The E-box DNA binding protein Sgc1p suppresses the gcr2 mutation, which is involved in transcriptional activation of glycolytic genes in Saccharomyces cerevisiae

AbstractGlycolytic gene expression is mediated by the Gcr1p-Gcr2p transcriptional activation complex. A screen for multicopy suppressors of gcr2 yielded SGC1. SGC1’s suppression activity was specific to gcr2, it did not extend to gcr1. Disruption of SGC1 moderately affected glycolytic enzyme activities, although no growth defect was evident. Sgc1p exhibits a bHLH motif which is characteristic of E-box DNA-binding proteins. DNA footprinting experiments demonstrated Sgc1p’s ability to bind at an E-box. However, its binding specificity was less than 10-fold, which is also characteristic of E-box binding proteins. LexA fusion experiments demonstrated that Sgc1p has weak intrinsic activating activity independent of GCR1 and GCR2. We propose that Sgc1p binds at E-boxes of glycolytic genes and contributes to their activation

PROTEIN ENGINEERING OF HUMAN-LYSOZYME

A gene encoding human-lysozyme was chemically synthesized and expressed both in E. coli and S. cerevisiae. The gene product expressed in E.coli formed insoluble material and had no enzymatic activity . For the expression in S. cerevisiae a signal sequence of chicken-lysozyme was attached. Prehuman-lysozyme expressed in yeast was properly processed and secreted outside the cell. Amino acid residues of catalytic and recognition sites (Glu35, Asp53, Tyr63, Trp64, Trp109) of human-lysozyme were changed by site specific mutagenesis and their influence to the enzymatic activity was examined. The surface charge of the enzyme has great effects on enzymatic activity to charged substrates. By increasing or decreasing the surface charge of human-lysozyme the optimum ionic strength or PH was shifted

Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in Saccharomyces cerevisiae▿ †

The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography

Construction of a Library of Human Glycosyltransferases Immobilized in the Cell Wall of Saccharomyces cerevisiae

Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library. In increasing the enzymatic activities of immobilized glycosyltransferases, several approaches were found to be effective. Additional expression of yeast protein disulfide isomerase increased the expression levels and activities of polypeptide N-acetylgalactosaminyltransferases and other glycosyltransferases. PIR3 and/or PIR4 was more effective than PIR1 as a cell wall anchor when the Pir-glycosyltransferase fusions were expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Oligosaccharides such as Lewis x, Lewis y, and H antigen were successfully synthesized using this immobilized glycosyltransferase library, indicating that the Pir-fused glycosyltransferases are useful for the production of various human oligosaccharides

Molecular and Phenotypic Analysis of CaVRG4, Encoding an Essential Golgi Apparatus GDP-Mannose Transporter

Cell surface mannan is implicated in almost every aspect of pathogenicity of Candida albicans. In Saccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision. The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p. This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatus-specific mannosylation) is a fungus-specific process. To begin to study the role of Golgi mannosylation in C. albicans, we isolated the CaVRG4 gene and analyzed the effects of loss of its function. CaVRG4 encodes a functional homologue of the S. cerevisiae GDP-mannose transporter. CaVrg4p localized to punctate spots within the cytoplasm of C. albicans in a pattern reminiscent of localization of Vrg4p in the Golgi apparatus in S. cerevisiae. Like partial loss of ScVRG4 function, partial loss of CaVRG4 function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes. While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy of CaVRG4 under control of the methionine-repressible MET3 promoter, did not grow in the presence of methionine and cysteine, demonstrating that CaVRG4 is essential for viability. Mutant Candida vrg4 strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth. Because the VRG4 gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies

Comparison of Cell Wall Localization among Pir Family Proteins and Functional Dissection of the Region Required for Cell Wall Binding and Bud Scar Recruitment of Pir1p

We examined the localization of the Pir protein family (Pir1 to Pir4), which is covalently linked to the cell wall in an unknown manner. In contrast to the other Pir proteins, a fusion of Pir1p and monomeric red fluorescent protein distributed in clusters in pir1Δ cells throughout the period of cultivation, indicating that Pir1p is localized in bud scars. Further microscopic analysis revealed that Pir1p is expressed inside the chitin rings of the bud scars. Stepwise deletion of the eight units of the repetitive sequence of Pir1p revealed that one unit is enough for the protein to bind bud scars and that the extent of binding of Pir1p to the cell wall depends on the number of these repetitive units. The localization of a chimeric Pir1p in which the repetitive sequence of Pir1p was replaced with that of Pir4p revealed the functional role of the different protein regions, specifically, that the repetitive sequence is required for binding to the cell wall and that the C-terminal sequence is needed for recruitment to bud scars. This is the first report that bud scars contain proteins like Pir1p as internal components

The Cytoplasmic Region of α-1,6-Mannosyltransferase Mnn9p Is Crucial for Retrograde Transport from the Golgi Apparatus to the Endoplasmic Reticulum in Saccharomyces cerevisiae▿ †

In Saccharomyces cerevisiae, Och1p and Mnn9p mannosyltransferases are localized in the cis-Golgi. Attempts to live image Och1p and Mnn9p tagged with green fluorescent protein or red fluorescent protein, respectively, using a high-performance confocal laser scanning microscope system resulted in simultaneous visualization of the native proteins in a living cell. Our observations revealed that Och1p and Mnn9p are not always colocalized to the same cisternae. The difference in the dynamics of these mannosyltransferases may reflect differences in the mechanisms for their retention in the cis-Golgi, since it has been reported that Mnn9p cycles between the endoplasmic reticulum and the cis-Golgi whereas Och1p does not (Z. Todorow, A. Spang, E. Carmack, J. Yates, and R. Schekman, Proc. Natl. Acad. Sci. USA 97:13643-13648, 2000). We investigated the localization of chimeric proteins of Mnn9p and Och1p in sec12 and erd1 mutant cells. A chimeric protein, M16/O16, which consists of the N-terminal cytoplasmic region of Mnn9p and the transmembrane and luminal region of Och1p, behaved like Mnn9p, suggesting that the N-terminal cytoplasmic region is important for the intracellular dynamics of Mnn9p. This observation is supported by results from subcellular-fractionation experiments. Mutational analysis revealed that two arginine residues in the N-terminal region of Mnn9p are important for the chimeric protein to cycle between the endoplasmic reticulum and the Golgi apparatus