Antagonistic roles of PP2A-Pab1 and Etd1 in the control of cytokinesis in fission yeast

Abstract

In Schizosaccharomyces pombe, Etd1 is a positive regulator of the septation initiation network (SIN), a conserved GTPase-regulated kinase cascade that triggers cytokinesis. Here we show that a mutation in the pab1 gene, which encodes the B-regulatory subunit of the protein phosphatase 2A (PP2A), suppresses mutations in the etd1 gene. Etd1 is required for the function of the GTPase Spg1, a key regulator of SIN signaling. Interestingly, the loss of Pab1 function restored the activity of Spg1 in Etd1-deficient cells. This result suggests that PP2A-Pab1-mediated dephosphorylation inhibits Spg1, thus antagonizing Etd1 function. The loss of pab1 function also rescues the lethality of mutants of other genes in the SIN cascade such as mob1, sid1, and cdc11. Two-hybrid assays indicate that Pab1 physically interacts with Mob1, Sid1, Sid2, and Cdc11, suggesting that the phosphatase 2A B-subunit is a component of the SIN complex. Together, our results indicate that PP2A-Pab1 plays a novel role in cytokinesis, regulating SIN activity at different levels. Pab1 is also required to activate polarized cell growth. Thus, PP2A-Pab1 may be involved in coordinating polar growth and cytokinesis.

Figure 1.—

Identification of the etd1-1 suppressor gene, pab1. (A) Growth at 25° of wild-type (wt), ret1-4, etd1Δ (etd1∷ura4), and etd1Δ ret1-4 strains. These strains were grown at 37° (permissive temperature for etd1Δ) and replica-plated to assay for growth at 25° (restrictive temperature for etd1Δ). The results show that ret1-4 is able to suppress growth lethality of a null etd1Δ allele (left) and septation deficiencies of these mutant cells, as determined by DAPI-calcofluor staining (right panels). (B) Growth sensitivity of ret1-4 to 0.005% SDS assessed by serial dilution drop tests on plates (10-fold dilutions starting with 10⁴ cells). Wild-type strain (wt) was used as a control. This property allowed complementation cloning using a cDNA library driven by the thiamine-repressible nmt1 promoter at low levels of expression (with thiamine) in the pREP3x plasmid. The corresponding cDNA coded for the pab1 gene, and the ret1-4 mutation was renamed pab1-4. (C) In vivo localization of Pab1 using a GFP-pab1 construct in pab1Δ cells. GFP-Pab1 was imaged in living cells at 25° by time-lapse microscopy at 5-min intervals in the same focal plane. GFP-Pab1 expression was monitored from time 0 to full expression after nmt41x-GFP-pab1 induction, and GFP-Pab1 localization was the same in the wide range of expression levels assessed. (D) Association of GFP-Pab1 with the mitotic spindle. The Acb2-tomato construct was used for microtubule localization. Bars, 5 μm.

Figure 2.—

Negative regulation of cytokinesis by Pab1. (A) Serial dilution of cdc16-116 pab1-4 double mutant and the single mutants to test for potential additive effects of SIN inhibitors growing at 25°, 30°, 32°, 34°, and 36°. (B) pab1-4, cdc16-116, and pab1 cdc16-116 cells were DAPI- and calcofluor-stained after 8 hr incubation at 32° (first three panels from left). DAPI-calcofluor staining of cdc16-116 cell at 36° (restrictive temperature for this strain) is also shown (far right panel).

Figure 3.—

Effects of pab1-4 in Spg1 activity. (A) Cdc7-GFP was imaged by time-lapse microscopy at 5-min intervals on a single focal plane in living wild-type cells and in etd1Δ, pab1-4, and etd1Δ pab1-4 mutant cells as indicated. Fluorescence intensity was quantified (arbitrary units) and represented for each SPB (SPB-1 and SPB-2). Bar, 4 μm. (B) Average of the time that SPBs remained active. Time (minutes) from initial SPB activation until the SPB was switched off was determined for each SPB (SPB-1 and SPB-2) in 10 cells per strain. Error bars represent standard deviations.

Figure 4.—

Genetic interactions for pab1-4 with SIN mutants. (A) Growth of cdc15-140, spg1-B8, cdc7-24, cdc11-119, cdc14-118, sid2-250, cdc16-116, mob1-R4, and sid1-239 septation mutants at their permissive (25°) and restrictive (36°) temperature. Double mutants were used to assay for suppression of lethality of these septation mutants by pab1-4, as indicated. Wild type (wt) and pab1-4 strains were used as a control. (B) Growth of spg1-B8, cdc11-119, cdc7-24, cdc14-11, and sid2-250 septation mutants at their permissive (25°) and restrictive (36°) temperature, assessed by serial dilution drop tests on plates (10-fold dilutions starting with 10⁴ cells). Moderated overexpression of pab1 (nmt41x-driven expression in the absence of thiamine) was used to assay for suppression of lethality by an excess of Pab1 in these septation mutants, as indicated.

Figure 5.—

Physical interaction between Pab1 and SIN proteins. (A) Protein–protein interactions were examined using the yeast two-hybrid system. S. cerevisiae AH109 cells transformed with Gal4-binding domain-fused and Gal4 activation domain-fused genes were grown on SD without leucine, tryptophan, histidine, and adenine. (B) Interactions were also tested using a colorimetric assay to test for α-galactosidase activity. The constructions GA-T/GB-Lam and GA-T/GB-53 were used as negative and positive controls, respectively. (C) Western blot analysis of Pab1 (MycHis-pab1construct) and Mob1 (mob1-GFP construct) with anti-α-His and anti-α-GFP antibodies, respectively. Yeast extracts were purified using immobilized cobalt affinity from a Mob1-GFP strain expressing Myc6His-Pab1 protein from a plasmid. Lane 1: mob1-GFP strain; lane 2: wild-type strain expressing pREPMycHis-pab1; and lane 3: mob1-GFP strain expressing pREPMycHis-pab1. In lane 3, Mob1-GFP is copurified with Pab1 protein. (D) Model for the physical assembly of different SIN components and regulators in the SPB from Morrell et al. (2004). The Sid2-Cdc14 complex is shown to interact with Pab1.

Figure 6.—

Role for Pab1 in morphogenesis. (A) Actin staining using rhodamine-conjugated phalloidin (in red) and microtubule localization using Atb2-GFP fusion protein (in green) in wild-type (wt) and pab1-4 cells at 30°. (B) Suppression assay of etd1Δ cells lethality by scd1Δ, a null allele of the scd1 gene encoding a Cdc42 GEF required for morphogenesis. Growth of etd1Δ, etd1Δ pab1-4 (positive control), and etd1Δ scd1Δ cells was assessed by serial dilution drop tests on plates (10-fold dilutions starting with 10⁴ cells). (C) Microscopic images of F-actin structures (rhodamine-conjugated phalloidin staining) in pab1-4 mutant cells (control) and pab1-4 cells overexpressing wee1 (pREP3x-wee1, nmt1-driven expression after 18 hr of derepression at 25°). (D) Microscopic images of F-actin (rhodamine-conjugated phalloidin staining) in pab1-4 cdc2-33 cells at 25° (permissive temperature as a control) and after 6 hr incubation at 36° (restrictive temperature for cdc2-33). Bars, 5 μm.