A phosphorylation site regulates sorting of the vesicular acetylcholine transporter to dense core vesicles.
Vesicular transport proteins package classical neurotransmitters for regulated exocytotic release, and localize to at least two distinct types of secretory vesicles. In PC12 cells, the vesicular acetylcholine transporter (VAChT) localizes preferentially to synaptic-like microvesicles (SLMVs), whereas the closely related vesicular monoamine transporters (VMATs) localize preferentially to large dense core vesicles (LDCVs). VAChT and the VMATs contain COOH-terminal, cytoplasmic dileucine motifs required for internalization from the plasma membrane. We now show that VAChT undergoes regulated phosphorylation by protein kinase C on a serine (Ser-480) five residues upstream of the dileucine motif. Replacement of Ser-480 by glutamate, to mimic the phosphorylation event, increases the localization of VAChT to LDCVs. Conversely, the VMATs contain two glutamates upstream of their dileucine-like motif, and replacement of these residues by alanine conversely reduces sorting to LDCVs. The results provide some of the first information about sequences involved in sorting to LDCVs. Since the location of the transporters determines which vesicles store classical neurotransmitters, a change in VAChT trafficking due to phosphorylation may also influence the mode of transmitter release.
Krantz
D E
DE
Graduate Programs in Neuroscience, Cell Biology, and Biomedical Sciences, University of California at San Francisco School of Medicine, San Francisco, California 94143-0435, USA.
Waites
C
C
Oorschot
V
V
Liu
Y
Y
Wilson
R I
RI
Tan
P K
PK
Klumperman
J
J
Edwards
R H
RH
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000502:physiology* / Q000648:ultrastructure*; D018698:Glutamic Acid; D007930:Leucine; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides; D016716:PC12 Cells; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D012701:Serotonin / Q000378:metabolism; D013572:Synaptic Vesicles / Q000502:physiology / Q000648:ultrastructure; D050494:Vesicular Acetylcholine Transport Proteins; D050492:Vesicular Biogenic Amine Transport Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D011994:Recombinant Proteins; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D050492:Vesicular Biogenic Amine Transport Proteins; D033921:Vesicular Transport Proteins; D012701:Serotonin; D018698:Glutamic Acid; D012694:Serine; D011493:Protein Kinase C; D007930:Leucine