Identification of the endoplasmic reticulum targeting signal in vesicle-associated membrane proteins

The vesicle-associated membrane proteins (Vamp(s)) function as soluble N-et hylmaleimide-sensitive factor attachment receptor proteins in the intracell ular trafficking of vesicles. The membrane attachment of Vamps requires a c arboxyl-terminal hydrophobic sequence termed an insertion sequence. Unlike other insertion sequence-containing proteins, targeting of the highly homol ogous Vamp1 and Vamp2 to the endoplasmic reticulum requires ATP and a membr ane bound receptor. To determine if this mechanism of targeting to the endo plasmic reticulum extends to other Vamps, we compared the membrane binding of Vamp1 and Vamp2 with the distantly related Vamp8. Similar to the other V amps, Vamp8 requires both ATP and a membrane component to target to the end oplasmic reticulum, Furthermore, binding curves for the three Vamps overlap , suggesting a common receptor-mediated process. We identified a minimal en doplasmic reticulum targeting domain that is both necessary and sufficient to confer receptor-mediated, ATP-dependent, binding of a heterologous prote in to microsomes. Surprisingly, this conserved sequence includes four posit ively charged amino acids spaced along an amphipathic sequence, which unlik e the carboxyl-terminal targeting sequence in mitochondrial Vamp isoforms, is amino-terminal to the insertion sequence. Because Vamps do not bind to p hospholipid vesicles, it is likely that these residues mediate an interacti on with a protein, rather than bind to acidic phospholipids, Therefore, we suggest that a bipartite motif is required for the specific targeting and i ntegration of Vamps into the endoplasmic reticulum with receptor-mediated r ecognition of specifically configured positive residues leading to the inse rtion of the hydrophobic tail into the membrane.