To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

We have developed an approach using Bayesian networks to predict protein-protein interactions genome-wide in yeast. Our method naturally weights and combines into reliable predictions genomic features only weakly associated with interaction (e.g., messenger RNAcoexpression, coessentiality, and colocalization). In addition to de novo predictions, it can integrate often noisy, experimental interaction data sets. We observe that at given levels of sensitivity, our predictions are more accurate than the existing high-throughput experimental data sets. We validate our predictions with TAP (tandem affinity purification) tagging experiments. Our analysis, which gives a comprehensive view of yeast interactions, is available at genecensus.org/intint .

Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass ∼5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability-a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu. A global understanding of the molecular mechanisms underpinning cell biology necessitates an understanding not only of an organism's genome but also of the protein complement encoded within this genome (the proteome). In the past, data regarding an organism's proteome have typically been accumulated piecemeal through studies of a single protein or cell pathway. Genomic methodologies have altered this paradigm: a variety of approaches are now in place by which proteins may be directly analyzed on a proteome-wide scale. Chromatography-coupled mass spectrometry (Gygi et al. 1999;Washburn et al. 2001), large-scale two-hybrid screens (Uetz et al. 2000;Ito et al. 2001;Tong et al. 2002), immunoprecipitation/mass spectrometric analysis of protein complexes (Gavin et al. 2002;Ho et al. 2002), and protein microarray technologies (MacBeath and Schreiber 2000;Zhu et al. 2000Zhu et al. , 2001 are yielding unprecedented quantities of protein data. Recent genomic techniques combining microarray technologies with either chromatin immunoprecipitation (Ren et al. 2000;Iyer et al. 2001) or targeted DNA methylation (van Steensel et al. 2001) have been used to globally map binding sites of chromosomal proteins in vivo. Initiatives are even underway to automate and industrialize processes by which protein structures may be solved, potentially providing a library of structural...

We investigate the relationship of protein-protein interactions with mRNA expression levels, by integrating a variety of data sources for yeast. We focus on known protein complexes that have clearly defined interactions between their subunits. We find that subunits of the same protein complex show significant coexpression, both in terms of similarities of absolute mRNA levels and expression profiles, e.g., we can often see subunits of a complex having correlated patterns of expression over a time course. We classify the yeast protein complexes as either permanent or transient, with permanent ones being maintained through most cellular conditions. We find that, generally, permanent complexes, such as the ribosome and proteasome, have a particularly strong relationship with expression, while transient ones do not. However, we note that several transient complexes, such as the RNA polymerase II holoenzyme and the replication complex, can be subdivided into smaller permanent ones, which do have a strong relationship to gene expression. We also investigated the interactions in aggregated, genome-wide data sets, such as the comprehensive yeast two-hybrid experiments, and found them to have only a weak relationship with gene expression, similar to that of transient complexes. (Further details on genecensus.org/expression/interactions and bioinfo.mbb.yale.edu/expression/interactions.)