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Cdk8 of the RNA polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55 and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation. [ABSTRACT FROM AUTHOR]

Defects in the spindle assembly checkpoint (SAC) can lead to aneuploidy and cancer. Sphingolipids have important roles in many cellular functions including cell cycle regulation and apoptosis. However, defining specific mechanisms and functions of sphingolipids in cell cycle regulation is missing. Using analysis of concordance for synthetic lethality for the yeast sphingolipid phospholipase ISC1, we identified two groups of genes: The first comprises genes involved in chromosome segregation and stability (CSM3, CTF4, YKE2, DCC1, GIM4) as synthetically lethal with ISC1. The second group, to which ISC1 belongs, comprises genes involved in the spindle checkpoint (BUB1, MAD1, BIM1, KAR3), and they all share the same synthetic lethality with the first group. We demonstrate that spindle checkpoint genes act upstream of Isc1, and their deletion phenocopies that of ISC1. Reciprocally, ISC1 deletion mutants were sensitive to Benomyl, indicating a SAC defect. Similar to BUB1 deletion, ISC1 deletion prevents spindle elongation in hydroxyurea treated cells. Mechanistically, PP2A-Cdc55 ceramide-activated phosphatase was found to act downstream of Isc1, thus, coupling the spindle checkpoint genes and Isc1 to CDC55-mediated nuclear functions. [ABSTRACT FROM AUTHOR]

Cdc55, a regulatory B-subunit of protein phosphatase 2A (PP2A) complex, is essential for the spindle assembly checkpoint (SAC) in budding yeast, but the regulation and molecular targets of PP2A-Cdc55 have not been clearly defined or are controversial. Here, we show that an important target of Cdc55 in the SAC is the anaphase-promoting complex (APC) coupled with Cdc20 and that APC-Cdc20 is kept inactive by dephosphorylation by nuclear PP2A-Cdc55 when spindle is damaged. By isolating a new class of Cdc55 mutants specifically defective in the SAC and by artificially manipulating nucleocytoplasmic distribution of Cdc55, we further show that nuclear Cdc55 is essential for the SAC. Because the Cdc55-binding proteins Zds1 and Zds2 inhibit both nuclear accumulation of Cdc55 and SAC activity, we propose that spatial control of PP2A by Zds1 family proteins is important for tight control of SAC and mitotic progression. [ABSTRACT FROM AUTHOR]

Spindle disruption or DNA damage prevents sister chromatid separation through the activation of checkpoint pathways that inhibit anaphase entry by stabilizing the anaphase inhibitor Pds1. Mutation of CDC55, which encodes a B regulatory subunit of protein phosphatase 2A (PP2A), results in precocious sister chromatid separation when spindle is disrupted. Here we report that decreased Pds1 levels in Δcdc55 mutants contribute to sister chromatid separation in the presence of nocodazole, a microtubule-depolymerizing drug. However, in the presence of DNA damage, Δcdc55 mutant cells separate sister chromatids without noticeable decrease of Pds1 or cohesin Mcd1/Scc1 levels. Further analysis demonstrates that Δcdc55 mutants lose cohesion along the entire chromosomes when the spindle is disrupted. In contrast, separation of sister chromatids is limited to the centromeric regions in Δcdc55 cells after DNA damage. Moreover, mutation of TPD3, which encodes the A regulatory subunit of PP2A, also results in sister chromatid separation in DNA- or spindle-damage-arrested cells. These data suggest that PP2A regulates sister chromatid cohesion in Pds1-dependent and -independent manners. [ABSTRACT FROM AUTHOR]

• cdc55 null mutant grows slowly as pseudohyphae with some cells lacking the nucleus. • rts1 null mutant cells are round and enlarged and seem to undergo incomplete cell separation producing multinucleated cells. • Strong chitin deposition occurs at the septum of cdc55 null mutant cells and on the surface of rts1 null mutant cells. • Deletion of cdc55 exhibits severe defects in hyphal and biofilm formation, while deletion of rts1 is partially defective. • cdc55 and rts1 null mutants show reduced virulence in mice. Protein phosphatase-2A (PP2A) is a heterotrimeric enzyme composed of a catalytic subunit, a regulatory subunit, and a structural subunit. In Candida albicans , Cdc55 and Rts1 have been identified as possible regulatory subunits of PP2A containing the catalytic subunit Pph21 and structural subunit Tpd3. The Tpd3-Pph21 phosphatase regulates cell morphogenesis and division. However, the functions of Cdc55 and Rts1 remain unclear. Here, we constructed cdc55 Δ/Δ and rts1 Δ/Δ mutants and found that they exhibit different defects in multiple phenotypes although both show similar hyperphosphorylation of the septin Sep7 and aberrant septin organization. Under yeast growth conditions, the cdc55 Δ/Δ mutant grows slowly as pseudohyphae with some cells lacking the nucleus, while rts1 Δ/Δ cells are round and enlarged and seem to undergo incomplete cell separation producing multinucleated cells. Strong chitin deposition occurs at the septum of cdc55 Δ/Δ cells and on the surface of rts1 Δ/Δ cells, which likely contributes to increased susceptibility to caspofungin. Also, cdc55 Δ/Δ exhibits severe defects in hyphal and biofilm formation, while rts1 Δ/Δ is partially defective. Both mutants show reduced virulence in mice, suggesting that PP2A-B subunits could serve as potential antifungal targets. [ABSTRACT FROM AUTHOR]