Specific transport proteins mediate the packaging of neurotransmitters into secretory vesicles and consequently require targeting to the appropriate intracellular compartment. To identify residues in the neuronspecific vesicular monoamine transporter (VMAT2) responsible for endocytosis, we examined the effect of amino (NH 2 -) and carboxyl (COOH-)-terminal mutations on steady state distribution and internalization. Deletion of a critical COOH-terminal domain sequence (AKEEKMAIL) results in

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... ulation of VMAT2 at the plasma membrane and a 50% reduction in 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 sequence functions as an internalization signal when transferred to the plasma membrane protein Tac, and the mutation of the isoleucine-leucine pair also abolishes internalization of this protein. The closely related vesicular acetylcholine transporter (VAChT) contains a similar di-leucine sequence within the cytoplasmic COOH-terminal domain that when mutated results in accumulation of VAChT at the plasma membrane. The VAChT di-leucine sequence also confers internalization when appended to two other proteins and in one of these chimeras, conversion of the di-leucine sequence to dialanine reduces the internalization rate by 50%. Both VMAT2 and VAChT thus use leucine-based signals for efficient endocytosis and as such are the first synaptic vesicle proteins known to use this motif for trafficking.