The phosphotyrosyl phosphatase activator, Ncs1p (Rrd1p), functions with Cla4p to regulate the G(2)/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 cla4-75 double 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. ncs1Delta cla4Delta 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 swe1Delta. 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 and SIT4 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 both NCS1 and NOH1 were inviable and arrested as unbudded cells, implying that PTPA function is required for proper G(1) progression.

FIG. 1

Schematic representation of the basis for the genetic screen used to isolate mutants that are synthetically lethal in the absence of CLA4. X_i (activator of STE20), X_ii (component of a non-STE20-related pathway), and Y_i (target of STE20) represent classes of mutants that are hypothesized to be recovered by the genetic screen.

FIG. 2

Phenotypic analysis of ncs1Δ::LEU2 cla4Δ strains. (A) Loss of NCS1 is lethal in the absence of CLA4. Strains SY3357 (wild type), SY3364 (ncs1Δ::LEU2 cla4Δ::TRP1 <pRS316ADE8CLA4>), SY3365 (ncs1Δ::LEU2 cla4Δ::TRP1 <YCpHIS3cla4-75>), and SY3360 carrying the cla4-75 allele (cla4Δ::TRP1 <YCpHIS3cla4-75>) were grown in synthetic medium at 25°C to an OD₆₀₀ of 1.0 to 2.0. A serial dilution (1/10) was performed starting with 10,000 cells. Cells were spotted onto prewarmed YEPD plates at 25 and 37°C and allowed to grow for 3 days. (B) Morphological examination of ncs1Δ::LEU2 cla4Δ::TRP1 strains. Strains SY3364, SY3365, and SY3360 (carrying YCpHIS3cla4-75) were grown in synthetic medium at 25°C and shifted to 37°C for 4 h.

FIG. 3

CLA4 functions are required for viability in ncs1Δ cells. (A) Cla4p lacking the PAK domain cannot support viability in the absence of Ncs1p. Strains SY3364, SY3365, SY3380 (cla4Δ::TRP1 ura3::cla4ΔPAK-MYC::URA3 <YCpHIS3cla4-75>), and SY3379 (ncs1Δ::LEU2 cla4Δ::TRP1 ura3::cla4ΔPAK-MYC::URA3 <YCpHIS3cla4-75>) (sectors 1 to 4, respectively) were grown at 25°C on YEPD medium; single colonies were assayed for growth by streaking onto prewarmed YEPD medium and incubating for 3 days at 37°C. (B) Cells lacking Ncs1p require an active Cla4p kinase. Yeast strain SY3378 (ncs1Δ::HIS3 cla4Δ::TRP1 <pRS316ADE8CLA4>) was transformed with pRS315 (sector a), pRS315ADE8cla4K549R (which encodes a kinase-dead Cla4p), (sector b), or pRS315ADE8CLA4 (sector c), and the ability of pRS315ADE8cla4K549R to substitute for pRS316ADE8CLA4 was assayed by growth on synthetic complete medium lacking leucine (left) or supplemented with 5′-FOA (right) for 3 days at 30°C.

FIG. 4

The cla4Δ ncs1Δ::LEU2 defect results in a G₂ delay that is suppressed by deletion of SWE1. (A) Strains SY3357, SY3360, SY3392, and SY3391 were grown to mid-log phase in YEPD medium at 30°C. The cells were fixed, stained with propidium iodide, and subjected to FACS analysis. (B) Strains expressing wild-type SWE1 (SY3362, SY3378, SY3368, and SY3504) or lacking SWE1 (SY3392 carrying pRS316ADE8CLA4, SY3403, SY3404, and SY3505) were constructed and grown on YEPD medium at 25°C. After 3 days, dilutions of cells were spotted onto rich medium (YEPD) or onto synthetic complete medium supplemented with 5′-FOA and incubated for 3 days at 25°C. (C) Deletion of CLA4 increases the phosphotyrosine content of Cdc28p. Whole-cell extracts were prepared from exponentially growing strains SY3357, SY3390, SY3393, SY3360, and SY3398. Cdc28p was enriched from the extracts using p13^SUC1-conjugated agarose beads. The beads were washed, and the eluted proteins were analyzed by Western analysis using antibodies that recognize phosphotyrosine (4G18) and the PSTAIRE amino acid motif of Cdc28p. The band that appears in the Cdc28Y19F sample of the upper panel (antiphosphotyrosine blot) differs in mobility from Cdc28p. We therefore conclude that it is not Cdc28p but another protein that is phosphorylated on a tyrosine residue(s), but only in Cdc28Y19F cells.

FIG. 5

Cla4p binds to Cdc28p. Cdc28p was enriched from whole-cell extracts of strains SY3409 and SY3357 (lanes 2 and 3, respectively) using p13^SUC1-agarose beads or from extracts of SY3357 carrying pSF19 (CDC28-HA) (lane 4), SY3409 (lane 5), and SY3409 carrying pSF19 (lane 6), using HA antibody-conjugated agarose. Neither Cdc28p nor Cla4-MYC could be enriched from extracts using undecorated agarose beads (lane 1; SY3409). Top, Western (immunoprecipitation [IP]) analysis of Cla4-MYCp associated with Cdc28p-coated beads; middle, amount of Cla4-MYCp present in the whole-cell extract (WCE); bottom, amount of Cdc28p associated with the agarose beads.

FIG. 6

Schematic representation of NCS1 null alleles and phenotypic analysis of ncs1Δ::LEU2 noh1Δ::HIS3 double-mutant strains. (A) Schematic representation of the NCS1 alleles, ncs1Δ::LEU2 and ncs1Δ::HIS3, used throughout this study. The putative translation start site is shown with an arrow. The termination codon is denoted by an asterisk. (B) Analysis of ncs1Δ noh1Δ double mutants. Tetrads dissected from the meiotic progeny of diploid MATa/MATα ncs1Δ::LEU2/NCS1 NOH1/noh1Δ::HIS3 were placed on YEPD medium and incubated at 30°C for 3 days. (C) Phenotypic analysis of cells lacking PTPA function. The budding index (percent budded cells/total number of cells) of asynchronously growing cultures was measured for strains SY3357, SY3383, and SY3382 after growth of cultures in YEPD medium at 25°C followed by a shift to 37°C for 5 h. WT, wild type.

FIG. 7

In vitro association of PTPA proteins with Sit4p. (A) Schematic representation of bacterially expressed MBP-Sit4p fusion proteins used in the assay. 1, MBP; 2, MBP–full-length Sit4p (F); 3, MBP–N-terminal regulatory domain of Sit4p (amino acids 1 to 164) (N); 4, MBP–C-terminal catalytic domain of Sit4p (amino acids 161 to 302) (C). (B) Yeast PTPA proteins associate with the MBP-Sit4p fusion proteins, as shown by Western analysis using the HA antibody 12CA5 as a probe (top) and the α-MBP antibody (New England Biolabs, Inc.) as a loading control) (bottom). WCE, whole-cell extract.

FIG. 8

Ncs1p coimmunoprecipitates with Sit4p from whole-cell extracts (WCE). Lane 1, SY3357 (wild type; WCE); lane 2, SY3357 (wild type; immunoprecipitate [IP]); lane 3, SY3394 (ncs1Δ) carrying YEp13NCS1-3xMYC (IP); lane 4, SY3394 (ncs1Δ) carrying YEp13NCS1-3xMYC (WCE); lane 5, SY3394 (ncs1Δ) carrying YEp13NCS1-3xMYC plus YEp24PPH21-HA (IP); lane 6, SY3394 (ncs1Δ) carrying YEp13NCS1-3xMYC plus YEpURA3ADH-SIT4-HA (IP).