Calvin S. McLaughlin

A sampling of the yeast proteome.

ABSTRACT 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.

Cell-cycle regulation of yeast histone mRNA

The levels of H2A and H2B mRNAs as a function of cell-cycle stage were determined by hybridization methods. The analysis was extended to H3 and H4 mRNAs by in vitro translation. Cells were partitioned into cell-cycle stages either by centrifugal elutriation or by G1 synchronization with the yeast mating pheromone, alpha factor. The data lead to the following conclusions. First, histone mRNA can be detected in significant quantities only in S-phase cells. Second, the point of maximal accumulation of histone mRNA is not coincident with the point of maximal DNA synthesis; rather, histone mRNA begins accumulating very early in S, reaching a maximum when less than one half of the DNA has replicated. From this point in the cell cycle the histone mRNA levels decrease, reaching basal levels at the end of S. Third, in spite of the fact that the rate of histone mRNA accumulation is not coincident with the rate of DNA synthesis, the two processes are coupled; inhibition of DNA synthesis results in an extremely rapid disappearance of histone mRNA that is much shorter than the normal histone mRNA half-life. Fourth, there is no visible accumulation of mRNA precursors at any cell-cycle stage. We can conclude that, in yeast, histone mRNA levels are tightly and coordinately regulated throughout cell division and that this regulation most likely occurs at both transcriptional and posttranscriptional levels. We also show that the two genetically unlinked H2B genes present in yeast are both expressed at comparable levels and are regulated. The regulation is probably sequence-specific, since genes in close proximity to the histones are not subject to cell-cycle control.

Induction of heat shock proteins and thermotolerance by ethanol in Saccharomycescerevisiae

Abstract The temperature sensitivity of Saccharomyces cerevisiae and the conditions of moderate heat pretreatment required to induce thermotolerance are established. Ethanol is identified as an inducer of heat shock proteins and an inducer of thermotolerance.

Identification of ten genes that control ribosome formation in yeast.

SummaryTwenty-three temperature-sensitive mutants of Saccharomyces cerevisiae, all of which undergo a rapid cessation of net RNA accumulation following a shift from the permissive (23°) to the restrictive temperature (36°), have been characterized. Genetic studies demonstrate that these mutants belong to ten different complementation groups and that, in most cases, their properties are the result of a single, recessive mutation in a nuclear gene. Although the mutants were isolated for heat sensitivity, mutants from 2 of the complementation groups are cold sensitive (at 13°) as well. The mutants continue to synthesize protein, including an enzyme, alkaline phosphatase, for two to four hours following a shift from 23° to 36°, suggesting that they are capable of messenger RNA synthesis and the translation of messenger RNA with fidelity at the restrictive temperature. The small amount of RNA that is synthesized in these mutants at the restrictive temperature has been examined on sucrose gradients and by acrylmide gel electrophoresis; in addition, the RNA components in polyribosomes have been fractionated by a new technique that separates messenger RNA from ribosomal RNA. As a result of these analyses we conclude that these mutants are strongly inhibited in the accumulation of 5S, 7S, 17S, and 25S RNA components but are only slight if at all inhibited in the synthesis of messenger RNA and 4S RNA. The results reported here define ten genes, designated rna 2 through rna 11, that play an essential role in the formation or maturation of ribosomes in yeast.

Structure-function relationship in the 12, 13-epoxytrichothecenes novel inhibitors of protein synthesis

Abstract An important group of mycotoxins, the 12,13-epoxytrichothecenes (Fig. 1), are among the most active inhibitors of protein synthesis in eucaryotic cells. Some of these compounds inhibit a step required for the initiation of protein synthesis, while other compounds inhibit a step required for the termination or elongation of protein synthesis. Structure-function studies on the various derivatives indicate which areas of the molecule are important in the inhibition of initiation and termination or elongation.

Binding of trichodermin to mammalian ribosomes and its inhibition by other 12,13-epoxytrichothecenes

SummaryTrichodermin binds to ribosomes from rabbit reticulocyte polysomes with an apparent Ka of 9.2 × 105 and with a maximum binding ratio of 0.44 molecules per ribosome. Trichodermin also binds firmly to HeLa cell polysomes under conditions where it is inhibiting protein synthesis. Purified HeLa 60S ribosomal subunits showed only a slight ability to bind trichodermin, and no binding was found with 40S subunits. Seventeen 12,13-epoxytrichothecenes were tested for their ability to interfere with [3H] trichodermin binding to reticulocyte ribosomes. All compounds known to inhibit ribosomal functions required for elongation-termination or initiation processes exhibited significant positive interference with the binding of trichodermin.

The effect of cycloheximide upon polyribosome stability in two yeast mutants defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis

SummaryThe effect of cycloheximide upon protein synthesis, RNA metabolism, and polyribosome stability was investigated in the parent and in two temperature-sensitive mutant yeast strains defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis. Cycloheximide at high concentrations (100 μg/ml) severely inhibits but does not completely stop protein synthesis (Fig. 1); the incorporation of 14C-amino acids into polyribosome-associated nascent polypeptide chains continues at a slow but measurable rate (Figs. 2 and 3). Polyribosome structures are stable in the parent strain at 36° whether or not cycloheximide is present (Fig. 5). However, in Mutant ts- 136, a mutant defective in messenger as well as in stable RNA production, polyribosomes decay at the restrictive temperature (36° C) at the same rate whether or not cycloheximide is present (Fig. 5). Thus the maintenance of polyribosome structures is dependent upon the continued synthesis of messenger RNA even under conditions of extremely slow polypeptide chain elongation. In mutant ts- 187, a mutant defective in the initiation of polypeptide chains, all of the polyribosomes decay to monoribosomes within 2 minutes after a shift to the restrictive temperature; cycloheximide completely prevents this decay demonstrating that this mutant is capable of continued messenger RNA synthesis at 36° C. Consistent with these observations is the fact that a newly synthesized heterogeneously sedimenting RNA fraction continues to enter polyribosomes in the presence of cycloheximide whereas the entrance of newly synthesized ribosomal RNA is severely inhibited (Figs. 7, 8, 9). The decay or lack of decay of polyribosomes at the restrictive temperature is, therefore, a rapid and discriminating test for the analysis of mutants defective in macromolecule synthesis. Mutants which exhibit a decay of polyribosomes in the presence of cycloheximide are likely to be defective directly or indirectly in the synthesis of messenger RNA whereas mutants in which decay is prevented or slowed by cycloheximide are likely to be defective in some factor required for the association of ribosomes and messenger RNA.

Monocistronic messenger RNA in yeast

Abstract We have determined the rate of polypeptide chain synthesis on different size polysomes in yeast. The completion time for the average polypeptide chain in vivo at 23 °C is two minutes by this technique and is in good agreement with values we have determined by other independent methods. These kinetic experiments indicate that the average size of a nascent polypeptide chain on a polysome is directly related to the size of the polysome. This demonstrates that in the simple eucaryotic organism, Saccharomyces cerevisiae , mRNA is monocistronic in the sense that each mRNA molecule codes for one protein molecule which is released intact from the ribosome upon completion. The pattern of amino acid incorporation into Escherichia coli polysomes is distinctly different. These findings have a number of interesting implications for the genetics of the lower eucaryotes and indicate that the cellular mechanisms of control and co-ordination in yeast may differ from those found in procaryotes and may be similar to cellular mechanisms of control for mammalian cells.

The extrachromosomal control of nonsense suppression in yeast: an analysis of the elimination of [psi+] in the presence of a nuclear gene PNM.

SummaryWhen a [psi-] strain of yeast mutates to [psi+], the efficiency of suppression by certain ochre suppressors is increased. The [psi+] phenotype is inherited extrachromosomally. There is a nuclear gene, PNM, which, when mutant, causes loss of the [psi+] phenotype. PNM- is dominant to PNM+ and a heterozygous diploid gradually loses the ability over successive generations, to produce PNM+ [psi+] spores. This paper describes the kinetics of this elimination and the data obtained are discussed in relation to two models of the molecular nature of the [psi] genetic determinant—one considering the [psi] determinant as an autonomous nucleic acid, the other treating the possibility that the [psi] nucleic acid is that which codes for rRNA in the nuclear genome.

Altered ribosomal protein S11 from the SUP46 suppressor of yeast

Abstract The dominant suppressor SUP46 of the yeast Saccharomyces cerevisiae was shown to act on a wide range of mutations (preceding paper by Ono et al., 1981). Masurekar et al. (1981) demonstrated that ribosomes from the SUP46 strain make an abnormally high rate of errors in a cell-free translation system. These findings indicated that SUP46 suppression was the result of abnormal ribosomes misreading mutant codons. We have used two-dimensional polyacrylamide gel electrophoresis to show that the S11 protein from the 40 S ribosomal subunit has an altered electrophoretic mobility. Thus the gene product of the SUP46 locus is either the S11 ribosomal protein or an enzyme that modifies the S11 protein. These results demonstrate that the altered S11 protein is responsible for the suppression by misreading.