Ste5 RING-H2 domain: Role in Ste4-promoted oligomerization for yeast pheromone signaling

Science

Volumn 278, Issue 5335, 1997, Pages 103-106

  Inouye, Carla ^a   Dhillon, Namrita ^a,b   Thorner, Jeremy ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

PHEROMONE;

AMINO ACID SEQUENCE; AMINO TERMINAL SEQUENCE; ARTICLE; CONFORMATIONAL TRANSITION; ENZYME ACTIVATION; NONHUMAN; OLIGOMERIZATION; PRIORITY JOURNAL; PROTEIN ASSEMBLY; SACCHAROMYCES CEREVISIAE; SIGNAL TRANSDUCTION;

ADAPTOR PROTEINS, SIGNAL TRANSDUCING; AMINO ACID SEQUENCE; BINDING SITES; CA(2+)-CALMODULIN DEPENDENT PROTEIN KINASE; CARRIER PROTEINS; DIMERIZATION; FUNGAL PROTEINS; GENETIC COMPLEMENTATION TEST; GLUTATHIONE TRANSFERASE; GTP-BINDING PROTEIN BETA SUBUNITS; GTP-BINDING PROTEINS; HETEROTRIMERIC GTP-BINDING PROTEINS; MOLECULAR SEQUENCE DATA; PEPTIDES; PHEROMONES; POINT MUTATION; POLYMERS; RECOMBINANT FUSION PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; SIGNAL TRANSDUCTION; TRANSFORMATION, GENETIC;

SACCHAROMYCES CEREVISIAE;

EID: 0030815533     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5335.103     Document Type: Article

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40. 600 nm = 0.6. The cultures were induced by the addition of Gal to a final concentration of 2% and incubated for an additional 2 to 3 hours. The cells were collected, lysed, clarified, and the resulting extracts were subjected to immunoprecipitation (15). The resulting immune complexes were resuspended in 1 × SDS-PAGE sample buffer, boiled for 5 min, and then resolved by SDS-PAGE. After transfer onto Immobilon-P membranes (Millipore) using semidry transfer apparatus (Bio-Rad), proteins were detected by immunoblotting with rabbit polyclonal antisera to Ste11, Ste7, Fus3, Ste4, and Ste5, as appropriate.
41. Qualitative mating tests were performed by patching the MATa strains to be tested on appropriate selective medium, and then replica-plating onto a lawn of DC17 on YP medium containing either 2% Gal/0.2% Suc or 2% Glc (depending on the promoter used for STE5 expression) at 30°C overnight. The resulting mating plates then were replica-plated onto a minimal medium [synthetic complete (SC)] (23) selective for diploids and further incubated at 30°C overnight.
42. 913 Met-Ser (where Met is the first residue of GST), thus yielding pCJ148. pCJ149, expressing the Ste5(C177A C180A)-GST fusion, was created from pCJ119 in an analogous fashion. The translation stop codon in both pCJ148 and pCJ149 is provided by the natural TAG at the end of the STE5 coding sequence.
43. MATa strains to be tested were grown in an appropriate selective medium (SC) (23) containing either 2% glucose (Glc) or 2% raffinose (Raf), depending on the promoter regulating STE5 expression. Strains carrying plasmids expressing STE5 constructs from the GAL1 promoter were induced by addition of galactose (Gal) to a final concentration of 2% and incubation for 60 min before dilution and were plated on a medium containing 2% Gal and 0.2% sucrose (Sue). Samples (0.2 ml) of serial dilutions of the MATa strains were mixed, in triplicate, with 0.6 ml of a culture of a MATα tester strain (DC17) that had been grown to midexponential phase in yeast extract-peptone (YP) (23) medium. Portions (0.4 ml) of these mixtures were plated on a medium lacking the appropriate supplements to select for diploids and incubated at 30°C for 36 to 40 hours. Corresponding dilutions of the MATa strains were also plated to determine the total number of viable haploids. Mating efficiencies were calculated as the ratio of diploid cells formed to the total number of input MATa haploids.
44. We thank J. Schultz, M. S. Hasson, R. L. Freedman, and E. T. Barfod for early contributions, L. Bardwell and J. G. Cook for critical reading of the manuscript, and T. Durfee for intellectual contributions, helpful discussions, and enthusiastic interest. Supported by grant GM21841 from NIH (J.T.), postdoctoral fellowship PF-3785 from the American Cancer Society (C.I.), by National Cancer Institute postdoctoral traineeship CA09041 and NRSA postdoctoral fellowship from NIH (N.D.), and by resources provided by the Berkeley campus Cancer Research Laboratory.