Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.In the yeast Saccharomyces cerevisiae, starvation for an amino acid or a defective aminoacyl-tRNA synthetase triggers increased transcription of over 40 genes encoding enzymes involved in amino acid biosynthesis. This response, known as general amino acid control, requires derepression of GCN4, a transcriptional activator which binds to the promoter regions of genes subject to the general control. GCN4 expression is increased at the translational level by a regulatory mechanism involving phosphorylation of translation initiation factor eIF2 by the protein kinase GCN2 (24). During the process of initiation, eIF2 delivers the initiator methionyl-tRNA (tRNA i Met ) to 40S ribosomal subunits in an eIF2/GTP/Met-tRNA i Met ternary complex and is released as an eIF2/GDP binary complex. eIF2 must be recycled to the GTP-bound state that is competent for ternary complex formation by the guanine nucleotide exchange factor eIF2B (22). Phosphorylation of the alpha subunit of eIF2 (eIF2␣) by GCN2 ...

Two elongation factors drive the ribosomal elongation cycle; elongation factor 1 alpha (EF-1 alpha) mediates the binding of an aminoacyl-tRNA to the ribosomal A site, whereas elongation factor 2 (EF-2) catalyzes the translocation reaction. Ribosomes from yeast and other higher fungi require a third elongation factor (EF-3) which is essential for the elongation process, but the step affected by EF-3 has not yet been identified. Here we demonstrate that the first and the third tRNA binding site (A and E sites, respectively) of yeast ribosomes are reciprocally linked; if the A site is occupied the E site has lost its binding capability, and vice versa, if the E site is occupied the A site has a low affinity for tRNAs. EF-3 is essential for EF-1 alpha-dependent A site binding of amino-acyl-tRNA only when the E site is occupied with a deacylated tRNA. The ATP-dependent activity of EF-3 is required for the release of deacylated tRNA from the E site during A site occupation.

Targeting of different cellular proteins for conjugation and subsequent degradation via the ubiquitin of the cellular proteins are Na-acetylated (7). As for the remaining free-N-termini proteins, the rules that govern removal of the initiator methionine residue by methionine aminopeptidase suggest that in most cases this residue is cleaved only when the penultimate residue is a "stabilizing" amino acid (8). Thus, proteins with exposed destabilizing N-termini appear to be sparse. (ii) The ubiquitin system degrades Na-acetylated proteins in a process that does not require removal ofthe modifying group and exposure ofa free N-terminal residue (9). We have previously shown that the degradation of certain Na-acetylated proteins requires a specific factor that is not required for the breakdown of free N-termini proteins. The factor, designated factor Hedva (FH), is required for the proteolysis of the core nucleosomal histone H2A, the cytoskeletal protein actin, and the lens protein a-crystallin (11). FH is a homodimer with a subunit molecular mass of46 kDa. Initial analysis of the mechanism of action of FH revealed that it is not involved in the conjugation process. Rather, it acts along with the 26S protease complex and stimulates degradation of conjugated H2A. The effect appears to be specific to this group of proteins, as the factor is not required for the degradation ofconjugates of several proteins with free N-termini, such as oxidized RNase A and lysozyme (11). Further analysis demonstrated that FH probably interacts with the conjugates prior to their degradation: incubation of conjugates in the presence of purified FH and the protease revealed a short, but significant, time lag that preceded initiation of degradation. The lag was completely abolished when FH was preincubated with the conjugates prior to the addition of the protease. These findings demonstrate that recognition of certain proteins and their targeting for degradation involves both conjugation ofubiquitin and degradation of the adducts by the 26S protease complex. MATERIALS AND METHODSPreparation of Ubiquitin-Conjugated Histone H2A. Multiply ubiquitinated histone H2A was prepared using 125I-H2A, ubiquitin, ATP, and purified El (ubiquitin-activating enzyme), E2 (14-kDa ubiquitin-carrier protein or ubiquitinconjugating enzyme), and E3a (ubiquitin-protein ligase) as described (11).

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