Nuclear Localization of the Saccharomyces cerevisiae HMG Protein NHP6A Occurs by a Ran-Independent Nonclassical Pathway

The Saccharomyces cerevisiae non-histone protein 6-A (NHP6A) is a member of the high-mobility group 1/2 protein family that bind and bend DNA of mixed sequence. NHP6A has only one high-mobility group 1/2 DNA binding domain and also requires a 16-amino-acid basic tail at its N-terminus for DNA binding. We show in this report that nuclear accumulation of NHP6A is strictly correlated with its DNA binding properties since only nonhistone protein 6 A–green fluorescent protein chimeras that were competent for DNA binding were localized to the nucleus. Despite the requirement for basic residues within the N-terminal segment for DNA binding and nuclear accumulation, this region does not appear to contain a nuclear localization signal. Moreover, NHP6A does not bind to the yeast nuclear localization signal receptor SRP1 and nuclear targeting of NHP6A does not require the function of the 14 different importins. Unlike histone H2B1 which contains a classical nuclear localization signal, entry of NHP6A into the nucleus was found to be independent of Ran as judged by coexpression of Ran GTPase mutants and was shown to occur at 0 °C after a 15-min induction. These unusual properties lead us to suggest that NHP6A entry into the nucleus proceeds by a nonclassical Ran-independent pathway.     

The dosage of chromatin proteins affects transcriptional silencing and DNA repair in Saccharomyces cerevisiae

Alterations in protein composition or dosage within chromatin may trigger changes in processes such as gene expression and DNA repair. Through transposon mutagenesis and targeted gene deletions in haploids and diploids of Saccharomyces cerevisiae, we identified mutations that affect telomeric silencing in genes encoding telomere-associated Sir4p and Yku80p and chromatin remodeling ATPases Ies2p and Rsc1p. We found that sir4/SIR4 heterozygous diploids efficiently silence the mating type locus HMR but not telomeres, and diploids heterozygous for yku80 and ies2 mutations are inefficient at DNA repair. In contrast, strains heterozygous for most chromatin remodeling ATPase mutations retain wild-type silencing and DNA repair levels. Thus, in diploids, chromatin structures required for DNA repair and telomeric silencing are sensitive to dosage changes.     

Functional analysis of a duplicated linked pair of ribosomal protein genes in Saccharomyces cerevisiae.

Ribosomal protein genes RP28 and S16A (RP55) are closely linked. Another set of this pair of genes exists in the genome (copy 2), genetically unlinked to copy 1. By using gene replacement techniques, we have shown that RP28 from copy 1 is required for vegetative growth and that the cells need S16A from copy 2 to achieve maximum growth rate.     

Heterologous expression of syntaxin 6 in Saccharomyces cerevisiae

The molecular mechanisms of vesicular protein transport in eukaryotic cells are highly conserved. Members of the syntaxin family play a pivotal role in the membrane fusion process. We have expressed rat syntaxin 6 and its cytoplasmic domain in wild-type and pep12 mutant strains of Saccharomyces cerevisiae to elucidate the role of the syntaxin 6-dependent vesicular trafficking step in yeast. Immunofluorescence microscopy revealed a punctate, Golgi-like staining pattern for syntaxin 6, which only partially overlapped with Pep12p in wild-type yeast cells. In contrast to Pep12p, syntaxin 6 was not mislocalized to the vacuole upon expression from 2 micron vectors, which might be attributed to conserved sorting and retention signals. Syntaxin 6 was not capable of complementing the sorting and maturation defects of the vacuolar hydrolase CPY in pep12 null mutants. No dominant negative effects of either syntaxin 6 or syntaxin 6 delta C overexpression on CPY sorting and maturation were observed in wild-type yeast cells. We conclude that syntaxin 6 and Pep12p do not act at the same vesicular trafficking step(s) in yeast and higher eukaryotes.     

Amino-terminal sequence of a Saccharomyces cerevisiae nuclear protein, NHP6, shows significant identity to bovine HMG1

Several nonhistone chromatin proteins (NHPs) have been isolated from Saccharomyces cerevisiae nuclei. They have molecular masses and amino acid compositions typical of the high mobility group (HMG) proteins from higher eukaryotic cells. Polyclonal antisera raised against two of the NHPs have been used in immunoblots of proteins from subcellular fractions of yeast to show that the NHPs are indeed nuclear. In addition, the amino-terminal amino acid sequences of several of the NHPs were determined. Importantly, the amino-terminal sequence of one of the proteins, NHP6, has significant (60%) identity with a stretch of amino acids in calf thymus HMG1.     

PET genes of Saccharomyces cerevisiae.

We describe a collection of nuclear respiratory-defective mutants (pet mutants) of Saccharomyces cerevisiae consisting of 215 complementation groups. This set of mutants probably represents a substantial fraction of the total genetic information of the nucleus required for the maintenance of functional mitochondria in S. cerevisiae. The biochemical lesions of mutants in approximately 50 complementation groups have been related to single enzymes or biosynthetic pathways, and the corresponding wild-type genes have been cloned and their structures have been determined. The genes defined by an additional 20 complementation groups were identified by allelism tests with mutants characterized in other laboratories. Mutants representative of the remaining complementation groups have been assigned to one of the following five phenotypic classes: (i) deficiency in cytochrome oxidase, (ii) deficiency in coenzyme QH2-cytochrome c reductase, (iii) deficiency in mitochondrial ATPase, (iv) absence of mitochondrial protein synthesis, and (v) normal composition of respiratory-chain complexes and of oligomycin-sensitive ATPase. In addition to the genes identified through biochemical and genetic analyses of the pet mutants, we have cataloged PET genes not matched to complementation groups in the mutant collection and other genes whose products function in the mitochondria but are not necessary for respiration. Together, this information provides an up-to-date list of the known genes coding for mitochondrial constituents and for proteins whose expression is vital for the respiratory competence of S. cerevisiae.     

Plasma-membrane phospholipid unsaturation affects expression of the general amino-acid permease in Saccharomyces cerevisiae Y185

Saccharomyces cerevisiae Y185, enriched in linoleyl residues and incubated for up to 4 h in derepression buffer, more rapidly acquired general amino-acid permease (GAP) activity, as measured by the rate of accumulation of L-alanine, compared with organisms enriched in oleyl residues. A GAP-less mutant incubated under the same conditions did not acquire further L-alanine-accumulating ability, irrespective of the nature of the fatty-acyl enrichment. During derepression, KT values for the GAP were virtually identical irrespective of the fatty-acyl enrichment, but Vmax values were greater for linoleyl residue-enriched organisms, particularly after 1 h in derepression buffer. During incubation in derepression buffer, organisms with either fatty-acyl enrichment did not differ in the size of the amino-N pool, the concentration of L-alanine in that pool, rates of protein synthesis and glucose fermentation, or rate and extent of incorporation of label from H2 32PO-4. Under conditions used to measure rates of L-alanine accumulation, organisms with either enrichment showed no evidence of metabolism of accumulated L-alanine.     

Gene expression and survival changes in Saccharomyces cerevisiae during suspension culture

This study explores the connection between changes in gene expression and the genes that determine strain survival during suspension culture, using the model eukaryotic organism, Saccharomyces cerevisiae. The Saccharomyces cerevisiae homozygous diploid deletion pool (HDDP), and the BY4743 parental strain were grown for 18 h in a rotating wall vessel (RWV), a suspension culture device optimized to minimize the delivered shear. In addition to the reduced shear conditions, the RWVs were also placed in a static position or in a shaker in order to change the amount of shear stress on the cells. Using simple linear regression, it was found that there were 140 differentially expressed genes for which >70% of the variation can be explained by shear stress alone. A significant number of these genes are involved in catalytic activity. In the HDDP, shear stress was associated with significant survival changes in 15 deletion strains (R(2>) > 0.7) Interestingly, both analyses uncovered changes in the ribosomal protein machinery. Comparing the changes in gene expression and strain survival under the different shear conditions allows for the insights into the molecular mechanisms behind the cells response to shear stress. This in turn can provide information for the optimization of suspension culture.     

The heterologous expression of polysaccharidase-encoding genes with oenological relevance in Saccharomyces cerevisiae

AIMS: The main objective of this study was to develop polysaccharide-degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking. METHODS AND RESULTS: Two yeast expression/secretion gene cassettes were constructed, namely (i) a pectinase gene cassette (pPPK) consisting of the endo-polygalacturonase gene (pelE) from Erwinia chrysanthemi and the pectate lyase gene (peh1) from Erwinia carotovora and (ii) a glucanase/xylanase gene cassette (pEXS) containing the endo-beta-1,4-glucanase gene (end1) from Butyrivibrio fibrisolvens and the endo-beta-1,4-xylanase gene (xynC) from Aspergillus niger. The commercial wine yeast strain, VIN13, was transformed separately with these two gene cassettes and checked for the production of pectinase, glucanase and xylanase activities. Pinot Noir, Cinsaut and Muscat d'Alexandria grape juices were fermented using the VIN13[pPPK] pectinase- and the VIN13[pEXS] glucanase/xylanase-producing transformants. Chemical analyses of the resultant wines indicated that (i) the pectinase-producing strain caused a decrease in the concentration of phenolic compounds in Pinot Noir whereas the glucanase/xylanase-producing strain caused an increase in phenolic compounds presumably because of the degradation of the grape skins; (ii) the glucanase/xylanase-producing strain caused a decrease in wine turbidity, especially in Pinot Noir wine, as well as a clear increase in colour intensity and (iii) in the Muscat d'Alexandria and Cinsaut wines, the differences between the control wines (fermented with the untransformed VIN3 strain) and the wines produced by the two transformed strains were less prominent showing that the effect of these polysaccharide-degrading enzymes is cultivar-dependent. CONCLUSIONS: The recombinant wine yeasts producing pectinase, glucanase and xylanase activities during the fermentation of Pinot Noir, Cinsaut and Muscat d'Alexandria grape juice altered the chemical composition of the resultant wines in a way that such yeasts could potentially be used to improve the clarity, colour intensity and stability and aroma of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspects of commercial-scale wine processing and clarification, colour extraction and stabilization, and aroma enhancement could potentially be improved by the use of polysaccharide-degrading wine yeasts without the addition of expensive commercial enzyme preparations. This offers the potential to further improve the price:quality ratio of wine according to consumer expectations.     

Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae

Metabolic pathway engineering in the yeast Saccharomyces cerevisiae leads to improved production of a wide range of compounds, ranging from ethanol (from biomass) to natural products such as sesquiterpenes. The introduction of multienzyme pathways requires precise control over the level and timing of expression of the associated genes. Gene number and promoter strength/regulation are two critical control points, and multiple studies have focused on modulating these in yeast. This MiniReview focuses on methods for introducing genes and controlling their copy number and on the many promoters (both constitutive and inducible) that have been successfully employed. The advantages and disadvantages of the methods will be presented, and applications to pathway engineering will be highlighted.     

Gene dosage effect of L-proline biosynthetic enzymes on L-proline accumulation and freeze tolerance in Saccharomyces cerevisiae

We have previously reported that L-proline has cryoprotective activity in Saccharomyces cerevisiae. A freeze-tolerant mutant with L-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding gamma-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo. Here, we found that the Asp154Asn mutant gamma-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex. We next examined the gene dosage effect of three L-proline biosynthetic enzymes, including Delta(1)-pyrroline-5-carboxylate reductase, which converts Delta(1)-pyrroline-5-carboxylate into L-proline, on L-proline accumulation and freeze tolerance in a non-L-proline-utilizing strain. Overexpression of the wild-type enzymes has no influence on L-proline accumulation, which suggests that the complex is very unstable in nature. However, co-overexpression of the mutant gamma-glutamyl kinase and the wild-type gamma-glutamyl phosphate reductase was effective for L-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not Delta(1)-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells. A high tolerance for freezing clearly correlated with higher levels of L-proline in yeast cells. Our findings also suggest that, in addition to its cryoprotective activity, intracellular L-proline could protect yeast cells from damage by oxidative stress. The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses.