Abstract: Cyclin-dependent kinases (Cdks) are key regulators of the cell division cycle. Pho85 is a multifunctional Cdk in budding yeast involved in aspects of metabolism, the cell cycle, cell polarity, and gene expression. Consistent with a broad spectrum of functions, Pho85 associates with a family of 10 cyclins and deletion of PHO85 causes a pleiotropic phenotype. Discovering the physiological substrates of protein kinases is a major challenge, and we have pursued a number of genomics approaches to reveal the processes regulated by Pho85 and to understand the root cause of reduced cellular fitness in pho85Delta mutant strains. We used a functional-genomics approach called synthetic genetic array (SGA) analysis to systematically identify strain backgrounds in which PHO85 is required for viability. In parallel, we used DNA microarrays to examine the genome-wide transcriptional consequences of deleting PHO85 or members of the Pho85 cyclin family. Using this pairwise approach coupled with phenotypic tests, we uncovered clear roles for Pho85 in cell integrity and the response to adverse growth conditions. Importantly, our combined approach allowed us to ascribe new aspects of the complex pho85 phenotype to particular cyclins; our data highlight a cell integrity function for the Pcl1,2 subgroup of Pho85 Cdks that is independent of a role for the Pho80-Pho85 kinase in the response to stress. Using a modification of the SGA technique to screen for suppressors of pho85Delta strain growth defects, we found that deletion of putative vacuole protein gene VTC4 suppressed the sensitivity of the pho85Delta strain to elevated CaCl(2) and many other stress conditions. Expression of VTC4 is regulated by Pho4, a transcription factor that is inhibited by the Pho80-Pho85 kinase. Genetic tests and electron microscopy experiments suggest that VTC4 is a key target of Pho4 and that Pho80-Pho85-mediated regulation of VTC4 expression is required for proper vacuole function and for yeast cell survival under a variety of suboptimal conditions. The integration of multiple genomics approaches is likely to be a generally useful strategy for extracting functional information from pleiotropic mutant phenotypes.