During the process of vesicle trafficking, membrane topology is normally conserved - that is, material found in the extracellular (exofacial) leaflet of the plasma membrane is topologically conserved. For example, it remains associated with the exofacial leaflet during endocytosis and after fusion with organelles. The exofacial leaflet is thus the inner leaflet of vesicles and the interior leaflet of organelles such as the ER. Proteins and lipids associated with the cytofacial leaflet (cytoplasmic side) remain associated with the cytofacial leaflet of endocytic vesicles, and upon fusion remain localized with the cytoplasmic leaflet. Surprisingly, during translocation of caveolin and cholesterol between the plasma membrane and the ER, caveolin-1 "flips" across the membrane from the cytofacial leaflet and becomes associated with the exofacial leaflet of the ER [6,45] rather than the cytosolic leaflet.
Early studies which identified caveolin as a transmembrane protein may have been based on this same process of flipping, as this appears to occur when caveolin is newly synthesized. Initially, caveolin is transported to the cell surface with cholesterol, but then appears outside the cell, and is subsequently found localized in the cytofacial leaflet of the plasma membrane in caveolae. This suggests that caveolin-1 has a role in cholesterol trafficking across membranes from one leaflet to another, and that this flipping function is active at multiple membranes where cholesterol is transported. Another protein family has been identified that exhibits this same flipping behavior; this family - the engrailed family of homeoproteins -associates with caveolae when expressed in COS cells [46,47]. The engrailed protein, as well as other homeodomain proteins, contains a short domain that has been identified as a potential flip domain, which may allow their crossing from one layer to the other in membranes rich in cholesterol [46,47]. Robenek et al. have recently further characterized the crossing of caveolin-1 from one to another during endocytosis using ultrathin cryosectioning, freeze fracture, and immunogold labeling . During the cycling of caveolin between the plasma membrane and intracellular sites, caveolin switches from the cytofacial leaflet to the exofacial leaflet, and is exclusively localized within the exofacial leaflet in the ER . As mentioned above, overexpression of caveolins or expression of specific caveolin mutants causes formation of lipid droplets marked by caveolin localized to the ER, as lipids accumulated and budded off from the cytofacial leaflet due to the accumula tion of caveolins at the ER [32,33]. Another study extended these observations to examine the relationship between caveolin-1 and lipid droplet biogenesis originating in association with the ER . Caveolin-1 was found localized to the surface of droplets, and also extensively localized to the interior of the droplets, which exhibited a highly organized lamellar structure. However, caveolin-1 was localized strictly in association with the exofacial leaflet of the ER. The model of lipid budding as lipids accumulate and aggregate within the cytofacial leaflet would result in droplets lacking caveolin. There may be differences due to cell type, and droplets produced for different functional reasons may involve different pathways in their formation. Lipid droplets that are transient, rapidly induced and critical to inflammatory prostaglandin production are likely to differ from lipid droplets in adipocytes. The specific proteins and mechanisms regulating their formation and breakdown are also likely to differ. Much remains to be defined before we understand the basic mechanisms regulating lipid storage and specialized lipid-metabo-lizing organelles, and caveolin has been localized to both types of droplets in specific cell types.
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