Using a combination of tandem affinity purification tagging and mass spectrometry, we characterized a novel, evolutionarily conserved protein phosphatase 4 (PP4)-containing complex (PP4cs, protein phosphatase 4, cisplatin-sensitive complex) that plays a critical role in the eukaryotic DNA damage response. PP4cs is comprised of the catalytic subunit PP4C; a known regulatory subunit, PP4R2; and a novel protein that we termed PP4R3. The Saccharomyces cerevisiae PP4R3 ortholog Psy2 was identified previously in a screen for sensitivity to the DNAdamaging agent and anticancer drug cisplatin. We demonstrated that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicited cisplatin hypersensitivity. Reversible protein phosphorylation is a highly conserved, essential regulatory mechanism involved in a host of cellular processes. Yet, while the phosphorylation of regulatory molecules by kinases has been studied intensively, their subsequent dephosphorylation is much less well understood. In eukaryotes, dephosphorylation on serine/threonine residues is effected by two distinct groups of functionally diverse phosphatases, the phosphoprotein M (represented by a sole member in higher eukaryotes, PP2C) and PPP 1 families (1 PP2A often functions as a standard trimeric complex with a catalytic (C) subunit (encoded by two genes in mammals) associated with one of many regulatory (or B) subunits via one of two adaptor (A) molecules (4, 5). The regulatory and adaptor subunits are thought to confer substrate specificity to the complex (5).In contrast to PP2A, the supramolecular architecture and subunit composition of PP4 multiprotein complexes remains largely unknown. Two mammalian PP4 regulatory subunits were previously identified (here termed PP4R1 and PP4R2, gene names PPP4R1 and PPP4R2; Refs. 6 and 7). Although PP4R1 shares some sequence homology with the PP2A adaptor proteins (PPP2R1A and PPP2R1B), it does not bridge PP4C and PP4R2; PP4R1 and PP4R2 display mutually exclusive PP4C interactions (Refs. 6 and 7; and see below). Other PP4C-interacting partners have also been reported (e.g. Refs. 8 and 9), but whether these proteins represent bona fide regulatory subunits or phosphatase substrates and how these binding proteins may affect PP4 activity are unclear.To gain a better understanding of the composition, function, and regulation of PP4, we systematically analyzed mammalian and yeast PP4C-interacting proteins. In doing so, we From the ‡Institute for

Interpreting genome sequences requires the functional analysis of thousands of predicted proteins, many of which are uncharacterized and without obvious homologs. To assess whether the roles of large sets of uncharacterized genes can be assigned by targeted application of a suite of technologies, we used four complementary protein-based methods to analyze a set of 100 uncharacterized but essential open reading frames (ORFs) of the yeast Saccharomyces cerevisiae. These proteins were subjected to affinity purification and mass spectrometry analysis to identify copurifying proteins, two-hybrid analysis to identify interacting proteins, fluorescence microscopy to localize the proteins, and structure prediction methodology to predict structural domains or identify remote homologies. Integration of the data assigned function to 48 ORFs using at least two of the Gene Ontology (GO) categories of biological process, molecular function, and cellular component; 77 ORFs were annotated by at least one method. This combination of technologies, coupled with annotation using GO, is a powerful approach to classifying genes.

Kinetochores attach the replicated chromosomes to the mitotic spindle and orchestrate their transmission to the daughter cells. Kinetochore–spindle binding and chromosome segregation are mediated by the multi-copy KNL1Spc105, MIS12Mtw1 and NDC80Ndc80 complexes that form the so-called KMN network. KMN–spindle attachment is regulated by the Aurora BIpl1 and MPS1Mps1 kinases. It is unclear whether other mechanisms exist that support KMN activity during the cell cycle. Using budding yeast, we show that kinetochore protein Cnn1 localizes to the base of the Ndc80 complex and promotes a functionally competent configuration of the KMN network. Cnn1 regulates KMN activity in a spatiotemporal manner by inhibiting the interaction between its complexes. Cnn1 activity peaks in anaphase and is driven by the Cdc28, Mps1 and Ipl1 kinases.