The decision of whether or not to initiate the cell cycle is crucial to the control of cell proliferation. Also, once the cell cycle has been initiated, many processes need to be regulated in a precise temporal and spacial program. To date, protein kinases have been shown to play a key role in regulation of the cell cycle. However, very little is known about regulation of the cell cycle by protein phosphatases. The Saccharomyces cerevisiae SIT4 protein phosphatase is required for the G1 to S stage transition. Strains containing temperature sensitive mutations in SIT4 give a cell cycle arrest phenotype as large unbudded uninucleate G1 cells at the nonpermissive temperature. Reciprocal shift experiments show that entry into the cell cycle from G1 requires SIT4 and that this is the only time in the cell cycle that SIT4 is required. The following specific aims are proposed to determine how the activity of SIT4 is regulated and what specific functions SIT4 performs in the G1/S transition. We will determine how the phosphatase activity of SIT4 is regulated during the cell cycle and if the association of SIT4 with two proteins, p43 and p56, serves to modulate the activity of SIT4. Since mutations in SIT4 interact genetically with and alter some of the same functions as both the RAS/cAMP-dependent protein kinase and the CDC28 protein kinase pathways, we will determine the role of SIT4 in relation to these two pathways. Since mutations in SIT4 were originally isolated as transcriptional suppressors, we will try to define the role of SIT4 in transcription by RNA polymerase II. Using genetic approaches, we have identified putative regulators and substrates of SIT4 (encoded by the PTS and SIS genes). The SIS1 protein is essential for viability and is a particularly interesting protein since it is homologous to the bacterial dnaJ protein. Therefore, SIS1 may mediate a cell cycle regulated dissociation of a protein from a protein/protein complex. Since one of the roles of bacterial dnaJ protein is during initiation of DNA replication, we will determine if SIS1 is required for initiation of DNA replication in yeast. We will analyze in detail the functions of the PTS and SIS gene products and how they relate to the function of SIT4.
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