Ste5 RING-H2 Domain: Role in Ste4-Promoted Oligomerization for Yeast Pheromone Signaling
10.1126/science.278.5335.103
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Ste5 is a scaffold for the mitogen-activated protein kinase (MAPK) cascade components in a yeast pheromone response pathway. Ste5 also associates with Ste4, the β subunit of a heterotrimeric guanine nucleotide–binding protein, potentially linking receptor activation to stimulation of the MAPK cascade. A RING-H2 motif at the Ste5 amino terminus is apparently essential for function because Ste5(C177S) and Ste5(C177A C180A) mutants did not rescue the mating defect of a ste5Δ cell. In vitro Ste5(C177A C180A) bound each component of the MAPK cascade, but not Ste4. Unlike wild-type Ste5, the mutant did not appear to oligomerize; however, when fused to a heterologous dimerization domain (glutathione S-transferase), the chimeric protein restored mating in an ste5Δ cell and an ste4Δ ste5Δ double mutant. Thus, the RING-H2 domain mediates Ste4-Ste5 interaction, which is a prerequisite for Ste5-Ste5 self-association and signaling.

Bibliography

Inouye, C., Dhillon, N., & Thorner, J. (1997). Ste5 RING-H2 Domain: Role in Ste4-Promoted Oligomerization for Yeast Pheromone Signaling. Science, 278(5335), 103–106.

Authors 3
  1. Carla Inouye (first)
  2. Namrita Dhillon (additional)
  3. Jeremy Thorner (additional)
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  41. Qualitative mating tests were performed by patching the MAT a 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. The GST fusions were generated as follows. A 640-bp fragment encoding S. japonicum GST was generated by PCR with a pGEX vector (Pharmacia) as the template and appropriate primers to install Ase I sites at both the 5′ and 3′ ends of the product and inserted into the Nde I site in-frame and downstream of codon 913 of the STE5 coding sequence in pCJ117 (22) creating a junction (5′-CATAATATGTCC-3′) encoding His 912 Asn 913 MetSer (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. MAT a 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 (Suc). Samples (0.2 ml) of serial dilutions of the MAT a strains were mixed in triplicate with 0.6 ml of a culture of a MAT α 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 MAT a 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 MAT a 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.
Dates
Type When
Created 23 years ago (July 27, 2002, 5:44 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 12:01 a.m.)
Indexed 2 weeks, 4 days ago (Aug. 5, 2025, 8:37 a.m.)
Issued 27 years, 10 months ago (Oct. 3, 1997)
Published 27 years, 10 months ago (Oct. 3, 1997)
Published Print 27 years, 10 months ago (Oct. 3, 1997)
Funders 0

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@article{Inouye_1997, title={Ste5 RING-H2 Domain: Role in Ste4-Promoted Oligomerization for Yeast Pheromone Signaling}, volume={278}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.278.5335.103}, DOI={10.1126/science.278.5335.103}, number={5335}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Inouye, Carla and Dhillon, Namrita and Thorner, Jeremy}, year={1997}, month=oct, pages={103–106} }