ABSTRACT
Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the budding yeast Wee1 kinase, Swe1, also restrains the metaphase-to-anaphase transition by preventing Cdk1 phosphorylation and activation of the mitotic form of the anaphase-promoting complex/cyclosome (APC(Cdc20)). Deletion of SWE1 or its opposing phosphatase MIH1 (the budding yeast cdc25(+)) altered the timing of anaphase onset, and activation of the Swe1-dependent morphogenesis checkpoint or overexpression of Swe1 blocked cells in metaphase with reduced APC activity in vivo and in vitro. The morphogenesis checkpoint also depended on Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). cdc55Δ checkpoint defects were rescued by mutating 12 Cdk1 phosphorylation sites on the APC, demonstrating that the APC is a target of this checkpoint. These data suggest a model in which stepwise activation of Cdk1 and inhibition of PP2A(Cdc55) triggers anaphase onset.
Subject(s)
Anaphase/physiology , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Metaphase/physiology , Protein-Tyrosine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , CDC2 Protein Kinase/genetics , Cell Cycle Proteins/genetics , Genes, cdc/physiology , Phosphorylation/physiology , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Protein-Tyrosine Kinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Ubiquitination/physiology , ras-GRF1/genetics , ras-GRF1/metabolismABSTRACT
We report the characterization of two new theta-replicating plasmids of Streptococcus thermophilus (pSMQ-312b and pSMQ-316) as well as the further analysis of pSMQ-308. The nucleotide sequences of pSMQ-312b and pSMQ-316 were determined and both contained 6710 bp. In fact, the two sequences were identical, despite that the plasmids were isolated from two different S. thermophilus strains as demonstrated by pulsed-field gel electrophoresis. Comparative analyses indicated that the two plasmids were highly related to the previously characterized S. thermophilus plasmid pSMQ-308 (8144 bp). Plasmid stability tests showed that pSMQ-312b/316 was more stable in LM17 medium while pSMQ-308 was the most stable in milk. The presence of the plasmids did not modify the acidification profile of the S. thermophilus strains during growth in milk and under time-temperature conditions mimicking an industrial process. These theta-replicating plasmids are unique genetic material for the construction of stable cloning vectors for industrially relevant strains of S. thermophilus.