The Phosphotyrosyl Phosphatase Activator, Ncs1p (Rrd1p), Functions with Cla4p To Regulate the G₂/M Transition in Saccharomyces cerevisiae

Abstract

The Saccharomyces cerevisiae p21-activated kinases, Ste20p and Cla4p, have individual functions but appear to share an essential function(s) as well because a strain lacking both kinases is inviable. To learn more about the shared function, we sought new mutations that were lethal in the absence of CLA4. This approach led to the identification of at least 10 complementation groups designated NCS (need CLA4 to survive). As for ste20 cla4-75 mutants, most ncs cla4-75double mutants were defective for septin localization during budding. One group, NCS1/RRD1 (YIL153w), did not confer this defect, however, and we investigated its function further.ncs1Δ cla4Δ cells arrested with elongated buds and short mitotic spindles. The morphological defects and lethality were suppressed by mutations that abrogate the cell cycle morphogenetic checkpoint, CDC28Y19F or swe1Δ. The connection to the cell cycle may be direct, as we detected a Cla4p-Cdc28p complex. NCS1 encodes a protein with significant similarity to a mammalian phosphotyrosyl phosphatase activator (PTPA) regulatory subunit for type 2A protein phosphatases (PP2As). Genetic and biochemical evidence suggested that the phosphatase Sit4p is a target for Ncs1p. First, CLA4 andSIT4 were synthetically lethal. Second, Ncs1p and its yeast paralog, Noh1p (Rrd2p), bound to the catalytic domain of Sit4p in vitro, and Ncs1p could be immunoprecipitated with Sit4p but not with another PP2A (Pph21p) from yeast cell extracts. Strains lacking bothNCS1 and NOH1 were inviable and arrested as unbudded cells, implying that PTPA function is required for proper G₁ progression.

ACKNOWLEDGMENTS

We thank Charlie Boone, Kim Arndt, Danny Lew, Kim Nasmyth, Fred Cross, Michael Stark, Megan Keniry, Kunliang Guan, and Xiaoli Zhan for providing plasmids, Mike Snyder for providing the yeast transposon library, and John Pringle, John Chant, Mike Marusich, and the University of Oregon Monoclonal Antibody Facility for providing antibodies. We are forever grateful to Daciana Margineantu, April Goehring, Diamond Bob Deschenes, Laurie Graham, Liz Conibear, and Tom Stevens for technical assistance, advice, and reagents. We also thank members of the Sprague lab for helpful comments and discussions. FACS analysis was performed by the University of Iowa Flow Cytometry Facility. DNA sequencing was performed by Yanling Wang.

This work was supported by National Research Service Award GM-18002-03 (to D.A.M.) and by grant GM30027 from the National Institutes of Health (to G.F.S.).