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W Tanner

Publications and source records attributed to W Tanner.

At least 55 records · Page 3Linked to original sources
PubMed1993

Unidirectional arginine transport in reconstituted plasma-membrane vesicles from yeast overexpressing CAN1.

Amino acids are accumulated in Saccharomyces cerevisiae by strictly unidirectional influx systems. To see whether cellular compartmentation causes this unusual amino-acid-transport behaviour, arginine transport was studied in plasma-membrane vesicles. The arginine permease gene CAN1 was overexpressed in S. cerevisiae RH218a and in a permease-deficient mutant RS453 (can1). Reconstituted plasma-membrane vesicles from these transformants, energized by incorporated cytochrome-c oxidase, showed 3-4-fold increased rates of arginine uptake compared to vesicles from wild-type cells. The KT values were 32.5 microM in vesicles from wild-type and 28.6 microM in vesicles from transformed cells; the corresponding in vivo values were 17.5 microM and 11.4 microM, respectively. It could be demonstrated that unidirectional arginine transport and accumulation also exist in vesicles; thus, unidirectional influx is not related to cellular compartmentation.

Amino Acid Transport Systems↗
PubMed1993

A yeast gene encoding a putative RNA helicase of the "DEAD"-box family.

An unknown open reading frame from Saccharomyces cerevisiae was identified and sequenced. The predicted amino acid sequence shows high homology to the DEAD-box family of proteins. Gene disruption revealed that the gene is not essential for yeast but necessary for normal cell growth.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-beta-1,3-glucanase.

One of the major proteins of the Saccharomyces cerevisiae cell wall, a beta-glucanase (BGL2 gene product), has been isolated and purified to homogeneity under conditions for preserving enzyme activity. The study of enzyme properties of the protein revealed that it is an endo-beta-1,3-glucanase and not an exoglucanase as reported previously (F. Klebl and W. Tanner, J. Bacteriol. 171:6259-6264, 1989). The examination of the glucanase structure showed that the lower apparent molecular mass of the protein (29 kDa) compared with what was calculated from the amino acid sequence of the enzyme (33.5 kDa) is due to anomalous migration in sodium dodecyl sulfate gels and not to posttranslational processing of the polypeptide chain. Of two potential N glycosylation sites at Asn-202 and Asn-284, only the latter site is glycosylated. The overproduction of the beta-glucanase from the high-copy-number plasmid brought about a significant decrease in the growth rate of transformed yeast cells.

Amino Acid Sequence↗
PubMed1991

Glucose increases the expression of the ATP/ADP translocator and the glyceraldehyde-3-phosphate dehydrogenase genes in Chlorella.

A presumably full-length cDNA clone of the mitochondrial ATP/ADP translocator (AAT) of Chlorella kessleri has been isolated and sequenced. The expression of the AAT gene is highly increased in the presence of D-glucose (14 mM). At least nine more genes are activated when autotrophically grown Chlorella cells switch to heterotrophic growth. Among these is the HUP1 gene coding for the hexose transporter (Sauer, N., Caspari, T., Klebl, F., and Tanner, W. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7949-7952) and, as also shown in this paper, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. When glucose or the nonmetabolizable analogue 6-deoxyglucose is added to the cells, an increased expression of GAPDH or AAT is observed after 10 or 30 min, respectively. Hexose uptake mutants (HUP1-) do not respond to sugars in this way, which indicates that either the inducer has to be internalized or that the HUP1 translocator is part of the signal transduction mechanism.

Amino Acid Sequence↗
PubMed1991

Protein O-glycosylation in Saccharomyces cerevisiae. Purification and characterization of the dolichyl-phosphate-D-mannose-protein O-D-mannosyltransferase.

The enzyme dolichyl-phosphate-D-mannose:protein O-D-mannosyltransferase has been solubilized from Saccharomyces cerevisiae membranes and its mannosyltransferase activity demonstrated using short peptides. The specific activity of the protein was enriched 130-fold before it was further purified by native and SDS gel chromatography. A 92-kDa band correlated well with the enzyme activity; an antibody raised against this protein precipitated the mannosyltransferase. The 92-kDa band was hydrolysed to 84 kDa after treatment with endoglycosidase F, indicating that the protein is a glycoprotein which may contain four carbohydrate chains. The purified mannosyltransferase is distinctly influenced in transfer specificity by amino acids next to serine and threonine within the acceptor peptides. Thus acidic amino acids strongly inhibit acceptor activity as do glycine and proline residues as amino-terminal and carboxy-terminal neighbours, respectively.

Dolichol Monophosphate Mannose↗
PubMed1991

Saccharomyces cerevisiae a- and alpha-agglutinin: characterization of their molecular interaction.

An O-glycosylated protein of approximately 18 kDa responsible for mating type specific agglutination has been isolated from Saccharomyces cerevisiae a cells, purified to homogeneity and via peptide sequences the gene was cloned by PCR. An open reading frame codes for a protein of 69 amino acids. A minimum of five serine and five threonine residues of the mature protein are glycosylated. alpha-Agglutinin is a highly N-glycosylated protein of approximately 250 kDa. Both purified agglutinins form a specific 1:1 complex in vitro. Pretreatment of alpha-agglutinin, but not of alpha-agglutinin, with diethylpyrocarbonate (DEPC) prevents formation of the complex; treatment of alpha-agglutinin in the presence of alpha-agglutinin protects the former from DEPC inactivation. By carboxy terminal shortening of the alpha-agglutinin gene and by replacing three of its eight histidyl residues by arginine, the active region of alpha-agglutinin for interaction with alpha-agglutinin has been defined. Neither the N- nor the O-linked saccharides of the two agglutinins seem to be essential for their interaction.

Base Sequence↗
PubMed1991

The Chlorella H+/hexose cotransporter gene.

The complete genomic sequence of the inducible Chlorella kessleri H+/hexose cotransporter (HUP1) has been obtained from two overlapping clones isolated from a lambda gt10 library. The HUP1 gene is interrupted by 14 introns with the first intron being located in the 5'-untranslated part of the gene. The average intron length is 220 bp, yielding a very regular intron/exon pattern in the gene. The codon usage in this gene is strongly biased with a clear preference for C and a strong suppression of A. A consensus sequence for a putative algal polyadenylation sequence is shown and compared with other algal cDNA sequences.

Base Sequence↗
PubMed1990

The protean manifestations of hemorrhagic fever with renal syndrome. A retrospective review of 26 cases from Korea.

Twenty-six cases of hemorrhagic fever with renal syndrome from 1981 to 1986 were retrospectively reviewed to determine the scope of clinical presentation and the unique complications of the illness. The diagnosis was confirmed by detection of Hantaan virus antibody in 25 cases and by characteristic autopsy findings in 1 case. The illness could be classified into three distinct clinical subgroups. Fever was universally present. Two patients presented with intractable shock and diffuse hemorrhage and died within 6 days from multi-organ system failure, mimicking the clinical picture of overwhelming sepsis. Eighteen patients presented with acute renal failure with an illness lasting a mean of 21 days (range, 10 to 36 days). Resolution of thrombocytopenia heralded recovery of renal function. At discharge, the serum creatinine level was normal in 13 patients; 5 patients had evidence of minimal renal dysfunction. Acute pulmonary edema requiring hemodialysis and retroperitoneal hemorrhage were the major complications in this subgroup. Six patients had an undifferentiated febrile illness with normal renal function. Fever, thrombocytopenia, abnormal urinalysis, hypertransaminasemia, and a benign clinical course characterized the third clinical pattern. The recent availability of serodiagnostic methods to detect Hantavirus group antibody facilitates the diagnosis of hemorrhagic fever with renal syndrome. Application of this test in the described clinical settings will identify unsuspected cases, broaden the knowledge of the geographic distribution of Hantavirus infection, and increase physician awareness of its protean manifestations.

Acute Kidney Injury↗
PubMed1990

Functional expression of the Chlorella hexose transporter in Schizosaccharomyces pombe.

Schizosaccharomyces pombe cells were transformed with an S. pombe expression vector containing a full-length cDNA of the Chlorella hexose transporter. The transformed cells accumulated 3-O-methylglucose up to 10-fold, whereas wild-type S. pombe and control transformants could only equilibrate this sugar analogue. In a pH-jump experiment, in which extracellular pH was lowered by 1.9 units, the accumulation ratio was increased in transformed cells but not in control cells. This result indicates that the gene product, Chlorella H+/glucose-symporter protein, and a pH gradient suffice for active sugar uptake. Km values for glucose, 6-deoxyglucose, and 3-O-methylglucose of 1.5 x 10(-5) M, 2.7 x 10(-4) M, and 1.0 x 10(-3) M, respectively, were identical in Chlorella and in S. pombe cells transformed with Chlorella cDNA and approximately 100-fold lower than those of the endogenous transport system of S. pombe.

Journal Article↗
PubMed1989

The hexose carrier from Chlorella. cDNA cloning of a eucaryotic H+-cotransporter.

The cDNA coding for the inducible H+/hexose cotransporter of Chlorella kessleri has been cloned and sequenced. It was isolated by differential screening of a cDNA library prepared from glucose-induced cells. The increase in expression of the gene correlates quantitatively with the increase in uptake activity due to induction; it is not expressed in a hexose transport mutant. An open reading frame allows for a membrane protein of 533 amino acids with a relative molecular mass of 57 kDa. This protein is highly homologous to the human and rat glucose transporters catalyzing facilitated diffusion and to the bacterial H+/pentose cotransporters. It is not related to the H+/lactose cotransporter of E. colli and to the mammalian Na+/glucose cotransporter.

Base Sequence↗
PubMed1989

Purification of the inducible alpha-agglutinin of S. cerevisiae and molecular cloning of the gene.

The alpha-agglutinin responsible for mating type-specific agglutination of S. cerevisiae alpha-cells has been purified to homogeneity. The glycoprotein released from the cell surface under mild conditions has a relative molecular mass of 200 to 300 kDa as determined by SDS-gel electrophoresis. The protein moiety corresponds to 68.2 kDa. With an oligonucleotide corresponding to the N-terminal amino acid sequence, the alpha-agglutinin gene has been cloned and sequenced. From the DNA sequence, a protein of 631 amino acids with 12 potential N-glycosylation sites is predicted. The carboxy terminal one-third of the protein is not required for agglutination activity.

Agglutination↗
PubMed1989

Cloning of the glutamine:fructose-6-phosphate amidotransferase gene from yeast. Pheromonal regulation of its transcription.

The activity of the amino sugar-synthesizing enzyme L-glutamine:D-fructose-6-phosphate aminotransferase (EC 2.6.1.16) in haploid a cells of Saccharomyces cerevisiae increases 1.7-fold after alpha factor addition. The gene (the gene should be called GFA1 for glutamine:fructose-6-phosphate amidotransferase) for the enzyme has been cloned by complementing the gcn1 mutation (Whelan, W. L., and Ballou, C. E. (1975) J. Bacteriol. 125, 1545-1557). Its expression is increased 2-3 times within 15 min when the mating pheromone is added. The gene codes for a protein of 716 amino acids in length. It is highly homologous (64%) to the corresponding gene of Escherichia coli, except for a sequence coding for 83 amino acids (numbers 204-286), which is lacking in E. coli. The amino-terminal region of the coding sequence also shows a high degree of homology to the corresponding sequence of the E. coli and S. cerevisiae L-glutamine:phosphoribosylpyrophosphate amidotransferase. In the promotor region of the S. cerevisiae L-glutamine:D-fructose-6-phosphate amidotransferase gene the heptanucleotide "TGAAACA," shown to be required for pheromone control of transcription (Kronstad, J. W., Holly, J. A., and MacKay, V. L. (1987) Cell 50, 369-377), is present six times.

Amino Acid Sequence↗
PubMed1989

Molecular cloning of a cell wall exo-beta-1,3-glucanase from Saccharomyces cerevisiae.

A major protein of Saccharomyces cerevisiae cell walls is a 29-kilodalton glycoprotein which shows lectinlike binding to beta-1,3-glucan and chitin. It was solubilized by heating isolated cell walls at 90 degrees C and purified to homogeneity by running two high-pressure liquid chromatography columns. With the sequence information of the N terminus and seven peptides, two oligonucleotides were synthesized and the gene was cloned. Its sequence is similar to those of two plant beta-glucanases, and the protein was shown to possess beta-1,3-exoglucanase activity with laminarin as substrate. Haploid yeast cells contained one copy of the gene (BGL2). Gene disruption did not result in a phenotype.

Amino Acid Sequence↗
PubMed1988

Purification and characterization of the inducible a agglutinin of Saccharomyces cerevisiae.

A cell surface glycoprotein induced by the mating pheromone alpha factor in Saccharomyces cerevisiae a cells has been purified to homogeneity. At 4 x 10(-9) M it strongly inhibits mating-type-specific agglutination between a and alpha cells. The protein is solely O-glycosylated. It consists of 29% carbohydrate and its apparent molecular mass is 22 kd on SDS gels. After HF treatment it behaves like a protein of 13 kd; therefore its true molecular mass probably is close to 18 kd. Mild periodate treatment destroys the biological activity of the purified protein. The protein contains one cysteine, no arginine, and 27% of the amino acids are serine and threonine residues, two thirds of which are glycosylated. With a polyclonal antibody the glycoprotein can already be detected at the cell surface 15 min after pheromone addition. The inducible antigen is not expressed in a specific phase of the cell cycle; it first appears exclusively on the growing bud. Mother cells express the antigen on their surface only after the daughter cells have separated; it is then localized at the tip of the pear-shaped 'shmoo'. Using the secretory ts-mutant sec 18 is shown that a mannosylated precursor of a agglutinin accumulates at the endoplasmic reticulum.

Antigens, Fungal↗
PubMed1987

Protein glycosylation in yeast.

S. cerevisiae contains many mannose-rich glycoproteins that possess N- and O-linked carbohydrate chains, and both types may even occur on one and the same protein. The steps in the synthesis of asparagine-linked chains begin with assembly and transfer of the lipid-linked precursor to protein in a way common to all eucaryotes. Subsequent modifications lead to mannosyl extensions of various lengths, but complex type carbohydrate structures are not formed. Oligosaccharides O-linked to serine/threonine consist exclusively of mannose in S. cerevisiae. The mannose residue attached directly to the protein is transferred from Dol-P-Man in a unique way, which has been observed so far for fungal cells only. The cellular localization of the glycosylation reactions is summarized and the problem of transmembrane translocation of the sugar precursors at the ER and the Golgi is discussed. Some aspects of secretory (sec) and asparagine linked glycosylation (alg) mutants have been covered, and the various hypotheses related to the possible functions of this costly protein modification process are discussed. The article may also be helpful for those, who want to exploit the yeast's protein synthesizing machinery by genetically manipulating the cells.

Asparagine↗
PubMed1986

Synthesis of an O-glycosylated cell surface protein induced in yeast by alpha factor.

A number of cell surface glycoproteins can be specifically and completely released from intact cells of Saccharomyces cerevisiae with 0.5% mercaptoethanol. Among these proteins is one with a molecular mass of 22 kDa, which is synthesized only in haploid a cells treated with the peptide mating pheromone alpha factor. This protein could be radiolabeled in vivo with [2-(3)H]mannose, [(14)C]phenylalanine, and [(35)S]sulfate. Its synthesis and export to the cell surface were not inhibited by tunicamycin. beta-Elimination released almost all radioactivity from the [2-(3)H]mannose-labeled protein, 36% of its radioactivity being recovered subsequently as mannose and 43% as a dimannoside. Evidence is presented that the 22-kDa O-glycosylated protein is a mating-type specific a cell agglutinin.

Journal Article↗
PubMed1986

Effects of Ca on Amino Acid Transport and Accumulation in Roots of Phaseolus vulgaris.

Ca(2+) stimulates the uptake of alpha-aminoisobutyric acid (AIB) into excised or intact Phaseolus vulgaris L. roots by a factor of two. In roots depleted of Ca(2+) by preincubation with ethylenediaminetetraacetate, ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, or streptomycin, the stimulatory effect is 7- to 10-fold. In the presence of Ca(2+), roots accumulate AIB more than 100-fold; Ca(2+)-depleted roots only equilibrate with AIB. Radioautography shows [(14)C]AIB to be present in all cells after 90 min. Although Ca(2+)-depleted roots lose accumulated [(14)C]AIB about 10 times faster than roots supplied with Ca(2+), this increased efflux is not the main cause for the decrease in net uptake observed. The latter is rather due to a less negative membrane potential Deltapsi in Ca(2+) depleted roots (-120 mV --> -50 mV). The basic feature explaining all the results of Ca(2+) deficiency is an increase in general membrane permeability. No indication of a specific regulatory function of Ca(2+) in membrane transport of roots has been obtained.

Journal Article↗