In yeast, progression through G1 is dependent on the function of the Ras/cAMP-dependent protein kinase (cAPK) pathway. Although some of the molecular details of this pathway have been described, almost nothing is understood about the cell cycle-specific protein, or processes, under cAPK control. By identifying and characterizing the gene products that mediate Ras/cAPK control, we aim to gain an understanding of how yeast cells coordinate entry into the cell-division cycle with macrovascular synthesis. The biochemical and functional identity between the yeast Ras proteins and their mammalian ras oncogene counterparts is intriguing in light of their respective roles in cell-cycle control. Moreover, because G1 control is found in almost all eukaryotes, with exceptions including tumorigenic cell lines, information we gain will likely be of broad, general interest. We will address the following, specific experimental aims: 1) The YAK1 gene has been genetically identified as a Ras suppressor. To understand the role of Yak1 in cell-cycle regulation, attempts will be made to assign a phenotype to strains bearing yak1 deletions. Biochemical experiments have shown that YAK1 encodes a kinase that is activated upon G1 arrest, and an immunocomplex assay will be exploited to study the mechanism of this activation. Extracts will be prepared from cells resident in different stages of the cell cycle, as well as strains containing YAK1 alleles bearing alterations (constructed in vitro or isolated in a genetic screen) of the putative regulatory domain. Finally, gene products that interact with Yak1, as regulators or substrates, will be identified by a genetic selection that relies on the conditional toxicity of Yak1. 2) It stands to reason that one or more cAPK substrates must mediae the cell-cycle regulatory activity of the Ras/cAPK pathway. To identify these substrates and their metabolic processes, two genetic screens will be employed. The first will identify mutations that confer a cell-cycle arrest phenotype to strains with low cAPK activity (synthetic lethals). The second will exploit the """"""""slow-growth"""""""" phenotype of strains lacking all cAPK and Yak1 activity (tpk1 tpk2 tpk3 yak1). Mutations and suppressors that fulfill the genetic criteria for genes whose products play a normal role in cell-cycle control will be cloned and characterized further by standard molecular analysis.
