Mutation of the protein-O-mannosyltransferase enhances secretion of the human urokinase-type plasminogen activator in Hansenula polymorpha

Human urokinase-type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein-O-mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene (HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O-glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C-terminal KDEL retention signal.     

The sequence of a 16691bp segment of Saccharomyces cerevisiae chromosome IV identifies the DUN1, PMT1, PMT5, SRP14 and DPR1 genes,and five new open reading frames

As part of the European BIOTECH programme, the nucleotide sequence of a 16691bp fragment from the left arm of chromosome IV of Saccharomyces cerevisiae has been deduced. Analysis of the sequence reveals the presence of 13 open reading frames (ORFs) larger than 100 codons. Five of these were previously identified as genes DUN1, PMT1, PMT5, SRP14 and DPR1. One putative protein, D2371p, contains an ATP-GTP binding site, and shares homology to the ArsA component of an Escherichia coli arsenical pump. No significant homology to any known protein has been found for the other ORFs. D2378p contains a zinc finger domain. The nucleotide sequence has been deposited at EMBL, with Accession Number X95644.     

Sequencing of a 17·6 kb segment on the right arm of yeast chromosome VII reveals 12 ORFs,including CCT,ADE3 and TR-I genes,homologues of the yeast PMT and EF1G genes,of the human and bacterial electron-transferring flavoproteins (β-chain) and of the Escherichia coli phosphoserine phosphohydrolase,and five new ORFs

A 17·6 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains twelve open reading frames (ORFs) longer than 100 amino acids. Three genes had already been cloned and sequenced: CCT, ADE3 and TR-I. Two ORFs are similar to other yeast genes: G7722 with the YAL023 (PMT2) and PMT1 genes, encoding two integral membrane proteins, and G7727 with the first half of the genes encoding elongation factors 1γ, TEF3 and TEF4. Two other ORFs, G7742 and G7744, are most probably yeast orthologues of the human and Paracoccus denitrificans electron-transferring flavoproteins (β chain) and of the Escherichia coli phosphoserine phosphohydrolase. The five remaining identified ORFs do not show detectable homology with other protein sequences deposited in data banks. The sequence has been deposited in the EMBL data library under Accession Number Z49133.     

Designer deletion and prototrophic strains derived from Saccharomyces cerevisiae strain W303‐1a

We report the construction of Saccharomyces cerevisiae strains isogenic to W303‐1a that are designed to allow efficient genetic analysis. To facilitate the generation of null alleles of target genes by PCR‐mediated gene disruption, we constructed designer deletion alleles of the ARG4, TRP1 and URA3 genes. In addition, a single pair of oligonucleotide primers were designed that can be used to amplify any of several marker genes for use in PCR‐mediated gene disruption. A new version of the ‘reusable’ hisG‐URA3‐hisG cassette was constructed for use in PCR‐mediated gene disruption. Finally, to facilitate the formation of isogenic diploids by selection, we constructed strains that contain combinations of wild‐type alleles of ADE2, HIS3, LEU2, TRP1 and URA3. Copyright © 1999 John Wiley & Sons, Ltd.     

Completion of the Saccharomyces cerevisiae Genome Sequence Allows Identification of KTR5, KTR6 and KTR7 and Definition of the Nine-Membered KRE2/MNT1 Mannosyltransferase Gene Family in this Organism

The KRE2/MNT1 mannosyltransferase gene family of Saccharomyces cerevisiae currently consists of the KRE2, YUR1, KTR1, KTR2, KTR3 and KTR4 genes. All six encode putative type II membrane proteins with a short cytoplasmic N-terminus, a membrane-spanning region and a highly conserved catalytic lumenal domain. Here we report the identification of the three remaining members of this family in the yeast genome. KTR5 corresponds to an open reading frame (ORF) of the left arm of chromosome XIV, and KTR6 and KTR7 to ORFs on the left arms of chromosomes XVI and IX respectively. The KTR5, KTR6 and KTR7 gene products are highly similar to the Kre2p/Mnt1p family members. Initial functional characterization revealed that some mutant yeast strains containing null copies of these genes displayed cell wall phenotypes. None was K1 killer toxin resistant but ktr6 and ktr7 null mutants were found to be hypersensitive and resistant, respectively, to the drug Calcofluor White. The sequences have been deposited in the GenBank data library under Accession Numbers Z71305; U39205; Z46728.©1997 John Wiley & Sons, Ltd.     

The Saccharomyces cerevisiae enolase‐related regions encode proteins that are active enolases

In addition to two genes (ENO1 and ENO2) known to code for enolase (EC4.2.1.11), the Saccharomyces cerevisiae genome contains three enolase‐related regions (ERR1, ERR2 and ERR3) which could potentially encode proteins with enolase function. Here, we show that products of these genes (Err2p and Err3p) have secondary and quaternary structures similar to those of yeast enolase (Eno1p). In addition, Err2p and Err3p can convert 2‐phosphoglycerate to phosphoenolpyruvate, with kinetic parameters similar to those of Eno1p, suggesting that these proteins could function as enolases in vivo. To address this possibility, we overexpressed the ERR2 and ERR3 genes individually in a double‐null yeast strain lacking ENO1 and ENO2, and showed that either ERR2 or ERR3 could complement the growth defect in this strain when cells are grown in medium with glucose as the carbon source. Taken together, these data suggest that the ERR genes in Saccharomyces cerevisiae encode a protein that could function in glycolysis as enolase. The presence of these enolase‐related regions in Saccharomyces cerevisiae and their absence in other related yeasts suggests that these genes may play some unique role in Saccharomyces cerevisiae. Further experiments will be required to determine whether these functions are related to glycolysis or other cellular processes. Copyright © 2012 John Wiley & Sons, Ltd.     

The SKS1 gene of Saccharomyces cerevisiae is required for long-term adaptation of snf3 null strains to low glucose

The SKS1 gene was originally identified as a multicopy suppressor of the growth defect of snf3 null mutations on low glucose concentrations. Snf3p is required for the rapid induction of HXT2 during growth on low substrate concentrations. Loss of Snf3p leads to a dramatic delay in expression of HXT2. Adaptation to low substrate concentrations does not occur in snf3 sks1 double null mutant strains, suggesting that SKS1 is required for the glucose-dependent expression of HXT2 in the absence of Snf3p activity. Over-expression of SKS1 leads to over-expression of Hxt2p, thus explaining the mechanism of suppression of the snf3 defect. SKS1 defines a novel, Snf3p-independent pathway for the expression of Hxt2p. Under certain growth conditions, over-expression of SKS1 itself leads to a growth defect which is diminished in snf3 hxt2 double mutants. This suggests that over-expression of Hxt2p at physiologically inappropriate times is detrimental to the cells. © 1998 John Wiley & Sons, Ltd.     

II. Yeast sequencing reports. The nucleotide sequence of TTP1, a gene encoding a predicted type II membrane protein

The DNA sequence of a 2967 bp fragment located near the centromere of chromosome II, between the CEN2 and FUR4 genes, was determined. The segment contains a new open reading frame of 1794 bp. The product encoded by the gene, designated TTP1, is a predicted type II membrane protein of 597 amino acid residues with a short cytoplasmic NH₂-terminus, a membrane-spanning region and a large COOH-terminal region containing three potential N-glycosylation sites. Gene disruption indicated that TTP1 is not essential for cell growth. The sequence has been deposited in the GenBank data library under Accession Number U05211.     

The S. cerevisiae nitrogen starvation-induced Yvh1p and Ptp2p phosphatases play a role in control of sporulation

Starvation for nitrogen in the absence of a fermentable carbon source causes diploid Saccharomyces cerevisiae cells to leave vegetative growth, enter meiosis, and sporulate; the former nutritional condition also induces expression of the YVH1 gene that encodes a protein phosphatase. This correlation prompted us to determine whether the Yvh1p phosphatase was a participant in the network that controls the onset of meiosis and sporulation. We found that expression of the IME2 gene, encoding a protein kinase homologue required for meiosis- and sporulation-specific gene expression, is decreased in a yvh1 disrupted strain. We also observed a decrease, albeit a smaller one, in the expression of IME1 which encodes an activator protein required for IME2 expression. Under identical experimental conditions, expression of the MCK1 and IME4 genes (which promote sporulation but do not require Ime1p for expression) was not affected. These results demonstrate the specificity of the yvh1 disruption phenotype. They suggest that decreased steady-state levels of IME1 and IME2 mRNA were not merely the result of non-specific adverse affects on nucleic acid metabolism caused by the yvh1 disruption. Sporulation of a homozygous yvh1 disruption mutant was delayed and less efficient overall compared to an isogenic wild-type strain, a result which correlates with decreased IME1 and IME2 gene expression. We also observed that expression of the PTP2 tyrosine phosphatase gene (a negative regulator of the osmosensing MAP kinase cascade), but not the PTP1 gene (also encoding a tyrosine phosphatase) was induced by nitrogen-starvation. Although disruption of PTP2 alone did not demonstrably affect sporulation or IME2 gene expression, sporulation was decreased more in a yvh1, ptp2 double mutant than in a yvh1 single mutant; it was nearly abolished in the double mutant. These data suggest that the YVH1 and PTP2 encoded phosphatases likely participate in the control network regulating meiosis and sporulation. Expression of YVH1 and PTP2 was not affected by nitrogen source quality (asparagine compared to proline) suggesting that nitrogen starvation-induced YVH1 and PTP2 expression and sensitivity to nitrogen catabolite repression are on two different branches of the nitrogen regulatory network.     

The yal017 gene on the left arm of chromosome I of Saccharomyces cerevisiae encodes a putative serine/threonine protein kinase

The DNA sequence of a region between the LTE1 and CYS3 genes on the left arm of chromosome I from Saccharomyces cerevisiae contains an open reading frame (ORF), YAL017, corresponding to the 5·0 kb FUN31 (F unction U nknown N ow) transcribed region. The predicted protein from this ORF contains 1358 amino acid residues with a molecular weight of 152 531, and an identifiable serine/threonine protein kinase catalytic domain. When compared with other yeast protein kinases, the Ya1017p kinase most resembles the SNF1 serine/threonine protein kinase which is involved in regulating sucrose fermentation genes. The Ya1017p kinase shows highest amino acid identities with two mammalian carcinoma-related serine/threonine protein kinases; PIM-1, which shows induced expression in T-cell lymphomas; and p78A1, whose expression is lost in human pancreatic carcinomas. Gene disruption of YAL017 indicates that it is non-essential for growth on glucose.     

Genomic disruption of six budding yeast genes gives one drastic example of phenotype strain-dependence

Using PCR to construct disruption cassettes, null alleles of six genes have been created in Saccharomyces cerevisiae. In a FY1679 background, no defects were detected in any of the haploid deletion mutants with respect to growth, gross morphology, or mating. A diploid FY1679-derived Δygl194c/Δygl194c homozygous disruptant displayed reduced sporulation. In contrast to the lack of phenotypic consequences of Δyol100w disruptions in the FY1679 background, in the CEN.PK2 strain even a heterozygous disruption of the same gene caused striking effects, very slow vegetative growth and highly impaired sporulation. Tetrad analysis showed YOL100w to be an essential gene in this strain. A copy of the YGL194c or the YOL100w wild-type gene borne on a centromeric episomal plasmid was introduced into a corresponding disruption mutant strain, and in both cases was found to partially complement the defects. © 1998 John Wiley & Sons, Ltd.     

N-glycosylation affects endoplasmic reticulum degradation of a mutated derivative of carboxypeptidase yscY in yeast

The endoplasmic reticulum (ER) of eukaryotic cells contains a quality control system, that is required for the proteolytic removal of aberrantly folded proteins that accumulate in this organelle. We used genetic and biochemical methods to analyse the involvement of N-glycosylation in the degradation of a mutant derivative of carboxypeptidase yscY in the ER of the yeast Saccharomyces cerevisiae. Our results demonstrate that N-glycosylation of this protein is required for its degradation since an unglycosylated species is retained stably in the ER. Cells that were devoid of the ER-processing α1,2-mannosidase showed reduced degradation of the glycosylated substrate protein. Disruption of CNE1, a gene encoding a putative yeast homologue for calnexin, did not exhibit any effects on the degradation of this substrate protein in vivo. Also, the α1,2-mannosidase-dependent reduction in the degradation rate did not show any correlation with the function of the CNE1 gene product. Our results suggest that the ER of yeast contains a glycosylation-dependent quality control system, as has been shown for higher eukaryotic cells.     

Suppression of sdh1 mutations by the SDH1b gene of Saccharomyces cerevisiae

Transformation of the respiratory-defective mutant (E264/U2) of Saccharomyces cerevisiae with a yeast genomic library yielded two different plasmids capable of restoring the ability of the mutant to grow on non-fermentable substrates. One of the plasmids (pG52/T3) contained SDH1 coding for the flavoprotein subunit of mitochondrial succinate dehydrogenase. The absence of detectable succinate dehydrogenase activity in mitochondria of E264/U2 and the lack of complementation of the mutant by an sdh11null strain indicated a mutation in SDH1. The second plasmid (pG52/T8) had an insert with reading frame (YJL045w) of yeast chromosome X coding for a homologue of SDH1. Subclones containing the SDH1 homologue (SDH1b), restored respiration in E264/U2 indicating that the protein encoded by this gene is functional. The expression of the two genes was compared by assaying the β-galactosidase activities of yeast transformed with plasmids containing fusions of lacZ to the upstream regions of SDH1 and SDH1b. The 100–500 times lower activity measured in transformants harbouring the SDH1b-lacZ fusion indicates that the isoenzyme encoded by SDH1b is unlikely to play an important role in mitochondrial respiration. This is also supported by the absence of any obvious phenotype in cells with a disrupted copy of SDH1b. © 1998 John Wiley & Sons, Ltd.     

XV. Yeast sequencing reports. Isolation and DNA sequence of the STE13 gene encoding dipeptidyl aminopeptidase

We have isolated a mutant which exhibits partial constitutivity for a -specific gene expression in α cells. The wild-type gene was cloned and demonstrated to be allelic to the STE13 gene, which encodes the dipeptidyl aminopeptidase involved in processing of the α-factor prepropheromone. Thus, the mating defect of the ste13 mutations in α cells may result both from the production of incompletely processed α-factor and from the increased expression of a -specific genes. The STE13 open reading frame of 931 amino acids contains a putative membrane-spanning segment near its amino terminus and is 31% identical to a second yeast dipeptidyl aminopeptidase (DAP2). A null mutant of STE13 has been constructed: it is viable and sporulation-proficient. The sequence has been deposited in the GenBank data library under Accession Number L21944.     

A family of vectors that facilitate transposon and insertional mutagenesis of cloned genes in yeast

This report describes two sets of plasmid vectors that facilitate the identification of regions of complementation in cloned genomic inserts via transposon or insertional mutagenesis. The first set contains ARS-H4 CEN6, a yeast selectable nutritional marker (HIS3, TRP1 or URA3), and neo for selection in Escherichia coli. These plasmids lack the Tn3 transposition immunity region present in pBR322 derived vectors, and are permissive recipients for Tn3 transposon mutagenesis. The second family of plasmids described facilitate gene disruption procedures performed in vitro. These vectors carry disruption cassettes that contain different yeast selectable markers (HIS3, LEU2, TRP1 or URA3) adjacent to the Tn5 neo gene. These genes can be excised as a cassette on a common restriction fragment and introduced into any desired restriction site with selection for kanamycin resistance.