Caveolae, flask-shaped invaginations of the plasma membrane, are particularly abundant in muscle cells. We have recently cloned a muscle-specific caveolin, termed caveolin-3, which is expressed in differentiated muscle cells. Specific antibodies to caveolin-3 were generated and used to characterize the distribution of caveolin-3 in adult and differentiating muscle. In fully differentiated skeletal muscle, caveolin-3 was shown to be associated exclusively with sarcolemmal caveolae. Localization of caveolin-3 during differentiation of primary cultured muscle cells and development of mouse skeletal muscle in vivo suggested that caveolin-3 is transiently associated with an internal membrane system. These elements were identified as developing transverse-(T)-tubules by double-labeling with antibodies to the α1 subunit of the dihydropyridine receptor in C2C12 cells. Ultrastructural analysis of the caveolin-3– labeled elements showed an association of caveolin-3 with elaborate networks of interconnected caveolae, which penetrated the depths of the muscle fibers. These elements, which formed regular reticular structures, were shown to be surface-connected by labeling with cholera toxin conjugates. The results suggest that caveolin-3 transiently associates with T-tubules during development and may be involved in the early development of the T-tubule system in muscle.

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells. INTRODUCTIONAnimal cells are continuously internalizing proteins and lipids of their plasma membrane via endocytosis. The internalized surface components enter a complex and dynamic membrane system, the early endosome, which plays a vital role in sorting endocytosed proteins to different destinations in the cell (Gruenberg and Maxfield, 1995). It is now clear that the early endosome comprises at least two functionally distinct compartments or subdomains (Ghosh et al., 1994;Ghosh and Maxfield, 1995;Gruenberg and Maxfield, 1995;Ullrich et al., 1996;Zacchi et al., 1998). Markers first enter the early sorting endosome, a complex organelle with tubular and multivesicular domains, where membrane proteins destined for degradation are sorted away from those proteins, such as the transferrin receptor, that are recycled back to the plasma membrane. Recycling proteins can then enter a second subcompartment, termed the recycling endosome, which has a tubular morphology and in many cell types is located in the pericentriolar area of the cell (Yamashiro et al., 1984;Dunn et al., 1989;Ghosh and Maxfield, 1995). Further complexity is added to this picture by the finding that fibroblasts, generally regarded as nonpolarized cells, may contain two sets of early endosomes analogous to those in polarized cells (Wilson and Colton, 1997). In these studies, endotubin, a membrane protein of the apical early endosomal compartment in neonatal rat intestine (Wilson et al., 1987), was heterologously expressed in normal rat kidney (NRK) cells and shown to associate with an apparently unique early endosomal compartment. This compartment was distinct from transferrin-containing early endosomes and was relatively insensitive to brefeldi...

Caveolins are integral membrane proteins which are a major component of caveolae. In addition, caveolins have been proposed to cycle between intracellular compartments and the cell surface but the exact trafficking route and targeting information in the caveolin molecule have not been defined. We show that antibodies against the caveolin scaffolding domain or against the COOH terminus of caveolin-1 show a striking specificity for the Golgi pool of caveolin and do not recognize surface caveolin by immunofluorescence. To analyze the Golgi targeting of caveolin in more detail, caveolin mutants were expressed in fibroblasts. Specific mutants lacking the NH2 terminus were targeted to the cis Golgi but were not detectable in surface caveolae. Moreover, a 32–amino acid segment of the putative COOH-terminal cytoplasmic domain of caveolin-3 was targeted specifically and exclusively to the Golgi complex and could target a soluble heterologous protein, green fluorescent protein, to this compartment. Palmitoylation-deficient COOH-terminal mutants showed negligible association with the Golgi complex. This study defines unique Golgi targeting information in the caveolin molecule and identifies the cis Golgi complex as an intermediate compartment on the caveolin cycling pathway.