t-SNARE activation through transient interaction with a Rab-like guanosine triphosphatase

Science

Volumn 276, Issue 5316, 1997, Pages 1255-1258

  Lupashin, Vladimir V ^a   Waters, M Gerard ^a  

^a PRINCETON UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

GUANOSINE TRIPHOSPHATASE; GUANOSINE TRIPHOSPHATE;

ARTICLE; ENDOPLASMIC RETICULUM; GOLGI COMPLEX; INTRACELLULAR TRANSPORT; MEMBRANE VESICLE; NONHUMAN; PRIORITY JOURNAL; PROTEIN ASSEMBLY; PROTEIN PROTEIN INTERACTION; PROTEIN TARGETING; RECEPTOR UPREGULATION; SACCHAROMYCES CEREVISIAE;

BIOLOGICAL TRANSPORT; CARRIER PROTEINS; ENDOPLASMIC RETICULUM; FUNGAL PROTEINS; GOLGI APPARATUS; GTP PHOSPHOHYDROLASES; GTP-BINDING PROTEINS; MEMBRANE PROTEINS; MODELS, BIOLOGICAL; MUNC18 PROTEINS; PLANT PROTEINS; PRECIPITIN TESTS; PROTEIN BINDING; QA-SNARE PROTEINS; RAB GTP-BINDING PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; SNARE PROTEINS; VESICULAR TRANSPORT PROTEINS;

EID: 0031001794     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5316.1255     Document Type: Article

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35. Cells were grown to mid-logarithmic phase at 23°C in either synthetic complete (SC) or SC-Ura media supplemented with 1% casamino acids, harvested by centrifugation (5000g, 5 min), converted to spheroplasts (10), regenerated during a 20-min incubation in YP media (1% yeast extract, 2% peptone) with 1% glucose and 0.8 M sorbitol at 23°C, and lysed (10). The lysate was centrifuged at 15,000g for 15 min at 4°C. A 0.5-mg amount of supernatant protein (usually about 100 μl) was diluted to 1 ml in IP buffer [150 mM NaCl, 1% Triton X-100, 15 mM tris-HCl, (pH 7.5)] and mixed gently for 14 hours at 4°C with 15 μl of protein A-Sepharose beads bearing covalently attached affinity-purified antibodies (28) as indicated in the figure legends. The beads were washed four times with 1 ml of IP butter at 23°C, and eluted with 40 μl of 2% SDS at 90°C. The eluates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (12% gel), electro-transferred onto polyvinylidene difluoride membrane, and probed with the indicated antibodies (28). Bound antibodies were visualized with chemiluminescent detection (Renaissance, DuPont NEN).
36. RSY255 and RSY271 spheroplasts (29) were incubated for 20 min at 23° or 38°C. sedimented, transferred to ice, and lysed in 20 mM Hepes-KOH (pH 7.0) and 150 mM potassium acetate with protease inhibitors (10) by gentle pipetting for 10 min. One-half volume of freshly prepared 10 mM 3,3′-dithiobis(sulfosuccinimidylpropionate) was added, and the reaction was incubated for 1 hour at 4°C with gentle mixing. The reaction was terminated with 1/10 volume of 1 M tris-HCl (pH 7.4) and incubation for 10 min at 4°C. The samples were adjusted to 1% SDS, heated to 90°C for 5 min, and diluted with nine volumes of IP buffer (29). The samples were centrifuged for 10 min at 15,000g, and 0.5 mg of soluble material was incubated for 4 hours at 23°C with 15 μl of protein A-Sepharose beads bearing covalently attached antibodies (28) to Sed5p. The samples were washed four times with IP buffer and eluted with 40 μl of 2% SDS at 90°C. The eluates were separated from the beads, 10 μl of 0.5 M dithiothreitol was added, and the samples were incubated for 20 min at 23°C. Samples (15 μl) were separated by SDS-PAGE (12% gel) and immunoblotted with antibodies (28) to Sed5p or Sly1p.
37. We thank S. Ferro-Novick, J. Rothman, R. Schekman, and H. D. Schmitt for materials, D. Hasara and I. D. Pokrovskaya for technical assistance, and C. Barlow, F. Hughson, and members of the Waters laboratory for discussions. Supported by NIH (GM48753) and the Lucille P. Markey Charitable Trust. M.G.W. is a Lucille P. Markey Biomedical Scholar.