Structural requirements for steady-state localization of the vesicular acetylcholine transporter.
The vesicular acetylcholine transporter (VAChT) regulates the amount of acetylcholine stored in synaptic vesicles. However, the mechanisms that control the targeting of VAChT and other synaptic vesicle proteins are still poorly comprehended. These processes are likely to depend, at least partially, on structural determinants present in the primary sequence of the protein. Here, we use site-directed mutagenesis to evaluate the contribution of the C-terminal tail of VAChT to the targeting of this transporter to synaptic-like microvesicles in cholinergic SN56 cells. We found that residues 481-490 contain the trafficking information necessary for VAChT localization and that within this region L485 and L486 are strictly necessary. Deletion and alanine-scanning mutants lacking most of the carboxyl tail of VAChT, but containing residues 481-490, were still targeted to microvesicles. Moreover, we found that clathrin-mediated endocytosis of VAChT is required for targeting to microvesicles in SN56 and PC12 cells. The data provide novel information on the mechanisms and structural determinants necessary for VAChT localization to synaptic vesicles.
Ferreira
Lucimar T
LT
Program in Molecular Pharmacology, Departamento de Farmacologia, Belo Horizonte, Brazil.
Santos
Magda S
MS
Kolmakova
Natalia G
NG
Koenen
Janaina
J
Barbosa
Jose
J
Gomez
Marcus V
MV
Guatimosim
Cristina
C
Zhang
Xiaodong
X
Parsons
Stanley M
SM
Prado
Vania F
VF
Prado
Marco A M
MA
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002966:Clathrin / Q000502:physiology; D004705:Endocytosis / Q000502:physiology; D006706:Homeostasis; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D011487:Protein Conformation; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism*; D014018:Tissue Distribution; D050494:Vesicular Acetylcholine Transport Proteins
D002966:Clathrin; D026901:Membrane Transport Proteins; C493460:Slc18a3 protein, mouse; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins