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PMID: 9335587 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Mutational analysis of STE5 in the yeast Saccharomyces cerevisiae: application of a differential interaction trap assay for examining protein-protein interactions.

Genetics ·Vol. 147 ·No. 2 ·1997-10-00 ·Pages 479-92

Inouye C, Dhillon N, Durfee T, Zambryski PC, Thorner J

Abstract

Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold that organizes the components of the mitogen-activated protein kinase (MAPK) cascade. A new method was developed to isolate missense mutations in Ste5 that differentially affect the ability of Ste5 to interact with either of two MAPK cascade constituents, the MEKK (Ste11) and the MEK (Ste7). Mutations that affect association with Ste7 or with Ste11 delineate discrete regions of Ste5 that are critical for each interaction. Co-immunoprecipitation analysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G protein beta subunit), and Fus3 (MAPK), confirmed that each mutation specifically affects the interaction of Ste5 with only one protein. When expressed in a ste5 delta cell, mutant Ste5 proteins that are defective in their ability to interact with either Ste11 or Ste7 result in a markedly reduced mating proficiency. One mutation that clearly weakened (but did not eliminate) interaction of Ste5 with Ste7 permitted mating at wild-type efficiency, indicating that an efficacious signal is generated even when Ste5 associates with only a small fraction of (or only transiently with) Ste7. Ste5 mutants defective in association with Ste11 or Ste7 showed strong interallelic complementation when co-expressed, suggesting that the functional form of Ste5 in vivo is an oligomer.

MeSH Terms
Adaptor Proteins, Signal Transducing Carrier Proteins Fungal Proteins/genetics,metabolism Point Mutation Protein Binding Saccharomyces cerevisiae/genetics,metabolism Saccharomyces cerevisiae Proteins
Chemicals
Adaptor Proteins, Signal Transducing Carrier Proteins Fungal Proteins STE5 protein, S cerevisiae Saccharomyces cerevisiae Proteins
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Inouye C
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Dhillon N
Durfee T
Zambryski P C
Thorner J
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Article Info
Journal
Genetics
Abbr.
Genetics
ISSN
0016-6731
DOI
10.1093/genetics/147.2.479
Published
1997-10-00
Pages
479-92
Language
English
Region
United States
NLM ID
0374636
PMCID
PMC1208172
Subset
IM
Grants
NCI NIH HHS · CA09041 · United States
NIGMS NIH HHS · GM-16915 · United States
NIGMS NIH HHS · GM-17573 · United States
External Links
PubMed source DOI: 10.1093/genetics/147.2.479 PMC Full Text View journal details
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