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Volumn 279, Issue 5351, 1998, Pages 696-700

Role of vesicle-associated syntaxin 5 in the assembly of pre-Golgi intermediates

Author keywords

[No Author keywords available]

Indexed keywords

SYNTAXIN;

EID: 0032579295     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5351.696     Document Type: Article
Times cited : (101)

References (48)
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  • 43
    • 6844221854 scopus 로고    scopus 로고
    • note
    • NRK cells on cover slips were infected at the restrictive temperature (39.5°C) with the tsO45 strain of VSV (30). The cells were subsequently shifted to either 15° or 32°C, then permeabilized with digitonin and probed with the appropriate primary antibodies followed by fluorescently labeled secondary antibodies (30). The primary antibodies used were directed against Syn5 (17), VSV-G (p5D4) (38), and α-1,2-mannosidase II (39).
  • 44
    • 6844227661 scopus 로고    scopus 로고
    • note
    • NRK cells were infected with tsO45 VSV for 4 hours at 39.5°C, then transferred to ice and permeabilized (18). Transport reactions containing semi-intact cells, gel-filtered cytosol, ATP mix, and uridine 5′-triphosphate-N-acetylglucosamine were performed for 60 min at 32°C and the membranes subsequently digested with endo D and β-N-acetylglucosaminidase (19). Syntaxin 5-specific reagents were preincubated in complete reaction cocktails on ice for 20 min before transfer to 32°C. Processing of VSV-G to the endo D-sensitive form was followed by quantitative immune blotting (14).
  • 45
    • 6844244893 scopus 로고    scopus 로고
    • note
    • Vesicle formation and two-stage assays were done as described (14) with the following modifications: (i) The membranes were resuspended in 0.25 M sucrose, 10 mM Hepes (pH 7.4) before the addition of salts and differential centrifugation; (ii) the medium speed spin was increased to 5 min in the two-stage assay; and (iii) stage 2 incubations contained gel-filtered cytosol (19). Endo D digestion was performed as described above.
  • 46
    • 6844244894 scopus 로고    scopus 로고
    • note
    • Immunoisolated ER-derived vesicles (14) were solubilized in 0.5% Triton X-100 buffers containing either ATP and EDTA (assembly) or MgATP (disassembly) (22). For immunoprecipitation experiments, the vesicle extracts (0.4 ml) were incubated with 0.5 mg of α-SNAP and Myc-NSF immobilized on anti-Myc-coupled magnetic beads. The immunoprecipitates were washed extensively before SDS-polyacrylamide gel electrophoresis (PAGE) and immune blotting. Analysis on 10 to 40% (w/v) glycerol density gradients was done as described (40). Fractions were collected and precipitated with 10% trichloroacetic acid before SDS-PAGE and immune blotting with enhanced chemiluminescence detection.
  • 47
    • 6844228738 scopus 로고    scopus 로고
    • note
    • 2, 20 mM Hepes (pH 7.4)], then resuspended in 4 ml of transport buffer plus PIC. The Golgi membranes were divided into 0.1-ml samples, frozen in liquid nitrogen, and stored at -80°C.
  • 48
    • 6844262231 scopus 로고    scopus 로고
    • note
    • We thank R. Scheller and J. Hay (Stanford University) for antibodies to mSec22b, rBet1, and membrin; J. Saraste (University of Bergen, Bergen, Norway) for anti-p58; and S. Whiteheart (University of Kentucky Medical Center) for recombinant α-SNAP, Myc-tagged NSF, and anti-NSF (6E6). Supported by NIH grant GM 42336 (to W.E.B.) and the National Cancer Institute (CA58689). Electron microscopy made extensive use of Core B in CA58689. T.R. is a recipient of a fellowship from the Muscular Dystrophy Association, C.D. is a recipient of a fellowship grant from the Deutsche Forschungsgemeinschaft, and S.B. is a recipient of a Cystic Fibrosis Foundation Fellowship.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.