2002 Fiscal Year Final Research Report Summary
Adenovirus-mediated expression and functional analyses of SNARE proteins in salivery gland cells
| Project/Area Number |
13671946
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional basic dentistry
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| Research Institution | Health Sciences University of Hokkaido |
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Principal Investigator |
TAKUMA Taishin Health Sciences University of Hokkaido, School of Dentistry, Professor, 歯学部, 教授 (40095336)
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| Co-Investigator(Kenkyū-buntansha) |
ARAKAWA Toshiya Health Sciences University of Hokkaido, School of Dentistry, Assistant Professor, 歯学部, 講師 (40306254)
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| Project Period (FY) |
2001 – 2002
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| Keywords | SNARE hypothesis / VAMP-2 / GFP / SNAP-23 / Exocytosis / Parotid acinar cell |
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| Research Abstract |
SNARE proteins are widely accepted to be involved in the docking and fusion process of intracellular vesicle trafficking. VAMP-2, syntaxin-4, and SNAP-23 are the plausible candidates of SNARE proteins for non-neuronal exocytosis. Thus, we examined the localization, protein-protein interaction, and intracellular trafficking of these proteins by expressing them as green fluorescent protein (GFP)- and FLAG-tagged fusion proteins in various cells, including HSY cells, a human parotid epithelial cell line. GFP-VAMP-2 was expressed strongly in the Golgi area and weakly on the plasma membrane. Although GFP-SNAP-23 seemed to be expressed universally in the cytosol, the GFP signal was clearly seen on the plasma membrane, when soluble GFP-SNAP-23 was removed by treatment with saponin. GFP-syntaxin-4 was undetectable on the plasma membrane but was strongly expressed on unusually large unidentified vesicles. GFP-syntaxin-4 without its transmembrane domain was still incompletely soluble and observed as aggregates. When syntaxin-4 and munc18c were coexpressed, syntaxin-4 was translocated at least in part to the plasma membrane. The protein-protein interaction between syntaxin-4 and VAMP-2 with their transmembrane domains was markedly inhibited by coexpression of munc18c. These results suggest that munc18c plays an important role for the trafficking of syntaxin-4 to its proper destination by preventing premature interactions with other proteins, including SNARE proteins.
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[Publications] Takuma, T, Arakawa, T, Okayama, M, Mizoguchi, I, Tanimura, A and Tajima, Y: "Trafficking of green fluorescent protein-tagged SNARE proteins in HSY cells"J. Biochemistry. 132. 729-735 (2002)
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