A role for the caveolin scaffolding domain in mediating the membrane attachment of caveolin-1. The caveolin scaffolding domain is both necessary and sufficient for membrane binding in vitro.

Here, we have created a series of caveolin-1 (Cav-1) deletion mutants to examine whether the membrane spanning segment is required for membrane attachment of caveolin-1 in vivo. One mutant, Cav-1-(1-101), contains only the cytoplasmic N-terminal domain and lacks the membrane spanning domain and the C-terminal domain. Interestingly, Cav-1-(1-101) still behaves as an integral membrane protein but lacks any known signals for lipid modification. In striking contrast, another deletion mutant, Cav-1-(1-81), behaved as a soluble protein. These results implicate caveolin-1 residues 82-101 (also known as the caveolin scaffolding domain) in membrane attachment. In accordance with the postulated role of the caveolin-1 scaffolding domain as an inhibitor of signal transduction, Cav-1-(1-101) retained the ability to functionally inhibit signaling along the p42/44 mitogen-activated protein kinase cascade, whereas Cav-1-(1-81) was completely ineffective. To rule out the possibility that membrane attachment mediated by the caveolin scaffolding domain was indirect, we reconstituted the membrane binding of caveolin-1 in vitro. By using purified glutathione S-transferase-caveolin-1 fusion proteins and reconstituted lipid vesicles, we show that the caveolin-1 scaffolding domain and the C-terminal domain (residues 135-178) are both sufficient for membrane attachment in vitro. However, the putative membrane spanning domain (residues 102-134) did not show any physical association with membranes in this in vitro system. Taken together, our results provide strong evidence that the caveolin scaffolding domain contributes to the membrane attachment of caveolin-1.