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J. Biol. Chem., Vol. 280, Issue 28, 26330-26338, July 15, 2005
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Lipid Droplets Gain PAT Family Proteins by Interaction with Specialized Plasma Membrane Domains*
From the
Leibniz-Institute for Arteriosclerosis Research, University of Münster, Münster, Germany and the ¶National Heart and Lung Institute, Imperial College, London SW3 6LY, United Kingdom
Received for publication, November 26, 2004 , and in revised form, April 27, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Proteins of the PAT family, named after perilipin, adipophilin, and TIP47 (tail-interacting protein of 47 kDa), are associated with lipid droplets and have previously been localized by immunofluorescence microscopy exclusively to the droplet surface. These proteins are considered not to be present in any other subcellular compartment. By applying the high resolution technique of freeze-fracture electron microscopy combined with immunogold labeling, we now demonstrate that in macrophages and adipocytes PAT family proteins are, first, distributed not only in the surface but also throughout the lipid droplet core and, second, are integral components of the plasma membrane. Under normal culture conditions these proteins are dispersed in the cytoplasmic leaflet of the plasma membrane. Stimulation of lipid droplet formation by incubation of the cells with acetylated low density lipoprotein leads to clustering of the PAT family proteins in raised plasma membrane domains. Fractures penetrating beneath the plasma membrane demonstrate that lipid droplets are closely apposed to these domains. A similar distribution pattern of labeling in the form of linear aggregates within the clusters is apparent in the cytoplasmic monolayer of the plasma membrane and the immediately adjacent outer monolayer of the lipid droplet. The aggregation of the PAT family proteins into such assemblies may facilitate carrier-mediated lipid influx from the extracellular environment into the lipid droplet. Lipid droplets appear to acquire their PAT proteins by interaction with plasma membrane domains enriched in these proteins.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Recent research has led to a new awareness that the lipid droplet is not merely a lipid storage body but a multi-functional organelle involved in lipid homeostasis, cell signaling, and intracellular vesicle trafficking, with potentially important roles in diseases including atherogenesis, diabetes, and obesity (1–6). This realization has made the study of proteins such as caveolin-1 and the PAT 1 family proteins (perilipin, adipophilin, and TIP47), which are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry (7–9). Despite this progress, however, virtually nothing is known about the mechanisms by which such proteins are incorporated into lipid droplets or, indeed, how lipid droplets themselves are actually assembled.
Current information on the localization of PAT family proteins is inconsistent. For example, although one early report described translocation of adipophilin to the plasma membrane in COS-7 cells during differentiation (10), more recent work has failed to confirm this finding (11). The general consensus, however, is that PAT family proteins occur exclusively in lipid droplets and not in any other subcellular compartment. Although some proteins such as cyclooxygenase (12), caveolin-2 (13), caveolin-1 (14, 15), and flotillins (16) have been localized throughout the lipid droplet core, the PAT family proteins are believed to be confined to the surface monolayer of the droplet and excluded from the core.
One factor limiting our current knowledge of PAT family proteins arises because previous findings on the distribution of these proteins have relied exclusively on immunofluorescence light microscopy (7, 17–20). To shed new light on the distribution and possible intracellular transit routes of these proteins, we have now applied freeze-fracture electron microscopy in combination with immunogold labeling (14, 15, 21), an approach that permits high resolution localization of proteins in planar views of membranes and lipid bodies. Immunofluorescence confocal microscopy, standard thin section electron microscopy, and immunogold cryo-electron microscopy were conducted in parallel to provide a comprehensive correlative morphological insight into the events associated with lipid droplet accumulation and the involvement of PAT family proteins.
To date, a key focus on the role of PAT family proteins has centered on the adipocyte. During differentiation of the adipocyte, adipophilin, which is initially expressed, is replaced by perilipin (17). Adipophilin is, however, expressed by differentiated macrophages, which also express the third protein of the PAT family, TIP47 (22). Moreover, macrophages can be readily stimulated to accumulate lipid in a similar manner to that of adipocytes. Thus, in seeking to determine whether there are common principles underlying the localization, dynamic reorganization, and potential roles for all three members of the PAT family, the present study examined both cell types.
Our findings show that PAT family proteins are not restricted to the lipid droplet surface as previously maintained but also clearly pervade the droplet core. We found that perilipin and caveolin-1 co-localize in lipid droplet cores of adipocytes and that adipophilin and TIP47 co-localize in lipid droplet cores of THP-1 cell-derived macrophages. We further demonstrate unequivocally that the PAT family proteins, in common with caveolin-1, are integral components of the plasma membrane. Under normal culture conditions, the PAT family proteins show a dispersed distribution on the cytoplasmic half of the plasma membrane. Incubation of macrophages and adipocytes with acetylated low density lipoprotein (AcLDL) stimulates their PAT family proteins, but not caveolin-1, to cluster on raised domains of the plasma membrane. The elevated domains are demonstrated to mark sites at which lipid droplets are closely pressed against the cytoplasmic surface of the plasma membrane. These findings suggest that the PAT family proteins are associated with lipid accumulation, functioning in a carrier-mediated fatty acid influx from the extracellular environment into the cell. Such findings further raise the possibility that the transit of PAT family proteins with their fatty acid cargo from specialized plasma membrane domains into lipid droplets functions as part of this influx mechanism.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Antibodies—A polyclonal antibody raised in guinea pigs against a synthetic polypeptide representing the N terminus (amino acids 1–16) of human TIP47 (GP30; Progen Biotechnik, Heidelberg, Germany) was used to detect TIP47. Adipophilin was immunolabeled using a mouse monoclonal antibody to a synthetic peptide representing the N terminus (amino acids 5–27) of human adipophilin (AP125; Progen Biotechnik). A polyclonal antibody raised in rabbit against a synthetic peptide corresponding to the N-terminal region of human perilipin A and B was used to detect perilipin (Dianova, Hamburg, Germany). Caveolin-1 was immunolabeled using a mouse monoclonal anti-caveolin-1 antibody (clone 2297; BD Transduction Laboratories). Irrelevant antibodies against Lamp-1, connexin43, and the inner nuclear membrane proteins LAP2
and emerin were used as controls. Cell Culture—Human THP-1 monocytes from the American Type Culture Collection (Manassas, VA) were cultured in suspension in RPMI 1640 medium containing the supplements recommended by Iwashima et al. (23), and differentiated to adherent macrophages by adding 100 µM phorbol 12-myristate 13-acetate to the medium for 3 days. Because normally cultured macrophages contain few lipid droplets, the cells were induced to accumulate lipid droplets before use by the addition of 50 µg/ml AcLDL at day 2 for 0, 6, 12, and 24 h as described by Hara et al. (24) and Gaus et al. (25).
3T3-L1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine fetal calf serum, 2 mM glutamine, 100 µg/ml penicillin, and 100 µg/ml streptomycin. Differentiation of 3T3-L1 cells to adipocytes was accomplished by incubating confluent monolayers of cells in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 0.5 mM isobutylmethylxanthine, 10 µg/ml insulin, and 10 µM dexamethasone with fresh medium changes every 24 h, followed by Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum with medium changes every 24 h for an additional 72 h (19). Both cell types were maintained at 37 °C in a 5% CO2 atmosphere. To induce lipid droplet formation in adipocytes, the cells were incubated in 50 µg/ml AcLDL as described for monocytes.
Immunofluorescence Light and Confocal Microscopy—Cultured THP-1 monocytes were differentiated to macrophages and lipid-loaded in chamber slides. They were rinsed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS at room temperature for 30 min. After extensive washing, the monocytes were incubated for 1 h in PBS containing 1% bovine serum albumin to block nonspecific binding and 0.05% Tween 20 for permeabilization. Macrophages were immunolabeled with anti-TIP47 and anti-adipophilin antibodies for 1 h, followed by washing and incubation with anti-guinea pig Cy3-conjugated and anti-mouse Cy2-conjugated secondary antibodies (Dianova) for 1 h. To visualize lipid droplets, the fluorophore BODIPY 493/503 (Invitrogen), which specifically stains neutral lipids (26), was dissolved in ethanol at 1 mg/ml and added to the secondary antibody solutions to a final concentration of 20 µg/ml. Nuclei were stained with Hoechst 33258 dye (Sigma Aldrich). The preparations were mounted in fluorescent mounting medium (DakoCytomation, Hamburg, Germany) and examined in a fluorescence microscope or confocal laser-scanning microscope (Zeiss, Jena, Germany).
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Thin Section Electron Microscopy—Lipid-laden macrophages and adipocytes were grown in flasks, fixed with 2% glutaraldehyde and 0.5% osmium tetroxide in PBS, and dehydrated with ethanol using standard procedures. A few milliliters of propylene oxide were added to the flasks, and the cells were removed by gently swirling and embedded in Epon using standard procedures. Thin sections were cut using an ultramicrotome and contrasted with uranyl acetate and lead citrate.
Ultrathin Cryosection Electron Microscopy—Lipid-laden macrophages and adipocytes were fixed in 1% paraformaldehyde in PBS, scraped from the culture vessels, prepared further for cryoimmunoelectron microscopy basically as outlined by Tokuyasu (27), and immunogold-labeled for PAT family proteins as described below.
Freeze-Fracture Replication—Cells were scraped from the culture vessels, centrifuged to remove excess medium, re-centrifuged briefly in 30% glycerol (<2 min), fixed in Freon 22 cooled with liquid nitrogen, and freeze-fractured in a BA310 freeze-fracture unit (Balzers AG, Balzers, Liechtenstein) at –100 °C. Replicas of the freshly fractured cells were immediately made by electron beam evaporation of platinum-carbon and carbon at angles of 38 and 90° and to thicknesses of 2 and 20 nm, respectively. The replicas were incubated overnight in 5% sodium dodecyl sulfate (21) to remove cellular material except for those molecules adhering directly to the replicas. They were then washed in distilled water and incubated briefly in 5% bovine serum albumin before immunolabeling.
Immunolabeling of Freeze-Fracture Replicas and Cryosections— Freeze-fracture replicas were immunogold-labeled with the following: 1) anti-TIP47 antibodies followed by a donkey anti-guinea pig 12-nm gold conjugate; 2) anti-adipophilin or anti-caveolin-1 antibodies followed by a goat anti-mouse 12- or 18-nm gold conjugate; and 3) anti-perilipin antibodies followed by a goat anti-rabbit 12- or 18-nm gold conjugate. Double immunogold labeling of replicas of adipocytes was carried out using a mixture of anti-perilipin and anti-caveolin-1 antibodies followed by a mixture of goat anti-rabbit 12-nm and goat antimouse 18-nm gold conjugates (both conjugates from Jackson Immuno Research, West Grove, VA). Double labeling of replicas of macrophages was carried out by a mixture of anti-TIP47 and anti-adipophilin antibodies followed by a mixture of donkey anti-guinea pig 12-nm and goat anti-mouse 18-nm gold conjugates.
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Ultrathin cryosections of macrophages were immunolabeled with anti-adipophilin followed by a goat anti-mouse 12-nm gold conjugate. Ultrathin cryosections of adipocytes were immunolabeled with anti-perilipin followed by a goat anti-rabbit 18-nm gold conjugate. Control specimens, prepared without the primary antibodies, were essentially free of gold particles. Antibodies against Lamp-1, connexin43, LAP2
, and emerin did not bind to replicas or cryosections of lipid droplets in adipocytes and macrophages. Electron Microscopy—Examination of thin sections, immunogold-labeled ultrathin cryosections, and immunogold-labeled freeze-fracture replicas was carried out using a Philips 410 transmission electron microscope. Observations on freeze-fracture/immunogold replicas were based on examination of >200 cells for each time point from three separate experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Lipid Droplet Accumulation—Normally cultured macrophages and adipocytes contain few lipid droplets. To induce lipid droplet formation, the cells were exposed to 50 µg/ml AcLDL for 6–24 h. Immunofluorescence confocal light microscopy of neutral lipids (BODIPY staining), adipophilin, and TIP47 demonstrated a time-dependent accumulation of lipid droplets and associated PAT family proteins in macrophages (Fig. 1). Conventional thin section electron microscopy confirmed progressive lipid droplet accumulation over the same time course in both macrophages (Fig. 2) and adipocytes (Fig. 3). Solitary lipid droplets and clusters of lipid droplets are apparent, some of which contain sheet-like structures. Some lipid droplets are seen lying adjacent to the plasma membrane; these may have a flattened shape at their sites of apposition, or the plasma membrane may appear elevated (Fig. 3A), thus increasing the area of association between the two. Cryosections viewed by electron microscopy reveal the lipid droplets as essentially electron-lucent vesicles with little internal structure in both macrophages (Fig. 4) and adipocytes (Fig. 5). Labeling for PAT family proteins in cryosections is predominantly seen at the surfaces of the droplets, as illustrated in Fig. 4 for adipophilin in macrophages and in Fig. 5 for perilipin in adipocytes.
Freeze Fracturing and Immunogold Labeling—Relating the freeze-fracture image data to the above findings requires a brief explanation of the principles of the technique. When frozen cells are fractured, the fracture preferentially splits membranes into their two constituent half-membrane leaflets along a plane between the hydrophobic tails of the phospholipids in the bilayer. In the case of the plasma membrane, one leaflet remains attached to the extracellular space (E-half), whereas the other leaflet remains attached to the cytoplasm or protoplasm (P-half). The view of the E-half revealed by freeze fracturing is termed the E-face, and that of the P-half is termed the P-face (28). Endocytotic vesicles arise by invagination of the plasma membrane, and the fracture faces of their inner (luminal) leaflet and those of all lysosomes and secretory granules are E-faces, homologous to the E-face of the plasma membrane. Similarly, the E-face of the Golgi cisternae, the endoplasmic reticulum (ER), and the nuclear envelope is the fracture face of the luminal leaflet that encloses the endoplasmic compartment. The fracture faces of the cytoplasmic leaflets of these intracellular membranes systems are, correspondingly, P-faces, equivalent to the P-face of the plasma membrane.
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The sodium dodecyl sulfate treatment applied to freeze-fracture replicas is designed to flush away cellular residues while retaining, for immunogold labeling, only those molecules adhering directly to the metal of the replicas. Labeling was carried out using primary antibodies against PAT family proteins and caveolin-1 with appropriate secondary antibodies coupled to colloidal gold. Viewed in the electron microscope, the electron-dense gold particles clearly mark the positions of these proteins, superimposed upon the en face membrane views and other ultrastructural features of the cell in replicated view.
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Whereas thin-sectioned and cryosectioned lipid droplets appear generally devoid of internal structure (Figs. 2, 3, 4), freeze-fracturing reveals multiple internal lamellae and amorphous appearing areas in the droplet cores (Figs. 6 and 13). Standard freeze-fracture nomenclature cannot readily be applied to lipid droplets, as these organelles do not have a limiting membrane enclosing a lumen. Thus, rather than P- or E-face designations, the views are more accurately described by their location within the lipid droplet and by the type of fracture through the droplet (i.e. convex, concave, or cross-fracture). However, because the outermost monolayer of the lipid droplet is thought to be derived from the cytoplasmic leaflet of the ER membrane, it is appropriate to refer to the fracture face seen when the outermost layer of the droplet is concavely fractured as the P-face-equivalent and to refer to its complementary view, seen in convex fractures, as the E-face equivalent.
Distribution of Perilipin and Caveolin-1 in Adipocytes— Freeze-fracture replicas of lipid droplets in adipocytes reveal a variety of appearances. Lipid droplets that are cross-fractured reveal a simple homogeneous content or multiple internal fracture faces. Concavely fractured droplets show a single surface layer representing the fracture face of the outermost monolayer of the lipid droplet (P-face equivalent). Droplets that are fractured in multiple layers exhibit an onion-like morphology (Fig. 6A). By double immunogold labeling, using gold markers of distinct sizes to simultaneously identify two different proteins in freeze-fracture views of lipid droplets, caveolin-1, and perilipin are seen to be present not only in the outermost monolayer (Fig. 6B) but also in several other fracture planes (Fig. 6A). Cross-fractured lipid droplets further reveal that labeling for caveolin-1 and perilipin occurs deep within and throughout the core of the lipid droplet (Fig. 6A). Mitochondria, ER, nuclear membranes, the Golgi, and the cytoplasmic matrix are free of labels. Controls in which the primary antibody is omitted show negligible labeling. No immunogold labeling on lipid droplets is found when antibodies against Lamp-1, connexin43, LAP2, and emerin are used.
Apart from the lipid droplets, abundant immunolabeling for caveolin-1 and perilipin is apparent in the plasma membrane. The perilipin label is found exclusively on the P-face of the plasma membrane; no label is seen on the E-face. Under normal culture conditions (i.e. without AcLDL in the culture medium), the perilipin label is widely dispersed across the plane of the membrane (Fig. 7A). During lipid loading of the adipocytes with AcLDL, however, a remarkable alteration in distribution takes place; the perilipin labeling becomes clustered in raised plasma membrane domains. Evidence for the start of this change is apparent at 6 h of AcLDL incubation (Fig. 7B), but the clusters become progressively larger, more abundant, and with a more densely packed label at 12 and 24 h. Over this period, the raised domains become more sharply delineated, with dimensions slightly larger than lipid droplets by 24 h (Fig. 7C). Correlative thin section electron microscopy and fluorescence light microscopy disclose a concomitant progressive increase in lipid droplet formation. Although considerable variation in the extent of cluster development is apparent in individual cells at any given time point because the cells are not synchronized, the trend toward larger, more closely aggregated, and more numerous clusters over time is clear from an examination of multiple samples.
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As with perilipin, the caveolin-1 label is also found on the P-face of the plasma membrane but not the E-face. Generally, caveolae are strongly marked on their rims, but caveolin-1 labeling occurs frequently in intervening plasma membrane regions. Double labeling of perilipin and caveolin-1 demonstrates that, under normal culture conditions, caveolin-1 retains this uniform distribution (Fig. 8A). However, after lipid loading the distribution of caveolin-1, in contrast to that of perilipin, remains virtually unchanged; caveolin-1 thus does not participate in the clustering process seen with perilipin (Fig. 8B).
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Favorable fractures permit simultaneous viewing of portions of the elevated plasma membrane domains that contain the perilipin clusters together with the immediately underlying cytoplasmic structures. Such fractures reveal lipid droplets that are evidently so closely apposed to the plasma membrane in these domains that they cause the plasma membrane to bulge (Fig. 9), corresponding to views seen by thin sectioning (Fig. 3A).
A conspicuous feature of the perilipin label on the P-face of the plasma membrane is its highly organized distribution pattern. Within each cluster of label are mini-clusters, often in the form of linear and curvilinear arrays, suggesting organization of the proteins into aggregates (Fig. 10A). Strikingly similar patterns are apparent on the outermost monolayer (P-face equivalent) of the lipid droplets (Fig. 10B). The resemblance of these distribution patterns in the immediately adjacent plasma membrane and lipid droplet monolayers, together with the intimate physical interaction between the lipid droplet and plasma membrane, suggests a close mechanistic and functional relationship between the two structures. Where labeling is seen in deeper layers of the droplet (e.g. the E-face equivalent complementary to the outer monolayer's P-face), the resemblance in distribution to the plasma membrane is less marked.
Distribution of Adipophilin and TIP47 in Macrophages—The PAT family proteins adipophilin and TIP47 are also components of the plasma membrane of macrophages. Labeling of adipophilin and TIP47 reveals that both proteins are exclusively localized and evenly distributed on the P-face of the plasma membrane, the E-face being completely devoid of labeling just as with perilipin in adipocytes. Lipid loading with AcLDL for 6, 12, and 24 h stimulates adipophilin to cluster in raised plasma membrane domains, following a similar sequence and time course as that seen with perilipin in adipocytes (Fig. 11). Double labeling of adipophilin and TIP47 demonstrates that both proteins are widely dispersed in the membrane plane under normal culture conditions (Fig. 12A) and located in the same clusters after lipid loading (Fig. 12B). Lipid droplets lie immediately beneath the elevated plasma membrane domains and clusters of label (Fig. 13), again, just as what was seen with perilipin in adipocytes. Both TIP47 and adipophilin appear as mini-clusters within the larger overall aggregates of labeled proteins in the plasma membrane and the lipid droplets. The example in Fig. 13 shows a convex fracture (E-face equivalent) of the complementary face to the outer monolayer of the lipid droplet. The mini-clustering pattern does not match that seen in the plasma membrane as closely as does that of the P-face of the outer monolayer (which lies immediately adjacent to the plasma membrane P-face in which its clusters are found). TIP47 and adipophilin, though visualized at the droplet surface, are also frequently found inside the lipid droplet core.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The key novel findings of the present study are as follows. 1) PAT family proteins occur throughout the lipid droplet rather than being confined to its surface. 2) PAT family proteins are conspicuously present in the plasma membrane. 3) Under conditions of lipid droplet formation, the PAT family proteins, normally dispersed in the plasma membrane, become organized into clusters. 4) The plasma membrane domains bearing clustered PAT family proteins are sites of close physical interaction with underlying lipid droplets. These findings have been made possible by the unique ability of the freeze-fracture/immunogold label technique to visualize at high resolution the distribution of PAT family proteins in planar views of membranes and lipid droplet layers. Application of this technique to both adipocytes and macrophages under conditions of lipid accumulation enabled common principles to be deduced for the behavior of PAT family proteins in general. Parallel application of confocal microscopy, thin section electron microscopy, and cryosection electron microscopy permitted verification of the rate of lipid droplet accumulation, the distribution of lipid droplets in the cell, and overviews of PAT family protein distribution at low resolution (confocal microscopy) or in two-dimensional views (cryosection electron microscopy).
Our findings from freeze-fracture immunogold labeling demonstrate unequivocally that the PAT family proteins and caveolin-1 are not confined to the droplet envelope as maintained previously (8, 29–31) and as cryosections might suggest, but clearly pervade the droplet core. We detected perilipin and caveolin-1 co-localized within the same lipid droplet cores of adipocytes, and we found adipophilin and TIP47 within the same lipid droplet cores of macrophages. Considerable heterogeneity of protein distribution was apparent from droplet to droplet. In addition, the antibodies labeled both the concave and convex faces of lipid layers in the lipid droplet cores, indicating that the proteins may be oriented in both inward facing and outward facing configurations. The detection of these lipid droplet-associated proteins pervading the droplet core in more than one cell type dispels the established concept that the proteins are exclusively components of the droplet surface and raises new questions regarding the mechanism by which they become associated with and penetrate within the lipid droplet.
The high resolution and sensitivity of freeze-fracture immunogold labeling has also enabled the first definitive demonstration that PAT family proteins are components of the plasma membrane. Previously, although adipophilin had been reported in the plasma membrane using standard immunofluorescence microscopy (10), a subsequent study was unable to confirm this conclusion (11). Such discrepancies may arise, at least in part, from the low resolution and other technical limitations inherent in standard immunofluorescence light microscopy as applied to lipid structures (19, 20).
Freeze-fracture immunogold labeling, as applied here, disclosed not only that PAT family proteins are truly components of the plasma membrane but also that they were labeled exclusively on the P-face and never on the E-face, indicating that their distribution is restricted to the cytoplasmic monolayer of the plasma membrane. In normally cultured adipocytes and macrophages the PAT family proteins are widely dispersed in the membrane plane. However, lipid loading of the cells leads to a dramatic re-organization of these proteins in the plane of the membrane; they cluster on raised plasma membrane domains. Double-labeling experiments revealed that this clustering is a specific response of the PAT family proteins because the distribution of caveolin-1 remains unaltered. Because, in freeze fracture, the fracture plane can skip between closely adjacent membranes or lipid layers, favorable fractures provided "windows" through which the structures immediately beneath the plasma membrane domains can be seen. This makes it possible to identify lipid droplets, closely apposed to the plasma membrane, as the structures responsible for the elevations of the plasma membrane domains in which the PAT family proteins are clustered. The discovery of an intimate spatial relationship between lipid droplets and clusters of PAT family proteins in the plasma membrane implies a functional relationship in the processes of subcellular lipid accumulation and the mechanism by which the proteins become incorporated into lipid droplets. Several investigations have established a role for adipophilin (10, 32, 33) and perilipin (34, 35) as free fatty acid transporters. One report showed that adipophilin translocated to the cell periphery in murine 1246 cells during differentiation (10) suggesting that adipophilin functions in carrier-mediated influx of free fatty acids from the extracellular environment to the surface of lipid droplets, supplying the source for triacylglycerol synthesis (36). Thus, our present finding that adipophilin and perilipin are organized in clusters in the plasma membrane may reflect a specialization that enhances the influx of free fatty acids by increasing the rate of uptake into the cells at times of lipid loading. The close apposition of the lipid droplets to the clusters in the plasma membrane may facilitate rapid shuttling of the free fatty acids to the surface of the lipid droplet. This process may be further promoted by the presence of matching aggregates of the proteins in the cytoplasmic monolayer of the plasma membrane and the immediately adjacent outermost monolayer of the lipid droplet. Compared with adipophilin and perilipin, little is known about the possible functions of TIP47; whether this protein might also be involved in the uptake of free fatty acids is unclear. However, similarities in the structure of PAT family proteins and in their localization and clustering in the plasma membrane as revealed in the present study are suggestive of common underlying functions in lipid transport and accumulation.
The current widely accepted model of lipid droplet formation assumes that lipid droplet-associated proteins are derived from proteins resident in the cytoplasmic leaflet of ER membranes, with their transfer to the surface of nascent lipid droplets occurring along with an ER-derived cytoplasmic leaflet, which becomes the lipid droplet envelope (8, 29, 37, 38). Our recent finding (15) that caveolin-1 is abundant in the endoplasmic leaflet of ER membranes, but absent in the cytoplasmic leaflet, challenges this notion; clearly, caveolin-1 molecules cannot gain access to the lipid droplet surface by lateral diffusion in the cytoplasmic membrane leaflet enveloping the lipid droplet because they are on the "wrong" side of the ER membrane. We concluded that the high affinity of lipids for caveolin-1 may itself promote extraction of caveolin-1 molecules from the endoplasmic leaflet of ER membranes and the subsequent partitioning of caveolin-1 into the lipid phase of the droplet (15). According to this model, caveolin-1 molecules may be transferred by the lipids themselves into the core of the forming droplet.
Evidence from reports of Diaz and Pfeffer (39), who described TIP47 as a cytosolic protein, Brasaemle et al. (40), who reported a lack of sedimentable membranes containing adipophilin, and Londos et al. (17), who discussed the synthesis of adipophilin and perilipin on free ribosomes, points to a non-ER pathway for access of the PAT family proteins to the lipid droplet. Our finding, in the present study, of conspicuous gold labeling for PAT family proteins in specialized plasma membrane domains focuses on a pivotal role for the plasma membrane in this pathway. Thus, taken with our previous findings (15), the present data suggest that whereas caveolin-1 is synthesized in the ER and transferred from the endoplasmic leaflet of the ER membrane to the lipid droplet by inundating lipids (15), the PAT family proteins, which are synthesized on free ribosomes in the cytoplasm, are first inserted in the cytoplasmic leaflet of the plasma membrane and transferred from there to closely juxtaposed lipid droplets. That the trafficking pathway of the PAT family proteins is first the plasma membrane followed by the lipid droplet and not vice versa is shown by the presence of the proteins in the plasma membrane under normal conditions in which lipid droplet formation is not stimulated. The clustering of the PAT family proteins under conditions of lipid droplet formation may not only facilitate lipid transfer into the droplet but also transfer of the proteins themselves. Whether PAT family protein transfer is an incidental by-product of the lipid transfer process or whether the presence of these proteins within the lipid droplet as well as the plasma membrane augments lipid transfer to the droplet is unclear. The remarkably similar distribution pattern of the complexes in the outermost monolayer of the lipid droplet with those in the plasma membrane hints at the possibility of a close functional linkage between the two. The question of whether, in addition to the plasma membrane, other pathways exist by which PAT proteins transit to the lipid droplet remains to be determined.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 492. 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.
To whom correspondence should be addressed: Dept. of Cell Biology and Ultrastructure Research, Leibniz-Institute for Arteriosclerosis Research, University of Münster, Domagkstr. 3, 48149 Münster, Germany. Tel.: 49-251-835-6426; Fax: 49-251-835-2998; E-mail: robenek{at}uni-muenster.de.
1 The abbreviations used are: PAT, perilipin, adipophilin, and TIP47; TIP47, tail-interacting protein of 47 kDa; AcLDL, acetylated low density lipoprotein; E-face, endoplasmic half of a membrane revealed by freeze fracturing; P-face, protoplasmic half of a membrane revealed by freeze fracturing; ER, endoplasmic reticulum; PBS, phosphate-buffered saline.
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ACKNOWLEDGMENTS |
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We thank Karin Schlattmann and Christina Köppler for competent and indispensable technical assistance.
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