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PMID: 12556475 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Effect of the pheromone-responsive G(alpha) and phosphatase proteins of Saccharomyces cerevisiae on the subcellular localization of the Fus3 mitogen-activated protein kinase.

Molecular and cellular biology ·Vol. 23 ·No. 4 ·2003-02-00 ·Pages 1135-50

Blackwell E, Halatek IM, Kim HJ, Ellicott AT, Obukhov AA, Stone DE

Abstract

The mating-specific G(alpha) protein of Saccharomyces cerevisiae, Gpa1, stimulates adaptation to pheromone by a mechanism independent of G(beta gamma) sequestration. Genetic evidence suggests that Gpa1 targets the Fus3 mitogen-activated protein kinase, and it has recently been shown that the two proteins interact in cells responding to pheromone. To test the possibility that Gpa1 downregulates the mating signal by affecting the localization of Fus3, we created a Fus3-green fluorescent protein (GFP) fusion protein. In vegetative cells, Fus3-GFP was found in both the cytoplasm and the nucleus. Pheromone stimulated a measurable increase in the ratio of nuclear to cytoplasmic Fus3-GFP. In contrast, the relative level of nuclear Fus3-GFP decreased as cells recovered from pheromone arrest and did not increase when cells adapted to chronic stimulus were challenged again. Accumulation of Fus3-GFP in the nuclei of stimulated cells was also inhibited by overexpression of either wild-type Gpa1, the E364K hyperadaptive mutant form of Gpa1, or the Msg5 dually specific phosphatase. The effects of Gpa1 and Msg5 on Fus3 are partially interdependent. In a genetic screen for adaptive defective mutants, a nonsense allele of the nucleocytoplasmic transport receptor, Kap104, was identified. Truncation of the Kap104 cargo-binding domain blocked the effect of both Gpa1(E364K) and Msg5 on Fus3-GFP localization. Based on these results, we propose that Gpa1 and Msg5 work in concert to downregulate the mating signal and that they do so by inhibiting the pheromone-induced increase of Fus3 in the nucleus. Kap104 is required for the G(alpha)/phosphatase-mediated effect on Fus3 localization.

MeSH Terms
Adaptation, Physiological/genetics Binding Sites Cell Division/genetics Cell Nucleus/genetics,metabolism Cytoplasm/genetics,metabolism Epistasis, Genetic GTP-Binding Protein alpha Subunits GTP-Binding Protein alpha Subunits, Gq-G11 Green Fluorescent Proteins Heterotrimeric GTP-Binding Proteins/genetics,metabolism Karyopherins/genetics,metabolism Luminescent Proteins/genetics,metabolism Mitogen-Activated Protein Kinases/genetics,metabolism Mutation Nucleocytoplasmic Transport Proteins/metabolism Pheromones/metabolism Protein Tyrosine Phosphatases/genetics,metabolism RNA-Binding Proteins/metabolism Recombinant Fusion Proteins/genetics,metabolism Saccharomyces cerevisiae/genetics,metabolism Saccharomyces cerevisiae Proteins/genetics,metabolism beta Karyopherins
Chemicals
GTP-Binding Protein alpha Subunits KAP104 protein, S cerevisiae Karyopherins Luminescent Proteins NAB2 protein, S cerevisiae Nucleocytoplasmic Transport Proteins Pheromones RNA-Binding Proteins Recombinant Fusion Proteins Saccharomyces cerevisiae Proteins beta Karyopherins Green Fluorescent Proteins FUS3 protein, S cerevisiae Mitogen-Activated Protein Kinases MSG5 protein, S cerevisiae Protein Tyrosine Phosphatases GPA1 protein, S cerevisiae GTP-Binding Protein alpha Subunits, Gq-G11 Heterotrimeric GTP-Binding Proteins
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Blackwell Ernest
Laboratory for Molecular Biology, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Halatek Izabel M
Kim Hye-Jin N
Ellicott Alexis T
Obukhov Andrey A
Stone David E
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.23.4.1135-1150.2003
Published
2003-02-00
Pages
1135-50
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC141143
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.23.4.1135-1150.2003 PMC Full Text View journal details
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