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.

The localization of proteins can give important clues about their function and help sort data from large-scale proteomic screens. Forty-five proteins were tagged with the GFP variant YFP. These proteins were chosen because they are encoded by genes that display strong cell cycle-dependent expression that peaks in G 1 . Most of these proteins localize to either the nucleus or to sites of cell growth. We are able to assign new cellular component GO terms to ASF2, TOS4, RTT109, YBR070C, YKR090W, YOL007C, YOL019W and YPR174C. We also have localization data for 21 other proteins. Noteworthy localizations were found for Rfa1p, a member of the DNA replication A complex, and Pri2p and Pol12p, subunits of the α-DNA polymerase : primase complex. In addition to its nuclear localization, Rfa1p assembled into cytoplasmic foci adjacent to the nucleus in cells during the G 1 -S phase transition of the cell cycle. Pri2 and Pol12 took on a beaded appearance at the G 1 -S transition and later in the cell cycle were enriched in the nuclear envelope. A new spindle pole body/nuclear envelope component encoded by YPR174 was identified. The cell cycle-dependent abundance of Tos4p mirrored Yox1p and these two proteins were the only proteins that were found exclusively at the G 1 -S phase of the cell cycle. A complete list of localizations, along with images, can be found at our website (http://www.yeastrc.org/cln2/).