The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins--and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect proxy for such information. To facilitate global protein analyses, we have created a Saccharomyces cerevisiae fusion library where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location. Through immunodetection of the common tag, we obtain a census of proteins expressed during log-phase growth and measurements of their absolute levels. We find that about 80% of the proteome is expressed during normal growth conditions, and, using additional sequence information, we systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 10(6) molecules per cell. Many of these molecules, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements.

A complete description of protein metabolism requires knowledge of the rates of protein production and destruction within cells. Using an epitope-tagged strain collection, we measured the halflife of >3,750 proteins in the yeast proteome after inhibition of translation. By integrating our data with previous measurements of protein and mRNA abundance and translation rate, we provide evidence that many proteins partition into one of two regimes for protein metabolism: one optimized for efficient production or a second optimized for regulatory efficiency. Incorporation of protein half-life information into a simple quantitative model for protein production improves our ability to predict steady-state protein abundance values. Analysis of a simple dynamic protein production model reveals a remarkable correlation between transcriptional regulation and protein half-life within some groups of coregulated genes, suggesting that cells coordinate these two processes to achieve uniform effects on protein abundances. Our experimental data and theoretical analysis underscore the importance of an integrative approach to the complex interplay between protein degradation, transcriptional regulation, and other determinants of protein metabolism.degradation ͉ proteomics ͉ cycloheximide ͉ epitope-tagged T he availability of whole-genome sequences and the advent of microarray technology have made global analyses of mRNA expression mainstream. However, most biological processes are mediated by proteins, which are subject to posttranscriptional regulation that is generally not observable at mRNA levels. A complete understanding of biological systems requires knowledge of protein properties, which is ultimately the goal of proteomics.Despite tremendous technical advances and effort in proteomics, the chemical heterogeneity of proteins and the large dynamic range of protein abundance make it challenging to establish global proteomic assays. This obstacle has been circumvented in the yeast Saccharomyces cerevisiae with the availability of two collections of yeast strains expressing epitope-tagged fusion proteins, one by using the tandem affinity purification (TAP) tag and a second employing the GFP (1, 2). In an initial study, we analyzed the TAP-tagged strain collection by Western blotting to quantify steady-state levels of protein abundance in actively dividing yeast cells. These data augmented previous efforts to quantify protein abundance by using mass spectrometry and 2D gel electrophoresis and provided a more comprehensive estimate of protein levels in a eukaryotic cell (3, 4).The availability of high-throughput protein abundance data has facilitated analysis of the relationship between protein abundance and mRNA levels. Although a statistically significant correlation is observed between these parameters, individual genes with similar mRNA levels can produce proteins with very different abundances. This complication makes it difficult to extrapolate from mRNA levels and microarray experiments to protein abundance. Three potential...

All genetic markers from phage T2 are partially excluded from the progeny of mixed infections with the related phage T4 (general, or phage exclusion). Several loci, including gene 56 of T2, are more dramatically excluded, being present in only ∼1% of the progeny. This phenomenon is referred to as localized marker exclusion. Gene 69 is adjacent to gene 56 of T4 but is absent in T2, being replaced by completely nonhomologous DNA. We describe SegF, a novel site-specific DNA endonuclease encoded by gene 69, which is similar to GIY-YIG homing endonucleases of group I introns. Interestingly, SegF preferentially cleaves gene 56 of T2, both in vitro and in vivo, compared with that of phage T4. Repair of the double-strand break (DSB) results in the predominance of T4 genes 56 and segF in the progeny, with exclusion of the corresponding T2 sequences. Localized exclusion of T2 gene 56 is dependent on full-length SegF and is likely analogous to group I intron homing, in which repair of a DSB results in coconversion of markers in the flanking DNA. Phage T4 has many optional homing endonuclease genes similar to segF, whereas similar endonuclease genes are relatively rare in other members of the T-even family of bacteriophages. We propose that the general advantage enjoyed by T4 phage, over almost all of its relatives, is a cumulative effect of many of these localized events.[Key Words: Gene conversion; homing endonuclease; phage exclusion; T-even bacteriophage]Received November 7, 2001; accepted December 12, 2001. Homing endonucleases are site-specific DNA endonucleases that are typically found encoded within introns and inteins (in-frame insertions that are removed by protein self-splicing). These endonucleases are required for initiation of a process known as homing (Dujon 1989), a phenomenon that was originally recognized as the unidirectional inheritance of the mitochondrial allele of Saccharomyces cerevisiae. Subsequently, it was discovered that the allele is an optional intron in the mitochondrial large (21S) subunit rRNA gene and that its pattern of inheritance is a consequence of gene conversion initiated by the endonuclease encoded within the intron (Jacquier and Dujon 1985). Homing is therefore described as an endonuclease-initiated process resulting in transfer of the intron and the endonuclease encoded within it, into the cognate intronless allele of the gene (Lambowitz and Belfort 1993).Intron-encoded homing endonucleases are phylogenetically widespread, occurring in Archaea, Bacteria, and Eukarya. They have been grouped into protein families based on conserved amino acid motifs: the LAGLI-DADG, GIY-YIG, His-Cys box, and H-N-H families. Recently, structural similarities between proteins having the His-Cys box and H-N-H motifs have led to the suggestion that these may be consolidated into a single group (Kü hlmann et al. 1999). Structural and biochemical information about representative members of these protein families has recently been reviewed (Chevalier and Stoddard 2001).Remarkably, despite this diversity,...