Localization and function of soluble N-ethylmaleimide-sensitive factor attachment protein-25 and vesicle-associated membrane protein-2 in functioning gastric parietal cells

Serhan Karvar; Xuebiao Yao; James M Crothers Jr; Yuechueng Liu; John G Forte

doi:10.1074/jbc.M207694200

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Localization and function of soluble N-ethylmaleimide-sensitive factor attachment protein-25 and vesicle-associated membrane protein-2 in functioning gastric parietal cells

Journal article

Open access

Peer reviewed

Localization and function of soluble N-ethylmaleimide-sensitive factor attachment protein-25 and vesicle-associated membrane protein-2 in functioning gastric parietal cells

Serhan Karvar, Xuebiao Yao, James M Crothers Jr, Yuechueng Liu and John G Forte

The Journal of biological chemistry, Vol.277(51), pp.50030-50035

12/20/2002

DOI: 10.1074/jbc.M207694200

PMID: 12386166

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Abstract

The soluble N-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the gastric parietal cell. Here we examined the cellular localization and functional importance of SNAP-25 in parietal cell cultures. Adenoviral constructs were used to express SNAP-25 tagged with cyan fluorescent protein, VAMP-2 tagged with yellow fluorescent protein, and SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25 Delta181-206). Membrane fractionation experiments and fluorescent imaging showed that SNAP-25 is localized to the apical plasma membrane. The expression of the mutant SNAP-25 Delta181-226 inhibited the acid secretory response of parietal cells. Also, SNAP Delta181-226 bound poorly in vitro with recombinant syntaxin-1 compared with wild type SNAP-25, indicating that pairing between syntaxin-1 and SNAP-25 is required for parietal cell activation. Dual expression of SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with yellow fluorescent protein revealed a dynamic change in distribution associated with acid secretion. In resting cells, SNAP-25 is at the apical plasma membrane and VAMP-2 is associated with cytoplasmic H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins co-localize on the apical plasma membrane. These data demonstrate the functional significance of SNAP-25 as a SNARE protein in the parietal cell and show the dynamic stimulation-associated redistribution of VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane.

Mutation

Adenoviridae - genetics

Animals

Blotting, Western

Cell Membrane - metabolism

Cells, Cultured

Cytoplasm - metabolism

Electrophoresis, Polyacrylamide Gel

Ethylmaleimide - pharmacology

Gene Deletion

Green Fluorescent Proteins

Guinea Pigs

H(+)-K(+)-Exchanging ATPase - chemistry

Luminescent Proteins - metabolism

Membrane Proteins - biosynthesis

Membrane Proteins - chemistry

Membrane Proteins - metabolism

Membrane Proteins - physiology

Microscopy, Fluorescence

Nerve Tissue Proteins - biosynthesis

Nerve Tissue Proteins - chemistry

Nerve Tissue Proteins - physiology

Parietal Cells, Gastric - metabolism

Protein Binding

Protein Structure, Tertiary

R-SNARE Proteins

Rabbits

Rats

SNARE Proteins

Synaptosomal-Associated Protein 25

Syntaxin 1

Time Factors

Vesicular Transport Proteins

Details

Title: Subtitle: Localization and function of soluble N-ethylmaleimide-sensitive factor attachment protein-25 and vesicle-associated membrane protein-2 in functioning gastric parietal cells
Creators: Serhan Karvar - University of California, Berkeley
Xuebiao Yao - University of California, Berkeley
James M Crothers Jr - University of California, Berkeley
Yuechueng Liu - University of Oklahoma Health Sciences Center
John G Forte - University of California, Berkeley
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.277(51), pp.50030-50035
DOI: 10.1074/jbc.M207694200
PMID: 12386166
ISSN: 0021-9258
eISSN: 1083-351X
Grant note: DK 56292 / NIDDK NIH HHS NS 35167 / NINDS NIH HHS DK 10141 / NIDDK NIH HHS DK 38972 / NIDDK NIH HHS
Language: English
Date published: 12/20/2002
Academic Unit: Internal Medicine
Record Identifier: 9984695834702771

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