A LEUCINE-BASED MOTIF MEDIATES THE ENDOCYTOSIS OF VESICULAR MONOAMINEAND ACETYLCHOLINE TRANSPORTERS

Specific transport proteins mediate the packaging of neurotransmitters into secretory vesicles and consequently require targeting to the app ropriate intracellular compartment, To identify residues in the neuron -specific vesicular monoamine transporter (VMAT2) responsible for endo cytosis, we examined the effect of amino (NH2-) and carboxyl (COOH-)-t erminal mutations on steady state distribution and internalization. De letion of a critical COOH terminal domain sequence (AKEEKMAIL) results in accumulation of VMAT2 at the plasma membrane and a 50% reduction i n endocytosis. Site directed mutagenesis shows that replacement of the isoleucine-leucine pair within this sequence by alanine-alanine alone reduces endocytosis by 50% relative to wild type VMAT2. Furthermore, the KEEKMAIL se quence functions as an internalization signal when tra nsferred to the plasma membrane protein Tac, and the mutation of the i soleucine-leucine pair also abolishes internalization of this protein. The closely related vesicular acetylcholine transporter (VAChT) conta ins a similar di leucine sequence within the cytoplasmic COOH-terminal domain that when mutated results in accumulation of VAChT at the plas ma membrane. The VAChT di-leucine sequence also confers internalizatio n when appended to two other proteins and in one of these chimeras, co nversion of the di-leucine sequence to dialanine reduces the internali zation rate by 50%. Both VMAT2 and VAChT thus use leucine-based signal s for efficient endocytosis and as such are the first synaptic vesicle proteins known to use this motif for trafficking.