and their relationship to other organelles are regulated by intricate mechanisms, and LDs in different functional states must coexist in the cell. The physiological significance of LDs, especially in relation to intracellular lipid homeostasis, should be clarified through further studies of their regulatory mechanisms.
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Acknowledgments |
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Footnotes |
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References |
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Barbero, P., Buell, E., Zulley, S. and Pfeffer, S. R. (2001). TIP47 is not a component of lipid droplets. J. Biol. Chem. 276, 24348-24351.
Bozza, P. T., Yu, W., Penrose, J. F., Morgan, E. S., Dvorak, A. M. and Weller, P. F. (1997). Eosinophil lipid bodies: specific, inducible intracellular sites for enhanced eicosanoid formation. J. Exp. Med. 186, 909-920.
Brasaemle, D. L., Barber, T., Wolins, N. E., Serrero, G., Blanchette-Mackie, E. J. and Londos, C. (1997). Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein. J. Lipid Res. 38, 2249-2263.[Abstract]
Cole, N. B., Murphy, D. D., Grider, T., Rueter, S., Brasaemle, D. and Nussbaum, R. L. (2002). Lipid droplet binding and oligomerization properties of the Parkinson's disease protein alpha-synuclein. J. Biol. Chem. 277, 6344-6352.
Fawcett, D. W. (1981). The Cell. Philadelphia, USA: W. B. Saunders.
Frolov, A., Petrescu, A., Atshaves, B. P., So, P. T., Gratton, E., Serrero, G. and Schroeder, F. (2000). High density lipoprotein-mediated cholesterol uptake and targeting to lipid droplets in intact L-cell fibroblasts. A single- and multiphoton fluorescence approach. J. Biol. Chem. 275, 12769-12780.
Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K. and Nomura, R. (2001). Caveolin-2 is targeted to lipid droplets, a new "membrane domain" in the cell. J. Cell Biol. 152, 1079-1085.
Fujimoto, Y., Itabe, H., Sakai, J., Makita, M., Noda, J., Mori, M., Higashi, Y., Kojima, S. and Takano, T. (2004). Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7. Biochim. Biophys. Acta 1644, 47-59.[CrossRef][Medline]
Fukumoto, S. and Fujimoto, T. (2002). Deformation of lipid droplets in fixed samples. Histochem. Cell Biol. 118, 423-428.[CrossRef][Medline]
Garcia, A., Sekowski, A., Subramanian, V. and Brasaemle, D. L. (2003). The central domain is required to target and anchor perilipin A to lipid droplets. J. Biol. Chem. 278, 625-635.
Garcia, A., Subramanian, V., Sekowski, A., Bhattacharyya, S., Love, M. W. and Brasaemle, D. L. (2004). The amino and carboxyl termini of perilipin a facilitate the storage of triacylglycerols. J. Biol. Chem. 279, 8409-8416.
Gibbons, G. F., Khurana, R., Odwell, A. and Seelaender, M. C. (1994). Lipid balance in HepG2 cells: active synthesis and impaired mobilization. J. Lipid Res. 35, 1801-1808.[Abstract]
Gocze, P. M. and Freeman, D. A. (1994). Factors underlying the variability of lipid droplet fluorescence in MA-10 Leydig tumor cells. Cytometry 17, 151-158.[Medline]
Hayashi, T. and Su, T. P. (2003). Sigma-1 receptors (sigma(1) binding sites) form raft-like microdomains and target lipid droplets on the endoplasmic reticulum: roles in endoplasmic reticulum lipid compartmentalization and export. J. Pharmacol. Exp. Ther. 306, 718-725.
Imamura, M., Inoguchi, T., Ikuyama, S., Taniguchi, S., Kobayashi, K., Nakashima, N. and Nawata, H. (2002). ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts. Am. J. Physiol. Endocrinol. Metab. 283, E775-E783.
Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y. and Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720-5728.
Kikuchi, M., Hatano, N., Yokota, S., Shimozawa, N., Imanaka, T. and Taniguchi, H. (2004). Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease. J. Biol. Chem. 279, 421-428.
Kobayashi, T., Stang, E., Fang, K. S., de Moerloose, P., Parton, R. G. and Gruenberg, J. (1998). A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 392, 193-197.[CrossRef][Medline]
Levine, T. (2004). Short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions. Trends Cell Biol. 14, 483-490.[CrossRef][Medline]
Liou, W., Geuze, H. J. and Slot, J. W. (1996). Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell Biol. 106, 41-58.[CrossRef][Medline]
Litvak, V., Shaul, Y. D., Shulewitz, M., Amarilio, R., Carmon, S. and Lev, S. (2002). Targeting of Nir2 to lipid droplets is regulated by a specific threonine residue within its PI-transfer domain. Curr. Biol. 12, 1513-1518.[CrossRef][Medline]
Liu, P., Ying, Y., Zhao, Y., Mundy, D. I., Zhu, M. and Anderson, R. G. (2004). Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 279, 3787-3792.
Londos, C., Brasaemle, D. L., Schultz, C. J., Segrest, J. P. and Kimmel, A. R. (1999). Perilipins, ADRP, and other proteins that associate with intracellular neutral lipid droplets in animal cells. Semin. Cell Dev. Biol. 10, 51-58.[CrossRef][Medline]
Lutcke, A., Parton, R. G., Murphy, C., Olkkonen, V. M., Dupree, P., Valencia, A., Simons, K. and Zerial, M. (1994). Cloning and subcellular localization of novel rab proteins reveals polarized and cell type-specific expression. J. Cell Sci. 107, 3437-3448.
McLauchlan, J. (2000). Properties of the hepatitis C virus core protein: a structural protein that modulates cellular processes. J. Viral Hepat. 7, 2-14.[CrossRef][Medline]
Miura, S., Gan, J. W., Brzostowski, J., Parisi, M. J., Schultz, C. J., Londos, C., Oliver, B. and Kimmel, A. R. (2002). Functional conservation for lipid storage droplet association among Perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium. J. Biol. Chem. 277, 32253-32257.
Murphy, D. J. (2001). The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog. Lipid Res. 40, 325-438.[CrossRef][Medline]
Murphy, D. J. and Vance, J. (1999). Mechanisms of lipid-body formation. Trends Biochem. Sci. 24, 109-115.[CrossRef][Medline]
Nakamura, N. and Fujimoto, T. (2003). Adipose differentiation-related protein has two independent domains for targeting to lipid droplets. Biochem. Biophys. Res. Commun. 306, 333-338.[CrossRef][Medline]
Nakamura, N., Akashi, T., Taneda, T., Kogo, H., Kikuchi, A. and Fujimoto, T. (2004). ADRP Is dissociated from lipid droplets by ARF1-dependent mechanism. Biochem. Biophys. Res. Commun. 322, 957-965.[CrossRef][Medline]
Ohashi, M., Mizushima, N., Kabeya, Y. and Yoshimori, T. (2003). Localization of mammalian NAD(P)H steroid dehydrogenase-like protein on lipid droplets. J. Biol. Chem. 278, 36819-36829.
Ostermeyer, A. G., Paci, J. M., Zeng, Y., Lublin, D. M., Munro, S. and Brown, D. A. (2001). Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage droplets. J. Cell Biol. 152, 1071-1078.
Ostermeyer, A. G., Ramcharan, L. T., Zeng, Y., Lublin, D. M. and Brown, D. A. (2004). Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets. J. Cell Biol. 164, 69-78.
Pichler, H., Gaigg, B., Hrastnik, C., Achleitner, G., Kohlwein, S. D., Zellnig, G., Perktold, A. and Daum, G. (2001). A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids. Eur. J. Biochem. 268, 2351-2361.
Pickett, C. B., Montisano, D., Eisner, D. and Cascarano, J. (1980). The physical association between rat liver mitochondria and rough endoplasmic reticulum. I. Isolation, electron microscopic examination and sedimentation equilibrium centrifugation analyses of rough endoplasmic reticulum-mitochondrial complexes. Exp. Cell Res. 128, 343-352.[CrossRef][Medline]
Pol, A., Luetterforst, R., Lindsay, M., Heino, S., Ikonen, E. and Parton, R. G. (2001). A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance. J. Cell Biol. 152, 1057-1070.
Pol, A., Martin, S., Fernandez, M. A., Ferguson, C., Carozzi, A., Luetterforst, R., Enrich, C. and Parton, R. G. (2004). Dynamic and regulated association of caveolin with lipid bodies: modulation of lipid body motility and function by a dominant negative mutant. Mol. Biol. Cell 15, 99-110.
Prattes, S., Horl, G., Hammer, A., Blaschitz, A., Graier, W. F., Sattler, W., Zechner, R. and Steyrer, E. (2000). Intracellular distribution and mobilization of unesterified cholesterol in adipocytes: triglyceride droplets are surrounded by cholesterol-rich ER-like surface layer structures. J. Cell Sci. 113, 2977-2989.
Rhodin, J. A. G. (1974). Histology, A Text and Atlas. New York: Oxford University Press.
Robenek, M. J., Severs, N. J., Schlattmann, K., Plenz, G., Zimmer, K. P., Troyer, D. and Robenek, H. (2004). Lipids partition caveolin-1 from ER membranes into lipid droplets: updating the model of lipid droplet biogenesis. FASEB J. 18, 866-868.
Rusinol, A. E., Cui, Z., Chen, M. H. and Vance, J. E. (1994). A unique mitochondria-associated membrane fraction from rat liver has a high capacity for lipid synthesis and contains pre-Golgi secretory proteins including nascent lipoproteins. J. Biol. Chem. 269, 27494-27502.
Tauchi-Sato, K., Ozeki, S., Houjou, T., Taguchi, R. and Fujimoto, T. (2002). The surface of lipid droplets is a phospholipid monolayer with a unique Fatty Acid composition. J. Biol. Chem. 277, 44507-44512.
Umlauf, E., Csaszar, E., Moertelmaier, M., Schuetz, G. J., Parton, R. G. and Prohaska, R. (2004). Association of stomatin with lipid bodies. J. Biol. Chem. 279, 23699-23709.
Vance, J. E. (1990). Phospholipid synthesis in a membrane fraction associated with mitochondria. J. Biol. Chem. 265, 7248-7256.
Voelker, D. R. (2003). New perspectives on the regulation of intermembrane glycerophospholipid traffic. J. Lipid Res. 44, 441-449.
Wolins, N. E., Rubin, B. and Brasaemle, D. L. (2001). TIP47 associates with lipid droplets. J. Biol. Chem. 276, 5101-5108.
Yu, W., Bozza, P. T., Tzizik, D. M., Gray, J. P., Cassara, J., Dvorak, A. M. and Weller, P. F. (1998). Co-compartmentalization of MAP kinases and cytosolic phospholipase A2 at cytoplasmic arachidonate-rich lipid bodies. Am. J. Pathol. 152, 759-769.[Abstract]