title: A leucine-based motif mediates the endocytosis of vesicular monoamine and acetylcholine transporters.
abstract: 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 neuron-specific vesicular monoamine transporter (VMAT2) responsible for endocytosis, we examined the effect of amino (NH2-) and carboxyl (COOH-)-terminal mutations on steady state distribution and internalization. Deletion of a critical COOH-terminal domain sequence (AKEEKMAIL) results in accumulation 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 di-alanine 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.
authors.affiliation: Departments of Neurology and Physiology, Graduate Programs in Neuroscience and Cell Biology, University of California School of Medicine, San Francisco, California 94143-0435, USA.
authors.forename: P K
authors.initials: PK
authors.lastname: Tan
authors.forename: C
authors.initials: C
authors.lastname: Waites
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: D E
authors.initials: DE
authors.lastname: Krantz
authors.forename: R H
authors.initials: RH
authors.lastname: Edwards
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D007930:Leucine / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides; D018377:Neurotransmitter Agents / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D050494:Vesicular Acetylcholine Transport Proteins; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D033921:Vesicular Transport Proteins
chemical_list: D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D050494:Vesicular Acetylcholine Transport Proteins; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D033921:Vesicular Transport Proteins; D007930:Leucine; D000109:Acetylcholine