EVIDENCE FOR MULTIPLE MECHANISMS FOR MEMBRANE-BINDING AND INTEGRATIONVIA CARBOXYL-TERMINAL INSERTION SEQUENCES

Subcellular localization of proteins with carboxyl-terminal insertion sequences requires the molecule be both targeted to and integrated int o the correct membrane, The mechanism of membrane integration of cytoc hrome b(5) has been shown to be promiscuous, spontaneous, nonsaturable , and independent of membrane proteins. Thus endoplasmic reticulum loc alization for cytochrome b5 depends primarily on accurate targeting to the appropriate membrane, Here direct comparison of this mechanism wi th that of three other proteins integrated into membranes via carboxyl -terminal insertion sequences [vesicle-associated membrane protein 1(V amp1), polyomavirus middle-T antigen, and Bcl-2] revealed that, unlike cytochrome b(5), membrane selectivity for these molecules is conferre d at least in part by the mechanisms of membrane integration, Bcl-2 me mbrane integration was similar to that of cytochrome bs except that in sertion into lipid vesicles was inefficient. Unlike cytochrome b(5) an d Bcl-2, Vamp1 binding to canine pancreatic microsomes was saturable, ATP-dependent, and abolished by mild trypsin treatment of microsomes. Surprisingly, although the insertion sequence of polyomavirus middle-T antigen was sufficient to mediate electrostatic binding to membranes, binding did not lead to integration into the bilayer. Together these results demonstrate that there are at least two different mechanisms f or correct membrane integration of proteins with insertion sequences, one mediated primarily by targeting and one relying on factors in the target membrane to mediate selective integration. Our results also dem onstrate that, contrary to expectation, hydrophobicity is not sufficie nt for insertion sequence-mediated membrane integration, We suggest th at the structure of the insertion sequence determines whether or not s pecific membrane-bound receptor proteins are required for membrane int egration.