Identification of yeast Rho1 p GTPase as a regulatory subunit of 1,3-β-glucan synthase

Science

Volumn 272, Issue 5259, 1996, Pages 279-281

  Qadota, Hiroshi ^a   Python, Christophe P ^b   Inoue, Shunsuke B ^c   Arisawa, Mikio ^c   Anraku, Yasuhiro ^a   Zheng, Yi ^d   Watanabe, Takahide ^c   Levin, David E ^b   Ohya, Yoshikazu ^a  

^a UNIVERSITY OF TOKYO   (Japan)

^b JOHNS HOPKINS UNIVERSITY   (United States)

^c NIPPON ROCHE RESEARCH CENTER   (Japan)

^d UNIVERSITY OF TENNESSEE HEALTH SCIENCE CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

1,3 BETA GLUCAN SYNTHASE; GLUCAN; GUANOSINE TRIPHOSPHATASE; GUANOSINE TRIPHOSPHATE; RECOMBINANT PROTEIN; RHO FACTOR;

ARTICLE; CELL WALL; CONTROLLED STUDY; ENZYME ACTIVITY; ENZYME SUBUNIT; NONHUMAN; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN LOCALIZATION; TEMPERATURE SENSITIVE MUTANT; YEAST CELL;

GLUCOSYLTRANSFERASES; GTP PHOSPHOHYDROLASES; GTP-BINDING PROTEINS; GUANOSINE 5'-O-(3-THIOTRIPHOSPHATE); GUANOSINE TRIPHOSPHATE; MEMBRANE PROTEINS; MULTIENZYME COMPLEXES; PROTEIN KINASE C; RECOMBINANT PROTEINS; RHO GTP-BINDING PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; SCHIZOSACCHAROMYCES POMBE PROTEINS; TEMPERATURE;

EID: 0029892037     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5259.279     Document Type: Article

References (30)

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13. Yeast strains YOC752 (rho1-2), YOC729 (rho1-3), YOC754 (rho1-4), YOC755 (rho1-5), and YOC764 (wild type) were used here YOC752, YOC729, and YOC755 were hypersensitive to Calcofluor white and echinocandin B at 23°C.
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15. YPH499 cells carrying plasmids with wild-type RHOT (pYO762), RHO1-G19V (pYO906) under the control of the GAL1 promoter, or vector alone (pYO761) were used. Cells were incubated in galactose medium for 10 hours, and GS activity associated with the membrane fraction was measured (3). Most of the GS activity from cells with pYO906 was independent of GTP-γ-S, whereas only 15 to 20% of the activity in the control strains was independent of GTP-γ-S.
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19. A temperature-sensitive pkc1 strain (SYT11-12A) and its isogenic wild-type strain (YS3-6D) [S Yoshida, E. Ikeda, I. Uno, H. Mitsuzawa, Mol. Gen. Genet 231, 337 (1992)] were grown in YPD (yeast extract-peptone-dextrose) at 23°C. A pkc1Δ strain (DL376) and its isogenic wild type (DL100) [D. E. Levin and E Bartlett-Heubusch, J. Cell Biol. 116, 1221 (1992)] were grown at 23°C in YPD containing 10% sorbitol GS activities were assayed at 23° and at 37°C.
20. A temperature-sensitive pkc1 strain (SYT11-12A) and its isogenic wild-type strain (YS3-6D) [S Yoshida, E. Ikeda, I. Uno, H. Mitsuzawa, Mol. Gen. Genet 231, 337 (1992)] were grown in YPD (yeast extract-peptone-dextrose) at 23°C. A pkc1Δ strain (DL376) and its isogenic wild type (DL100) [D. E. Levin and E Bartlett-Heubusch, J. Cell Biol. 116, 1221 (1992)] were grown at 23°C in YPD containing 10% sorbitol GS activities were assayed at 23° and at 37°C.
21. Mutants used were rho1-3 and rho1-5 carrying PKC1 on a multicopy plasmid (pYO910) or vector alone (pYO324).
22. The partially purified enzyme fraction (second product entrapment) was analyzed by immunoblotting with antibody to Pkc1p.
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25. 3H]glucose incorporation into glycogen.
26. Recombinant GST-Rho1p and GST-Cdc42p were expressed in Sf9 insect cells and purified as described [Y. Zheng, R Cerione, A. Bender, J Biol. Chem 269, 2369 (1994)]
27. A series of protein sample dilutions was analyzed by immunoblotting with guinea pig antiserum to Rho1p or mouse monoclonal antibody to Fks1p (T2B8) (3). Each antibody recognized a single band with an apparent molecular size of 25 kD (Rho1p) (13) or 200 kD (Fks1p) (3) on immunoblots of yeast whole membrane fraction. The amount of antigens was estimated by densitometry.
28. Goat antibody to mouse immunoglobulin G (IgG) coupled to agarose (20 μl; Sigma) was incubated with medium (500 μl) from cultures of cells producing the monoclonal antibody for 5 hours at 37°C. The agarose beads were washed five times with phosphate-buffered saline and twice with buffer A [0.4% CHAPS, 0.08% cholesteryl hemisuccinate, 50 mM tris-Cl (pH 7.5), 1 mM EDTA, 8 μM GTP-γ-S, and 33% glycerol]. Partially purified GS (1.8 μg) was added, and the reaction mixtures were further incubated for 2 hours at 37°C The beads were washed four times with buffer A, and the bound complexes were analyzed by immunoblotting with antiserum to Rho1p or monoclonal antibody to Fks1p (T2B8).
29. HARho1p may represent the presence of secretory intermediates.
30. We thank J. Mulholland and D Botstein for facilities to raise antibodies, Y Kamada and R. Cerione for discussion, A. Myers for plasmids, and B. J. Blacklock for Sf9 cells Supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (07740581 and 07254203) to Y.O., from NIH (GM48533) and the American Cancer Society (FRA-446) to D E. L., from the Japan Society for the Promotion of Science for Japanese Junior Scientists to H.Q., and from the Swiss National Science Foundation to C.C P.