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Volumn 289, Issue 5478, 2000, Pages 444-448

Rab1 recruitment of p115 into a cis-SNARE complex: Programming budding COPII vesicles for fusion

Author keywords

[No Author keywords available]

Indexed keywords

GUANOSINE TRIPHOSPHATASE; SNARE PROTEIN;

EID: 0034698202     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.289.5478.444     Document Type: Article
Times cited : (399)

References (69)
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    • The p115 mAbs used were 13F12 (specific for bovine p115; also known as mAb 115-5) and 3A10 (specific for rat p115) (63, 64). GST-Rab expression vectors and GST-SNARE expression vectors were generated using conventional techniques. EEA1 mAb was obtained from Transduction Laboratories (Lexington, KY). Antibodies to the cytoplasmic domains of membrin, rbet1, and sec22b were generated by injecting purified GST fusion proteins into the lymph nodes of female New Zealand white rabbits. Specific immunoglobulin G (IgG) antibodies were purified from serum by first passing a 36% ammonium sulfate cut over an Aminolink Plus (Pierce) GST column, to remove anti-GST IgGs, and then over a second Aminolink Plus column to which the specific antigen had been coupled. Antibodies were eluted in 1-ml fractions with 0.1 M glycine (pH 2.8) and 0.15 M NaCl directly into 0.1 ml of 1 M tris-HCl (pH 7.8).
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    • To measure COPII vesicle budding, we incubated ER microsomes containing VSV-G with cytosol and adenosine triphosphate (ATP) at 32°C. The released COPII vesicles were separated from ER microsomes by differential centrifugation and collected by high-speed centrifugation. The appearance of VSV-G in the high-speed pellet (HSP) is a direct measure of COPII vesicle budding from the ER (29). For measurement of fusion, COPII vesicles recovered in the HSP were incubated with purified Golgi membranes in a separate reaction and fusion was measured by processing of VSV-G to the endoglycosidase D (endo D)-sensitive form, a hallmark of transport to the cis-Golgi network (23, 29).
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    • The 42-kD recycling form of syntaxin5 (65) was principally recovered in the p115 complex.
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    • 7 M500 Dynabeads (Dynal, New York) coupled to p115 mAb 3A10. Immunoprecipitation was carried out for 2 hours at 4°C with rotation. Beads were washed four times for 5 min each with RIPA buffer without SDS, and bound proteins were eluted by boiling in SDS sample buffer supplemented with 10 mM DTT for 5 min. Amounts of SNAREs and SNARE-binding proteins recovered in the p115 complex on COPII vesicles (Fig. 4A, lane b) were determined by densitometry of immunoblots. Recovery was expressed as a percentage of the amount of specific protein recovered in the HSP of the normal in vitro incubation minus the background levels observed in the HSP in the control (Sar1-GDP-containing) incubation that prevents COPII vesicle formation. In the microsome control, the level of p115-SNARE associations was less than 0.1%.
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    • 3Cl and separated by SDS-polyacrylamide get electrophoresis (PAGE) followed by immunoblotting using p115 mAb 13F12.
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    • note
    • We thank G. Waters for p115 cDNA and p115 mAbs; G. Warren for p97 and p47 antibodies; R. Scheller for rbet1, membrin, and sec22 cDNAs; H. Plutner for excellent technical assistance; and P. Tan for help during the initial phase of this work. Supported by NIH grants GM 33301 and GM42336 and National Cancer Institute grant CA58689 (W.E.B.), a NIH National Research Service Award (B.D.M.), and a Wellcome Trust International Traveling Fellowship (B.B.A.).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.