A Leucine-based Motif Mediates the Endocytosis of Vesicular Monoamine and Acetylcholine Transporters*
- From the Departments of Neurology and Physiology, Graduate Programs in Neuroscience and Cell Biology, University of California School of Medicine, San Francisco, California 94143-0435
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.
Footnotes
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↵* The work was supported by an National Research Service Award from the National Institute of Mental Health (to P. T.), an institutional training grant from the National Institute of Neurological Disease and Stroke (to C. W.), a Young Investigator Award from the National Alliance for Research on Schizophrenic and Affective Disorders (to Y. L.), a postdoctoral fellowship from the Howard Hughes Medical Institute (to D. K.), and grants from National Institute on Drug Abuse and the National Institute of Neurological Disease and Stroke (to R. H. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Current address: Dept. of Cell Biology IMM11, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037.
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↵§ To whom correspondence should be addressed: Depts. of Neurology and Physiology, UCSF School of Medicine, Third and Parnassus Ave., San Francisco, CA 94143-0435. Tel./Fax: 415-502-5687; E-mail:edwards{at}itsa.ucsf.edu.
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↵1 The abbreviations used are: TGN, trans-Golgi network; AP, adaptor protein complex; CMF-PBS, calcium/magnesium-free phosphate-buffered saline; FITC, fluorescein isothiocyanate; HA, hemaglutinin; LDCV, large dense core vesicle; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PNS, post-nuclear supernatant; Tac, interleukin-2 receptor α-subunit; VAChT, vesicular acetylcholine transporter; VMAT, vesicular monoamine transporter; ELISA, enzyme-linked immunosorbent assay; CHO, Chinese hamster ovary; BSA, bovine serum albumin.
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↵2 P. K. Tan, C. Waites, Y. Liu, D. E. Krantz, and R. H. Edwards, unpublished observations.
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↵3 Y. Liu and R. H. Edwards, manuscript in preparation.
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↵4 D. Krantz and R. H. Edwards, unpublished results.
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- Received February 12, 1998.
- Revision received May 3, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
