Characterization of ypa1 and ypa2, the Schizosaccharomyces pombe orthologs of the peptidyl proyl isomerases that activate PP2A, reveals a role for Ypa2p in the regulation of cytokinesis

Abstract

The Schizosaccharomyces pombe septation initiation network (SIN) regulates cytokinesis. Cdc7p is the first kinase in the core SIN; we have screened genetically for SIN regulators by isolating cold-sensitive suppressors of cdc7-24. Our screen yielded a mutant in SPAC1782.05, one of the two fission yeast orthologs of mammalian phosphotyrosyl phosphatase activator. We have characterized this gene and its ortholog SPAC4F10.04, which we have named ypa2 and ypa1, respectively. We find that Ypa2p is the major form of protein phosphatase type 2A activator in S. pombe. A double ypa1-Δ ypa2-Δ null mutant is inviable, indicating that the two gene products have at least one essential overlapping function. Individually, the ypa1 and ypa2 genes are essential for survival only at low temperatures. The ypa2-Δ mutant divides at a reduced cell size and displays aberrant cell morphology and cytokinesis. Genetic analysis implicates Ypa2p as an inhibitor of the septation initiation network. We also isolated a cold-sensitive allele of ppa2, the major protein phosphatase type 2A catalytic subunit, implicating this enzyme as a regulator of the septation initiation network.

Figure 1

Characterization of suppressors of cdc7-24 that are cold sensitive. (A) Cells of the indicated genotype were grown to late exponential phase at 25° in YE medium. Serial 10-fold dilutions were made and spotted on YE agar plates at the indicated temperatures. The plates were incubated until the wild-type cells had formed colonies at the indicated temperature (2–5 days). (B) Exponentially growing cells of the indicated genotype were spread onto YE agar plates containing phloxin B. The plates were incubated at 19° for 3 days, and typical colonies were photographed. The green channel of the captured image is shown; dead cells are dark, as they are unable to exclude phloxin B. Bar, 10 μm. (C) The indicated mutants were grown to exponential phase at 25° in YE and then shifted to 29° for 6 hr or 36° for 5 hr. Cells were fixed, and stained with DAPI and calcofluor. Bar, 10 μm. (D) Mutants were grown to exponential phase at 25° in YE and then shifted to 32° for 6 hr. Cells were fixed and stained with DAPI and calcofluor. Bar, 10 μm.

Figure 2

Characterization of Ypa1p and Ypa2p. (A and B) Strains expressing Ypa1p-HA or Ypa2p-HA were synchronized as described in Materials and Methods. Protein samples were prepared at the indicated times (minutes) after release from the block. Western blots were probed with either 12CA5 (HA), or TAT-1 (α-tubulin) as indicated. Cell cycle progression data are represented by the percentage of septated cells, indicated at each time point. (C) Proteins were prepared from strains expressing Ypa1-HA (lane 2), Ypa2-HA (lane 3), or both alleles (lane 1). Serial dilution of the sample indicates that the Ypa2p-HA signal in lane 1 is at least 30 times stronger than the Ypa1p-HA signal (Figure S2A). (D) Cells of the indicated genotype were grown to exponential phase and GFP was visualized as described in Materials and Methods. Bar, 10 μm.

Figure 3

Analysis of the phenotypes of ypa1–Δ and ypa2–Δ. Cells of the indicated genotype were grown at 32° and then shifted to 19° for 18 hr. Cell were fixed and stained with DAPI and calcofluor. Bar, 10 μm throughout. (A) Analysis of ypa1–Δ and ypa2–Δ. The arrows in ypa2–Δ indicate cells that have delayed complete separation. (B) Analysis of ypa2–Δ ppa2–Δ. Note the presence of asymmetrically placed septa, cells with elongated “stretched” nuclei, indicative of problems with chromosome separation, and (inset) two multinucleate cells that have failed to separate. (C) Circled 1 indicates a representative cell with an eccentrically placed septum. Note that in these cells, the nuclei are also flattened and positioned at the cell periphery. The arrowed 2 indicates representative cells showing lagging chromosomes. The circled 3 (inset) shows a pair of cells that have failed to separate, one of which has initiated the subsequent mitosis. The circled 4 indicates a cell with aberrant morphology. (D) ppa2-6 cells at 32°. (E) Tetrads from a cross ypa1–Δ ppa2-6 were dissected at 32°. Colonies were photographed prior to replica plating. The genotype of the microcolony was inferred from the phenotypes after replica plating.

Figure 4

Analysis of the cytoskeleton of ypa1–Δ, ypa2–Δ, and ppa2-6. Mutants expressing either crn1-GFP (A–E) or GFP-atb2 (F–I) were grown to exponential phase at 32° and GFP was visualized. For the 19° images, cells were shifted to 19° for 18 hr prior to analysis. Bar, 10 μm throughout. The circled I is an interphase cell, the circled M is a mitotic cell, and the circled P is a cell with a postanaphase array.

Figure 5

Localization of Cdc7p-GFP and Sid2p-GFP in ypa2 and ppa2 mutants. Cells were grown to exponential phase at 25° and then shifted to 32° for 3 hr. They were fixed and analyzed as described in Materials and Methods. (A) Localization of Cdc7p-GFP in anaphase cells. The indicated strains were grown to exponential phase. Note the presence of Cdc7p on both SPBs in the ppa2–Δ and ypa2–Δ cells. See text for details. (B) Localization of Sid2p-GFP to the CAR is rescued in cdc7-24 ppa2–Δ and cdc7-24 ypa2–Δ mutants: Cells of the indicated genotypes were grown at the temperatures shown. Note the absence of Sid2p between the two anaphase nuclei in cdc7-24 at 32° (the nuclei are seen as dark patches, and the SPB signals are at the tip-proximal edge of the nucleus, indicating that the cell is in anaphase).