In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome.The sequence of the yeast genome has been determined (9). More recently, the number of mRNA molecules for each expressed gene has been measured (27,30). The next logical level of analysis is that of the expressed set of proteins. We have begun to analyze the yeast proteome by using two-dimensional (2D) gels.2D gel electrophoresis separates proteins according to isoelectric point in one dimension and molecular weight in the other dimension (21), allowing resolution of thousands of proteins on a single gel. Although modern imaging and computing techniques can extract quantitative data for each of the spots in a 2D gel, there are only a few cases in which quantitative data have been gathered from 2D gels. 2D gel electrophoresis is almost unique in its ability to examine biological responses over thousands of proteins simultaneously and should therefore allow us a relatively comprehensive view of cellular metabolism.We and others have worked toward assembling a yeast protein database consisting of a collection of identified spots in 2D gels and of data on each of these spots under various conditions (2,7,8,10,23,25). These data could then be used in analyzing a protein or a metabolic process. Saccharomyces cerevisiae is a good organism for this approach since it has a well-understood physiology as well as a large number of mutants, and its genome has been sequenced. Given the sequence and the relative lack of introns in S. cerevisiae, it is easy to predict the sequence of the primary protein product of most genes. This aids tremendously in identifying these proteins on 2D gels.There are three pillars on which such a database rests: (i) visualization of many protein spots simultaneously, (ii) quantification of the protein in each spot, and (iii) identification of the gene product for each spot. Our first efforts at visualization and identification for S. cerevisiae have been described elsewhere (7,8). Here we describe quantitative data for these proteins under a variety of experimental conditions. ade2-1 his3-11,15 leu2-3, 112 trp1-1 ura3-1 can1-100) was used (26). ϪMet YNB (yeast nitrogen base) medium was 1.7 g of YNB (Difco) per liter, 5 g of ammonium sulfate per liter, and adenine, uracil, and all amino acids except methionine; ϪMet ϪCys YNB medium was the same but wi...

Two-dimensional (2-D) gel electrophoresis can now be coupled with protein identification techniques and genome sequence information for direct detection, identification, and characterization of large numbers of proteins from microbial organisms. 2-D electrophoresis, and new protein identification techniques such as amino acid composition, are proteome research techniques in that they allow direct characterization of many proteins at the same time. Another new tool important for yeast proteome research is the Yeast Protein Database (YPD), which provides the sequence-derived protein properties needed for spot identification and tabulations of the currently known properties of the yeast proteins. Studies presented here extend the yeast 2-D protein map to 169 identified spots based upon the recent completion of the yeast genome sequence, and they show that methods of spot identification based on predicted isoelectric point, predicted molecular mass, and determination of partial amino acid composition from radiolabeled gels are powerful enough for the identification of at least 80% of the spots representing abundant proteins. Comparison of proteins predicted by YPD to be detectable on 2-D gels based on calculated molecular mass, isoelectric point and codon bias (a predictor of abundance) with proteins identified in this study suggests that many glycoproteins and integral membrane proteins are missing from the 2-D gel patterns. Using the 2-D gel map and the information available in YDP, 2-D gel experiments were analyzed to characterize the yeast proteins associated with: (i) an environmental change (heat shock), (ii) a temperature-sensitive mutation (the prp2 mRNA splicing mutant), (iii) a mutation affecting post-translational modification (N-terminal acetylation), and (iv) a purified subcellular fraction (the ribosomal proteins). The methods used here should allow future extension of these studies to many more proteins of the yeast proteome.

Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in Acya or Acrp mutants during starvation. Most of the cyciic AMP-dependent proteins were synthesized in the Acya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, ,l-galactosidase induction of several carbon starvation response gene fusions occurred only in a cyd+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coi require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since Acya or Acip mutants of either E. coli or Salmonella typhimurium survived starvation as well as thefr wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya-and cip-independent proteins were also induced during nitrogen starvation and, as shown previousl, during phosphate starvation. Proteins whose synthesis is independent of cya-and crp control are referred to as Pex (postexponential).Starving bacteria are of both fundamental and applied interest (14,15,21,27,28). We have shown that during the first 4 to 5 h of starvation for carbon substrates (glucose or succinate) approximately 30 proteins are induced in Escherichia coli K-12 (14, 15) and that these proteins are important in starvation survival (27,28,32). Starvation-induced proteins have different temporal patterns of synthesis-some are synthesized very transiently during starvation, whereas others have a broader peak of synthesis (15). Investigators in other laboratories have also shown that unique genes are switched on and new proteins are synthesized at the onset of starvation (10,33,34).To better understand the regulation of starvation protein synthesis, we have focused on the role of the signal molecule adenosine 3',5'-cyclic monophosphate (cAMP) and its receptor protein (CRP). Intracellular cAMP levels increase at the onset of carbon starvation (5,8,19,26), and there appears to be an inverse relationship between the energetic state of the cell and cAMP levels (8, 13). This raises the possibility that cAMP acts as a signal for starvation protein synthesis. We have therefore investigated the effect of the loss of cAMP control on starvation protein synthesis in E. coli. Isogenic E. coli strains deleted in the adenylate cyclase gene (Acya) or the gene encoding CRP (Acrp) were examined. The results indicate that although several starvation genes are positively regulated by cAMP, many others are not, and that the latter class of ge...

We identified six tropomyosin (Tm) isoforms in diploid human fibroblasts. We used computerized microdensitometry of 2-dimensional protein profiles to measure the relative rates of synthesis and abundance of the individual Tm isoforms and actin, the two major structural constituents of microfilaments. In carcinogen-transformed human fibroblasts (HuT-14), the rates of synthesis of three Tm isoforms (Tml, Tm2, and Tm6) were greatly decreased relative to normal diploid parental fibroblasts and to actin. In contrast, related nontumorigenic HuT fibroblasts which are "immortalized" and anchorage independent exhibited both slight down-regulation of Tml and Tm6 and 3.5-fold up-regulation of Tm3. Thus, Tm isoform switching from the predominance of the larger more avid Tm isoforms (Tml, Tm2, Tm3, and Tm6) to the smaller, less avid Tm isoforms (Tm4 and TmS) in microfilaments was a transformation-induced change correlated with tumorigenicity in human fibroblasts.

Two different mutant human j-actin genes have been introduced into normal diploid human (KD) fibroblasts and their immortalized derivative cell line, HuT-12, to assess the impact of an abnormal cytoskeletal protein on cellular phenotypes such as morphology, growth characteristics, and properties relating to the neoplastic phenotype. A mutant ,-actin containing a single mutation (Gly-244 --Asp-244) was stable and was incorporated into cytoskeletal stress fibers. Transfected KD cells which expressed the stable mutant I(-actin in excess of normal ,-actin were morphologically altered. In contrast, a second mutant ,-actin gene containing two additional mutations (Gly-36 -* Glu-36 and Glu-83 -* Asp-83, as well as Gly-244 -* Asp-244) did not alter cell morphology when expressed at high levels in transfected cells, but the protein was labile and did not accumulate in stress fibers. In both KD and HuT-12 cells, endogenous ,-and y-actin decreased in response to high-level expression of the stable mutant I-actin, in a manner consistent with autoregulatory feedback of actin concentrations. Since the percent decreases in the endogenous I8-and y-actins were equal, the ratio of net 0-actin (mutant plus normal) to y-actin was significantly increased in the transfected cells. Antisera capable of distinguishing the mutant from the normal epitope revealed that the mutant j-actin accumulated in stress fibers but did not participate in the formation of the actin filament-rich perinuclear network. These observations suggest that different intracellular locations differentially incorporate actin into cytoskeletal microfilaments. The dramatic impact on cell morphology and on P-actin/y-actin ratios in the transfected diploid KD cells may be related to the acquisition of some of the characteristics of cells that underwent the neoplastic transformation event that originally led to the appearance of the j-actin mutations.Actin is a ubiquitous, highly abundant protein that is responsible for a variety of cellular activities, including motility and the structural properties of the cytoplasm. This protein, highly conserved in evolution, forms complex structures in all eucaryotic cells by a combination of selfpolymerization and interactions with a host of binding proteins (reviewed in reference 37). These complex interactions have been the subject of intense biochemical and structural study but have not yet been amenable to genetic analysis. Few structural mutations in actin are known, as might be expected for a protein whose actions are so central to normal cell morphology and function. The recessive flightless mutants of Drosophila melanogaster show a disordered structure of the sarcomeres (18, 31) and induction of stress proteins (15,18,31) in the indirect flight muscles of homozygotes in which mutant alleles of a tissue-specific actin are expressed (18,31).Human fibroblasts which express mutant P-actins have been generated by the neoplastic transformation of diploid KD fibroblasts in vitro and the isolation of stable focusderived, neoplast...

The rapid progress in understanding the genes of the yeast Saccharomyces cerevisiae can be supplemented by two-dimensional (2-D) gel studies to understand global patterns of protein synthesis, protein modification, and protein degradation. The first step in building a protein database for yeast is to identify many of the spots on 2-D gels. We are using protein sequencing, overexpression of genes on high-copy number plasmids, and amino acid analysis to identify the proteins from 2-D gels of yeast. The amino acid analysis technique involves labeling yeast samples with different amino acids and using quantitative image analysis to determine the relative amino acid abundances. The observed amino acid abundances are then searched against the current database of 2600 known yeast protein sequences. At present about 90 proteins on our yeast maps have been identified, and the number is rising rapidly. With many known proteins on the map, it will soon be possible to use 2-D gel analysis to study regulatory pathways in normal and mutant yeast, with knowledge of many the protein products that respond to each genetic or environmental manipulation.

Several molecular theories of aging postulate that there are age-dependent changes in gene expression and that these changes contribute to the reduction in the viability of senescent cells. High-resolution, semiautomated, quantitative two-dimensional gel electrophoresis of many soluble proteins was used to test this hypothesis in Drosophila. Two-dimensional protein gel patterns were analyzed for each of three age groups of [(35)S]methionine-labeled adult male Drosophila melanogaster, which, except for their spermatocytes, consist entirely of fixed postmitotic cells. Seven relatively abundant polypeptides expressed in middle-aged (28-day-old) flies were absent in both young(10-day-old) and old (44-day-old) flies. Quantitative analyses of an additional 100 polypeptides were carried out by computer-assisted microdensitometry of fluorograms of the gel preparations. These analyses revealed a significant age-related heterogeneity in the quantitative distribution of radiolabel in these proteins. The data indicate that the qualitative pattern of gene expression is identical in young and old flies, but that profound quantitative changes occur in the expression of proteins during the Drosophila life-span.

Mutant human I8-actin genes were introduced into normal human (KD) fibroblasts and the derivative cell line HuT-12, which is immortalized but nontumorigenic, to test their ability to promote conversion to the tumorigenic state. Transfected substrains of fibroblasts that expressed abundant levels of mutant 0-actin (Gly-244 -> Asp-244) produced subcutaneous tumors in athymic mice after long latent periods (1.5 to 3 months). However, transfected substrains of KD fibroblasts retained their normal finite life span in culture and consequently were incapable of producing tumors. Substrains of HuT-12 cells transfected with the wild-type 0-actin gene and some transfected strains that expressed low or undetectable levels of mutant I-actin did not produce tumors. Cell lines derived from transfectant cell tumors always exhibited elevated synthesis of the mutant ,-actin, ranging from 145 to 476% of the level expressed by the transfected cells that were inoculated to form the tumor. In general, primary transfectant cells that expressed the highest levels of mutant ,-actin were more tumorigenic than strains that expressed lower levels. The tumor-derived strains were stable in tumorigenicity and produced tumors with shortened latent periods of only 2 to 4 weeks. These findings imply that the primary transfectant strains develop subpopulations of cells that are selected to form tumors because of their elevated rate of exogenous mutant 0-actin synthesis. Actin synthesis and accumulation of y-actin mRNA from the endogenous j(-and 'y-actin genes were diminished in tumor-derived strains, apparently to compensate for elevated mutant 0-actin synthesis and maintain the normal cellular concentration of actin.Synthesis of the transformation-sensitive tropomyosin isoforms was decreased along with mutant ,1-actin expression. Such modulations in tropomyosin synthesis are characteristically seen in transformation of avian, rodent, and human fibroblasts. Our results suggest that this mutant ,-actin contributes to the neoplastic phenotype of immortalized human fibroblasts by imposing a cytoarchitectural defect and inducing abnormal expression of cytoskeletal tropomyosins.