Fred Sherman

Getting started with yeast

Publisher Summary The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated. Yeast has only a slightly greater genetic complexity than bacteria and shares many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal bakers yeast are commercially available and can provide a cheap source for biochemical studies. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Cloned yeast sequences, accompanied by foreign sequences on plasmids, can therefore be directed at will to specific locations in the genome.

N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins

N(alpha)-terminal acetylation occurs in the yeast Saccharomyces cerevisiae by any of three N-terminal acetyltransferases (NAT), NatA, NatB, and NatC, which contain Ard1p, Nat3p and Mak3p catalytic subunits, respectively. The N-terminal sequences required for N-terminal acetylation, i.e. the NatA, NatB, and NatC substrates, were evaluated by considering over 450 yeast proteins previously examined in numerous studies, and were compared to the N-terminal sequences of more than 300 acetylated mammalian proteins. In addition, acetylated sequences of eukaryotic proteins were compared to the N termini of 810 eubacterial and 175 archaeal proteins, which are rarely acetylated. Protein orthologs of Ard1p, Nat3p and Mak3p were identified with the eukaryotic genomes of the sequences of model organisms, including Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, Mus musculus and Homo sapiens. Those and other putative acetyltransferases were assigned by phylogenetic analysis to the following six protein families: Ard1p; Nat3p; Mak3p; CAM; BAA; and Nat5p. The first three families correspond to the catalytic subunits of three major yeast NATs; these orthologous proteins were identified in eukaryotes, but not in prokaryotes; the CAM family include mammalian orthologs of the recently described Camello1 and Camello2 proteins whose substrates are unknown; the BAA family comprise bacterial and archaeal putative acetyltransferases whose biochemical activity have not been characterized; and the new Nat5p family assignment was on the basis of putative yeast NAT, Nat5p (YOR253W). Overall patterns of N-terminal acetylated proteins and the orthologous genes possibly encoding NATs suggest that yeast and higher eukaryotes have the same systems for N-terminal acetylation.

Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain.

Ubiquitin is ligated to L28, a component of the large ribosomal subunit, to form the most abundant ubiquitin-protein conjugate in S. cerevisiae. The human ortholog of L28 is also ubiquitinated, indicating that this modification is highly conserved in evolution. During S phase of the yeast cell cycle, L28 is strongly ubiquitinated, while reduced levels of L28 ubiquitination are observed in G1 cells. L28 ubiquitination is inhibited by a Lys63 to Arg substitution in ubiquitin, indicating that L28 is modified by a variant, Lys63-linked multiubiquitin chain. The K63R mutant of ubiquitin displays defects in ribosomal function in vivo and in vitro, including a dramatic sensitivity to translational inhibitors. L28, like other ribosomal proteins, is metabolically stable. Therefore, these data suggest a regulatory role for multiubiquitin chains that is reversible and does not function to target the acceptor protein for degradation.

RESPIRATION-DEFICIENT MUTANTS OF YEAST. II. BIOCHEMISTRY.

Abstract Investigations of various enzymic activities, respiratory capacity, and cytochrome content have been carried out on mutants of yeast which had: p genes resulting in the inability to utilize nonfermentable carbon sources for growth; the cy1 gene resulting in a partial deficiency of cytochrome c; the “loss” of the cytoplasmic factor (ϱ−) necessary for the synthesis of cytochromes a + a3 and b; and various combinations of these determinants. p4ϱ+ strains respired (but “ineffectually”) and had low concentrations of cytochromes a + a3 and b. p5ϱ+ strains were deficient in respiration and cytochromes a+a3. p1ϱ+, p6ϱ+, p7ϱ+, Pϱ− an all pϱ− strains were deficient in respiration and cytochromes a + a3 and b. Numerous strains, which had various alterations in the content of cytochromes a + a3, b, c, and c1 + b2, could be obtained by using various combinations of the p/P genes, cy1/CY gene and ϱ+/ϱ− cytoplasmic factor. The frequently occuring deficiency of cytochromes a + a3 and b, is discussed in relationship to mitochondrial structure.

Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast and humans

Nα-terminal acetylation is one of the most common protein modifications in eukaryotes. The COmbined FRActional DIagonal Chromatography (COFRADIC) proteomics technology that can be specifically used to isolate N-terminal peptides was used to determine the N-terminal acetylation status of 742 human and 379 yeast protein N termini, representing the largest eukaryotic dataset of N-terminal acetylation. The major N-terminal acetyltransferase (NAT), NatA, acts on subclasses of proteins with Ser-, Ala-, Thr-, Gly-, Cys- and Val- N termini. NatA is composed of subunits encoded by yARD1 and yNAT1 in yeast and hARD1 and hNAT1 in humans. A yeast ard1-Δ nat1-Δ strain was phenotypically complemented by hARD1 hNAT1, suggesting that yNatA and hNatA are similar. However, heterologous combinations, hARD1 yNAT1 and yARD1 hNAT1, were not functional in yeast, suggesting significant structural subunit differences between the species. Proteomics of a yeast ard1-Δ nat1-Δ strain expressing hNatA demonstrated that hNatA acts on nearly the same set of yeast proteins as yNatA, further revealing that NatA from humans and yeast have identical or nearly identical specificities. Nevertheless, all NatA substrates in yeast were only partially N-acetylated, whereas the corresponding NatA substrates in HeLa cells were mainly completely N-acetylated. Overall, we observed a higher proportion of N-terminally acetylated proteins in humans (84%) as compared with yeast (57%). N-acetylation occurred on approximately one-half of the human proteins with Met-Lys- termini, but did not occur on yeast proteins with such termini. Thus, although we revealed different N-acetylation patterns in yeast and humans, the major NAT, NatA, acetylates the same substrates in both species.

Nα-terminal Acetylation of Eukaryotic Proteins

The two cotranslational processes, cleavage of N-terminal methionine residues and N-terminal acetylation, are by far the most common modifications, occurring on the vast majority of eukaryotic proteins. Studies with the yeast Saccharomyces cerevisiae revealed three N-terminal acetyltransferases, NatA, NatB, and NatC, that acted on groups of substrates, each containing degenerate motifs. Orthologous genes encoding the three N-terminal acetyltransferases and the patterns of N-terminal acetylation suggest that eukaryotes generally use the same systems for N-terminal acetylation. The biological significance of this N-terminal modification varies with the particular protein, with some proteins requiring acetylation for function, whereas others do not.

Micromanipulation and dissection of asci.

Publisher Summary This chapter describes the micromanipulation and dissection of asci. Separation of the four ascospores from individual asci by micromanipulation is required for meiotic genetic analyses and for the construction of strains with specific markers. In addition, micromanipulation is used to separate zygotes from mass-mating mixtures and, less routinely, for positioning of vegetative cells and spores for mating purposes and for single-cell analyses. Micromanipulation can be implemented directly on the surfaces of ordinary petridishes filled with nutrient medium or in special chambers on thin agar slabs. The petridish is positioned so that the inoculum is in the microscope field over the microneedle. Examination of the streak should reveal the presence of the desired four-spored clusters as well as smaller clusters and vegetative cells. The relocation and transfer of ascospores, zygotes, and vegetative cells are almost exclusively carded out on agar surfaces with a fine glass microneedle mounted in the path of a microscope objective and controlled by a micromanipulator. Although specialized equipment and some experience are required to carry out these procedures, most workers can acquire proficiency with a few days of practice.

The diversity of acetylated proteins

Acetylation of proteins, either on various amino-terminal residues or on the ε-amino group of lysine residues, is catalyzed by a wide range of acetyltransferases. Amino-terminal acetylation occurs on the bulk of eukaryotic proteins and on regulatory peptides, whereas lysine acetylation occurs at different positions on a variety of proteins, including histones, transcription factors, nuclear import factors, and α-tubulin.

Phenotypic suppression of nonsense mutants in yeast by aminoglycoside antibiotics

STREPTOMYCIN, an aminoglycoside antibiotic, can reverse the mutant phenotypes of many nonsense and missense mutations in Escherichia coli and in bacteriophage T4. This phenomenon has been called phenotypic suppression, since the mutant phenotype returns after removal of the drug1. The most likely explanation for phenotypic suppression is that streptomycin promotes mistranslation in vivo, and that acceptable amino acids are inserted into the growing polypeptide chain at the site of the mutant codon. Consistent with this view is the observation that streptomycin causes E. coli ribosomes to mistranslate RNA in vitro2,3. Streptomycin and neomycin have however been found to have no effect in stimulating ribosomes from eukaryotic cells to mistranslate RNA in vitro4,5. A subclass of the aminoglycoside antibiotics has been shown6,7 to stimulate eukaryotic ribosomes to misread RNA. The highly active molecules are distinguished in that they contain the drug fragment paromamine (or 3′-deoxyparomamine). We have therefore examined the capacity of various aminoglycosides to suppress mutations phenotypically in the eukaryotic yeast, Saccharomyces cerevisiae. The results presented here show that paromomycin, which contains paromamine, is capable of phenotypic suppression of the nonsense mutations in S. cerevisiae.

Identification and sequence of the gene encoding cytochrome c heme lyase in the yeast Saccharomyces cerevisiae

Mitochondrial cytochrome c contains a heme group covalently attached through thioether linkages to two cysteinyl residues of the protein. We demonstrate here that the nuclear gene, CYC3, in the yeast Saccharomyces cerevisiae, encodes cytochrome c heme lyase (CCHL), the enzyme catalyzing the attachment of heme to apocytochrome c. Mitochondrial extracts from cyc3‐ mutants are deficient in CCHL activity compared with extracts from normal strains, whereas strains carrying multiple copies of the CYC3 gene exhibit high levels of the activity. The CYC3 gene was cloned by functional complementation of a cyc3‐ mutant using a previously isolated plasmid containing the gene PYK1, which is tightly linked to CYC3. An open reading frame encoding a protein of 269 amino acids was identified from the DNA sequence of a fragment encompassing the CYC3 gene, and the corresponding transcript shown to be approximately 0.9 kb in length. CCHL appears to be a single polypeptide chain which acts specifically on the two forms of cytochrome c, but not on cytochrome c1.