When cells are migrating, caveolin-1, the principal protein component of caveolae, is excluded from the leading edge and polarized at the cell rear. The dynamic feature depends on a specific sequence motif that directs intracellular trafficking of the protein. Deletion mutation analysis revealed a putative polarization domain at the N terminus of caveolin-1, between amino acids 32-60. Alanine substitution identified a minimal sequence of 10 residues ( 46 TKEIDLVNRD 55 ) necessary for caveolin-1 rear polarization. Interestingly, deletion of amino acids 1-60 did not prevent the polarization of caveolin-1 in human umbilical vein endothelial cells or wild-type mouse embryonic fibroblasts because of an interaction of Cav 61-178 mutant with endogenous caveolin-1. Surprisingly, expression of the depolarization mutant in caveolin-1 null cells dramatically impeded caveolae formation. Furthermore, knockdown of caveolae formation by methyl-â€-cyclodextrin failed to prevent wild-type caveolin-1 rear polarization. Importantly, genetic depletion of caveolin-1 led to disoriented migration, which can be rescued by full-length caveolin-1 but not the depolarization mutant, indicating a role of caveolin-1 polarity in chemotaxis. Thus, we have identified a sequence motif that is essential for caveolin-1 rear polarization and caveolae formation.Caveolae are specific microdomains of the plasma membrane that were discovered more than 50 years ago (1). In endothelial cells, numerous vesicles appeared to derive from the uniformly flask-shaped invaginations, suggesting the endocytic potential of caveolae (2). Although the function of caveolae as transport vesicles mediating endocytosis and transcytosis remained obscure (3, 4), the identification, cloning, and characterization of caveolar coat proteins, caveolins, has increased our knowledge of caveolae, and a good body of evidence implicates caveolae in a specialized form of delivery of membrane components, extracellular ligands, bacterial toxins, and nonenveloped virus in several cell types (5-8). The caveolae-mediated endocytic pathway differs from that mediated by clathrincoated pits. It is sensitive to protein kinase C inhibitors and cholesterol depletion (by filipin), and in some cells it is involved in the activation of protein tyrosine kinases (9). Phosphorylation at tyrosine 14 of caveolin-1 (Cav-1) 2 may be required for the internalization of caveolae. The mechanism controlling caveolae trafficking remains unclear, but it apparently involves both microtubule and actin cytoskeletons (10). Surprisingly, using Cav-1 as a marker for caveolae, recent studies demonstrate that caveolae are rich in a variety of signaling molecules, with the implication that caveolae may function in the regulation of signal transduction. Given these views, an attractive hypothesis would be whether caveolae could carry signaling machinery to different locations of the cell to spatially organize signaling events. Indeed, Anderson and colleagues (11) have shown recently that concomitant with the relocation o...