Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin-sensitive step and interaction with the AP-2 adaptor complex.

Published

Journal Article

The pathways by which synaptic vesicle proteins reach their destination are not completely defined. Here we investigated the traffic of a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) in cholinergic SN56 cells, a model system for neuronal processing of this cargo. GFP-VAChT accumulates in small vesicular compartments in varicosities, but perturbation of endocytosis with a dominant negative mutant of dynamin I-K44A impaired GFP-VAChT trafficking to these processes. The protein in this condition accumulated in the cell body plasma membrane and in large vesicular patches therein. A VAChT endocytic mutant (L485A/L486A) was also located at the plasma membrane, however, the protein was not sorted to dynamin I-K44A generated vesicles. A fusion protein containing the VAChT C-terminal tail precipitated the AP-2 adaptor protein complex from rat brain, suggesting that VAChT directly interacts with the endocytic complex. In addition, yeast two hybrid experiments indicated that the C-terminal tail of VAChT interacts with the micro subunit of AP-2 in a di-leucine (L485A/L486A) dependent fashion. These observations suggest that the di-leucine motif regulates sorting of VAChT from the soma plasma membrane through a clathrin dependent mechanism prior to the targeting of the transporter to varicosities.

Full Text

Duke Authors

Cited Authors

  • Barbosa, J; Ferreira, LT; Martins-Silva, C; Santos, MS; Torres, GE; Caron, MG; Gomez, MV; Ferguson, SSG; Prado, MAM; Prado, VF

Published Date

  • September 2002

Published In

Volume / Issue

  • 82 / 5

Start / End Page

  • 1221 - 1228

PubMed ID

  • 12358769

Pubmed Central ID

  • 12358769

International Standard Serial Number (ISSN)

  • 0022-3042

Digital Object Identifier (DOI)

  • 10.1046/j.1471-4159.2002.01068.x

Language

  • eng

Conference Location

  • England