Although caveolins normally reside in caveolae, they can accumulate on the surface of cytoplasmic lipid droplets (LDs). Here, we first provided support for our model that overaccumulation of caveolins in the endoplasmic reticulum (ER) diverts the proteins to nascent LDs budding from the ER. Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs. We used the fact that wild-type caveolin-1 accumulates in LDs after brefeldin A treatment or when linked to an ER retrieval motif to search for mutants defective in LD targeting. The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1. Certain Leu insertions blocked LD targeting, independently of hydrophobic domain length, but dependent on their position in the domain. We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1.

Caveolin-1, a structural protein of caveolae, is cleared unusually slowly from the Golgi apparatus during biosynthetic transport. Furthermore, several caveolin-1 mutant proteins accumulate in the Golgi apparatus. We examined this behavior further in this mutant study. Golgi accumulation probably resulted from loss of Golgi exit information, not exposure of cryptic retention signals, because several deletion mutants accumulated in the Golgi apparatus. Alterations throughout the protein caused Golgi accumulation. Thus, most probably acted indirectly, by affecting overall conformation, rather than by disrupting specific Golgi exit motifs. Consistent with this idea, almost all the Golgi-localized mutant proteins failed to oligomerize normally (even with an intact oligomerization domain), and they showed reduced raft affinity in an in vitro detergent-insolubility assay. A few mutant proteins formed unstable oligomers that migrated unusually slowly on blue native gels. Only one mutant protein, which lacked the first half of the N-terminal hydrophilic domain, accumulated in the Golgi apparatus despite normal oligomerization and raft association. These results suggested that transport of caveolin-1 through the Golgi apparatus is unusually difficult. The conformation of caveolin-1 may be optimized to overcome this difficulty, but remain very sensitive to mutation. Disrupting conformation can coordinately affect oligomerization, raft affinity, and Golgi exit of caveolin-1. INTRODUCTIONCaveolin-1 is an important component of the membraneembedded coat that surrounds caveolae (Rothberg et al., 1992;Fra et al., 1994;Smart et al., 1999;Fernandez et al., 2002). Although it is an integral membrane protein, caveolin-1 lacks conventional transmembrane domains. Instead, Nand C-terminal hydrophilic domains flank a central 33-residue hydrophobic domain (Smart et al., 1999). Both hydrophilic domains are located on the cytoplasmic side of the plasma membrane, and the hydrophobic domain has been suggested to form a helical hairpin embedded in the bilayer. A peptide corresponding to the last 20 residues of the N-terminal hydrophilic domain, a region named the caveolin scaffolding domain (CSD), can bind to membranes independently (Schlegel et al., 1999), at least in part because of the high number of aromatic residues in this domain (Arbuzova et al., 2000). The C-terminal domain also can associate with membranes independently, at least in part via three palmitate chains (Luetterforst et al., 1999).Caveolin-1, alone or with the related protein caveolin-2, forms large (Ͼ400 kDa), SDS-stable oligomers (Monier et al., 1995;Sargiacomo et al., 1995;Scheiffele et al., 1998). These oligomers are thought to associate with each other to form the filamentous structure of the caveolar coat (Fernandez et al., 2002). Oligomerization occurs soon after synthesis (Scheiffele et al., 1998). It is not known where along the secretory pathway caveolin oligomers assemble into caveolae, although pits with the distinctive morphology of caveolae have only been re...