The fission yeast genes pyp1+ and pyp2+ encode protein tyrosine phosphatases that negatively regulate mitosis (original) (raw)
Related papers
A fission-yeast gene encoding a protein with features of protein-tyrosine-phosphatases
Proceedings of the …, 1991
Degenerate oligonucleotide probes encoding sequences conserved among mammalian protein-tyrosinephosphatases (PTPases) were used to amplify DNA fragments from a Schizosaccharomyces pombe cDNA library by polymerase chain reaction (PCR) methods. A cloned PCR product predicted peptide sequences similar to those found in PTPases but not identical to any published sequences. A S. pombe gene, designated pyp1 , was identified in a cDNA library with this PCR probe, cloned, and sequenced. The sequence of the gene predicted a 550-amino acid protein with Mr 61,586, which includes amino acid sequences that are highly conserved in mammalian PTPases. Disruption of the pypl + gene resulted in viable cells. Overexpression of the pypi+ gene in S. pombe permitted detection of a protein of apparent Mr 63,000.
Experimental Cell Research, 2001
In Saccharomyces cerevisiae, PTPA is encoded by two genes, YPA1 and YPA2. In order to examine the biological role of PTPA as potential regulator of protein phosphatase 2A (PP2A), we compared the phenotypes of the ypa⌬ mutants with these of PP2A-deficient strains. While deletion of both YPA genes is lethal, deletion of YPA1 alone results in a phenotype resembling that of PP2A-deficient strains in specific aspects such as aberrant bud morphology, abnormal actin distribution, and similar growth defects under various growth conditions. These phenotypes were even more pronounced when YPA1 was deleted in a pph21⌬ genetic background. Moreover, ypa⌬ mutants are hypersensitive to nocodazole and show inappropriate mitotic spindle formation as previously described for mutants in the catalytic subunit of PP2A, suggesting that Ypa, like PP2A, has a function in mitotic spindle formation. These results are consistent with an in vivo role of Ypa as a regulator of PP2A. However, unlike a PP2A-deficient strain, ypa⌬ mutants do not show a G2 arrest. Therefore, Ypa does not seem to play a role in the regulation of PP2A at this stage of the cell cycle. These results imply that Ypa regulates a specific subset of PP2A functions, possibly by controlling the subunit composition of PP2A.
PLoS ONE, 2009
The availability of yeast strain collections expressing individually tagged proteins to facilitate one-step purification provides a powerful approach to identify proteins with particular biochemical activities. To identify novel exo-and endo-nucleases that might function in DNA repair, we undertook a proteomic screen making use of the movable ORF (MORF) library of yeast expression plasmids. This library consists of 5,854 yeast strains each expressing a unique yeast ORF fused to a tripartite tag consisting of His 6 , an HA epitope, a protease 3C cleavage site, and the IgG-binding domain (ZZ) from protein A, under the control of the GAL1 promoter for inducible expression. Pools of proteins were partially purified on IgG sepharose and tested for nuclease activity using three different radiolabeled DNA substrates. Several known nucleases and phosphatases were identified, as well as two new members of the histidine phosphatase superfamily, which includes phosphoglycerate mutases and phosphatases. Subsequent characterization revealed YDR051c/Det1 to be an acid phosphatase with broad substrate specificity, whereas YOR283w has a broad pH range and hydrolyzes hydrophilic phosphorylated substrates. Although no new nuclease activities were identified from this screen, we did find phosphatase activity associated with a protein of unknown function, YOR283w, and with the recently characterized protein Det1. This knowledge should guide further genetic and biochemical characterization of these proteins.
Eukaryotic Cell, 2007
In Schizosaccharomyces pombe, over 90% of transcription factor genes are nonessential. Moreover, the majority do not exhibit significant growth defects under optimal conditions when deleted, complicating their functional characterization and target gene identification. Here, we systematically overexpressed 99 transcription factor genes with the nmt1 promoter and found that 64 transcription factor genes exhibited reduced fitness when ectopically expressed. Cell cycle defects were also often observed. We further investigated three uncharacterized transcription factor genes (toe1 + -toe3 + ) that displayed cell elongation when overexpressed. Ectopic expression of toe1 + resulted in a G1 delay while toe2 + and toe3 + overexpression produced an accumulation of septated cells with abnormalities in septum formation and nuclear segregation, respectively. Transcriptome profiling and ChIP-chip analysis of the transcription factor overexpression strains indicated that Toe1 activates target genes of the pyrimidine-salvage pathway, while Toe3 regulates target genes involved in polyamine synthesis. We also found that ectopic expression of the putative target genes SPBC3H7.05c, and dad5 + and SPAC11D3.06 could recapitulate the cell cycle phenotypes of toe2 + and toe3 + overexpression, respectively. Furthermore, single deletions of the putative target genes urg2 + and SPAC1399.04c, and SPBC3H7.05c, SPACUNK4.15, and rds1 + , could suppress the phenotypes of toe1 + and toe2 + overexpression, respectively. This study implicates new transcription factors and metabolism genes in cell cycle regulation and demonstrates the potential of systematic overexpression analysis to elucidate the function and target genes of transcription factors in S. pombe. Available freely online through the author-supported open access option. Supporting information is available online at http://www.genetics.org/lookup/suppl/
Regulation of Schizosaccharomyces pombeWee1 Tyrosine Kinase
Journal of Biological Chemistry, 1997
Wee1 tyrosine kinase regulates mitosis by carrying out the inhibitory tyrosine 15 phosphorylation of Cdc2 M-phase inducing kinase. Schizosaccharomyces pombe Wee1 is a large protein, consisting of a C-terminal catalytic domain of ϳ350 amino acids preceded by a N-terminal domain of ϳ550 residues. The functional properties of the Wee1 N-terminal domain were investigated by expressing truncated forms of Wee1 in S. pombe. Both positive and negative regulatory domains were identified. Sequences important for Wee1 function were mapped to a central region (residues 363-408). This region is not required for kinase activity or nuclear localization, suggesting it may be involved in substrate recognition. The negative regulatory domain resides in the N-terminal third of Wee1, Wee1 constructs lacking this domain are more effective at delaying mitosis than wildtype Wee1. The negative regulatory domain contains clusters of potential Cdc2 phosphorylation sites. Investigations to monitor the abundance of Wee1 mRNA and protein during the cell cycle were also carried out.
Genetics
RNA polymerase I of Saccharomyces cerevisiae contains a small subunit, A12.2, encoded by RPA12, that was previously shown to be involved in the assembly and/or stabilization of the largest subunit, A190, of RNA polymerase I. To examine whether an equivalent subunit is present in another eukaryotic RNA polymerase I, we have cloned a Schizosaccahromyces pombe cDNA that is able to complement the rpa12 mutation in S. cerevisiae. The gene, named Sprpa12 + , encodes a polypeptide of 119 amino acids that shows 55% identity to S. cerevisiae A12. 2 over its entire length, including two zinc-®nger motifs. Disruption of the chromosomal Sprpa12 + gene shows that it is required for growth at higher temperatures but not at lower temperatures. Expression of Sprpa190 + /nuc1 + , which encodes the largest subunit of the S. pombe RNA polymerase I, from a multicopy plasmid can partially suppress the growth defect of the Sprpa12 disruptant at higher temperatures. These ®ndings suggest that A12.2 subunit is functionally and structurally conserved between S. cerevisiae and S. pombe. Finally, the analysis of mutants suggests that SpRPA12 requires the zinc-®nger domain in the Nterminal region but not the one in the C-terminal region for its function.