Biomedical subjects

W Tanner

Publications and source records attributed to W Tanner.

At least 19 recordsLinked to original sources

PubMed2003

Specific lipid requirements of membrane proteins--a putative bottleneck in heterologous expression.

Membrane proteins are mostly protein-lipid complexes. For more than 30 examples of membrane proteins from prokaryotes, yeast, plant and mammals, the importance of phospholipids and sterols for optimal activity is documented. All crystallized membrane protein complexes show defined lipid-protein contacts. In addition, lipid requirements may also be transitory and necessary only for correct folding and intercellular transport. With respect to specific lipid requirements of membrane proteins, the phospholipid and glycolipid as well as the sterol content of the host cell chosen for heterologous expression should be carefully considered. The lipid composition of bacteria, archaea, yeasts, insects,Xenopus oocytes, and typical plant and mammalian cells are given in this review. A few examples of heterologous expression of membrane proteins, where problems of specific lipid requirements have been noticed or should be thought of, have been chosen.

Animals↗

PubMed2001

Transpiration, a prerequisite for long-distance transport of minerals in plants?

The major "benefit" alleged to accrue from transpiration (the evaporative loss of water from plant surfaces) is that it is essential for the long-distance transport of mineral ions, but the possible interrelation between these two processes has rarely been tested. Transpiration was experimentally dissociated from mineral supply by growing sunflowers (Helianthus anuus) in hydroculture and providing mineral nutrients only during the nights. These plants grew as well as a control group that received nutrients only during the day and transpired 12-15 times more water during the exposure period. It thus appears that convective water transport in the xylem, brought about by root pressure and the resultant guttation, "growth water," and Münch's phloem counterflow is in itself sufficient for long-distance mineral supply and that transpiration is not required for this function.

Biological Transport↗

PubMed2001

Construction of phosphatidylethanolamine-less strain of Saccharomyces cerevisiae. Effect on amino acid transport.

A triple yeast mutant was constructed which lacks BST1, the gene for sphingosine lyase, besides the phosphatidylserine decarboxylases PSD1 and PSD2. In this yeast mutant, which can only be grown in the presence of exogenous ethanolamine, phosphatidylethanolamine can be depleted to very low levels. Under those conditions, respiration as well as glucose and 3-O-methylglucose uptake proceed unaffected. Plasma membrane ATPase is as active in these cells as that of control cells grown in the presence of ethanolamine. Drastically decreased, however, are H+/amino acid symporters. The activities of arginine (Can1p), proline (Put4p) and general amino acid permease (Gap1p) are decreased more than 20-fold. Amino acid transport in yeast is dependent on coupling to the proton motive force. It can be envisaged that phosphatidylethanolamine might play a role in this process or in the early steps of the secretion pathway common for all amino acid permeases or, eventually, it could affect the transport proteins directly at the plasma membrane Transformation of the triple mutant with a CEN plasmid harbouring BST1 wild-type gene totally reversed its phenotype to that observed in the double mutant.

Adenosine Triphosphatases↗

PubMed2000

The C-terminal tetrapeptide HWFW of the Chlorella HUP1 hexose/H(+)-symporter is essential for full activity and an alpha-helical structure of the C-terminus.

C-terminal tails of plant hexose/H(+)-symporters of the major facilitator superfamily contain a highly conserved motif of four amino acids: HWFW. A deletion of these four amino acids in the Chlorella HUP1 protein leads to a decrease in transport activity by a factor of 3-4. The mutated tail is highly sensitive to trypsin; it does not show alpha-helical conformation in contrast to the wild type C-terminal peptide with an alpha-helical content of at least 15%. The production of monoclonal antibody 416B8 recognizing an epitope within the central loop of HUP1 protein has been a prerequisite for the experiments described.

Amino Acid Sequence↗

PubMed2000

Properties of a reconstituted eukaryotic hexose/proton symporter solubilized by structurally related non-ionic detergents: specific requirement of phosphatidylcholine for permease stability.

Overexpression of the hexose/proton symporter HUP1 from Chlorella kessleri in S. cerevisiae permits a one-step purification via a biotinylation domain. Milligram amounts of the protein are obtained starting from 2 l of yeast culture. The HUP1 protein is used as a model eukaryotic membrane protein of the 'major facilitator superfamily' (MFS) to study specific lipid requirements for activity and stability. Testing two series of detergents revealed that n-nonyl-beta-D-glucoside (NG) and n-octyl-beta-D-glucoside (OG) solubilize the HUP1 protein efficiently. Only the use of NG resulted in long-term stabilization of the HUP1 protein in the absence of external lipids. When affinity purified protein was extracted with organic solvents, a stoichiometric amount of phosphatidyl choline, phosphatidyl ethanolamine and ergosterol in the ratio of close to 2:1 was detected. These lipids were only observed, however, when the protein purification was carried out in the presence of NG; no lipids were copurified with the HUP1 protein in the presence of OG. Of the three lipids copurified, phosphatidyl choline showed a crucial role in ensuring maximal HUP1 permease activity and stability when added back to the OG-protein. The requirement of phosphatidylcholine documents a specific effect of lipids on vectorial transport mediated by a eukaryotic membrane protein of the MFS family.

3-O-Methylglucose↗

PubMed2000

The Chlorella hexose/H(+)-symporters.

The physiology, molecular biology, and biochemistry of the inducible hexose uptake protein of Chlorella kessleri is reviewed. The protein encoded by the HUP1 gene is the most intensively studied membrane transporter of plants. Responsible for substrate accumulation up to 1500-fold, it translocates one proton together with one hexose, and the cell invests 1 ATP per sugar transported. Kinetics suggest that substrate accumulation is mainly brought about by a large delta Km (Kminside >> Kmoutside). The HUP1 protein (534aa) consists of 12 transmembrane helices of which at least helices I, V, VII, and XI interact with the sugar during translocation and participate in lining the transport path through the membrane. The helix packing might very well be identical to the one suggested for the E. coli lac permease, although the mechanism for transport and proton coupling that has been suggested for lac permease (Kaback, 1997) certainly does not hold for the Chlorella symporter; both are distantly related members, however, of the MFS-family of transporters. HUP1 has been functionally expressed in Schizosaccharomyces pombe, Saccharomyces cerevisiae, Escherichia coli, Volvox carteri, and in Xenopus oocytes.

Amino Acid Sequence↗

PubMed1999

O-Glycosylation of Axl2/Bud10p by Pmt4p is required for its stability, localization, and function in daughter cells.

Cells of the yeast Saccharomyces cerevisiae choose bud sites in a manner that is dependent upon cell type: a and alpha cells select axial sites; a/alpha cells utilize bipolar sites. Mutants specifically defective in axial budding were isolated from an alpha strain using pseudohyphal growth as an assay. We found that a and alpha mutants defective in the previously identified PMT4 gene exhibit unipolar, rather than axial budding: mother cells choose axial bud sites, but daughter cells do not. PMT4 encodes a protein mannosyl transferase (pmt) required for O-linked glycosylation of some secretory and cell surface proteins (Immervoll, T., M. Gentzsch, and W. Tanner. 1995. Yeast. 11:1345-1351). We demonstrate that Axl2/Bud10p, which is required for the axial budding pattern, is an O-linked glycoprotein and is incompletely glycosylated, unstable, and mislocalized in cells lacking PMT4. Overexpression of AXL2 can partially restore proper bud-site selection to pmt4 mutants. These data indicate that Axl2/Bud10p is glycosylated by Pmt4p and that O-linked glycosylation increases Axl2/ Bud10p activity in daughter cells, apparently by enhancing its stability and promoting its localization to the plasma membrane.

Amidohydrolases↗

PubMed1999

Expression of eukaryotic plasma membrane transporter HUP1 from Chlorella kessleri in Escherichia coli.

To study the effect of sterols on the activity of the eukaryotic plasma membrane transporter, the hexose-proton symporter HUP1 from the unicellular alga Chlorella kessleri was expressed in Escherichia coli, a prokaryotic microorganism containing virtually no sterols. Under certain conditions, the recombinant protein was partially active in this prokaryotic organism. The heterologously produced HUP1p was purified from membrane fractions of E. coli and reconstituted in an in vitro system. The presence of ergosterol during solubilization, purification and reconstitution resulted in an increased activity of the reconstituted protein. Its activity, however, was 5-6 times lower as compared to the activity of HUP1p produced in Saccharomyces cerevisiae membranes and solubilized, purified, and reconstituted under the same conditions as above.

Biological Transport↗

PubMed1999

Protein O-mannosylation.

Protein O-mannosylation, originally observed in fungi, starts at the endoplasmic reticulum with the transfer of mannose from dolichyl activated mannose to seryl or threonyl residues of secretory proteins. This reaction is catalyzed by a family of protein O-mannosyltransferases (PMTs), which were first characterized in Saccharomyces cerevisiae. The identification of this evolutionarily conserved PMT gene family has led to the finding that protein O-mannosylation plays an essential role in a number of physiologically important processes. Focusing on the PMT gene family, we discuss here the main aspects of the biogenesis of O-linked carbohydrate chains in S. cerevisiae, Candida albicans, and other fungi. We summarize recent work utilizing pmt mutants that demonstrates the impact of protein O-mannosylation on protein secretion, on maintenance of cell wall integrity, and on budding. Further, the occurrence of PMT orthologs in higher eukaryotes such as Arabidopsis, Drosophila and mammals is reported and discussed.

Animals↗

PubMed1999

Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the Pir protein family) in stability of the Saccharomyces cerevisiae cell wall.

The Saccharomyces cerevisiae cell wall contains more than 20 identified mannoproteins. Some of them can be released from the wall by hot SDS/mercaptoethanol treatment and are, therefore, considered as disulphide-linked or non-covalently attached to wall structural components. A number of covalently linked cell wall proteins are released after SDS extraction. They can be divided into these extractable by glucanases and those which can be released with 30 mM NaOH. The SDS-extractable proteins either possess enzymatic activities or are homologues of enzymes, mainly glucanases. Nothing is known, however, about the function of covalently linked proteins. In order to investigate the role of NaOH-extractable cell wall proteins, genes encoding all four identified members of this family of Pir proteins, CCW5, CCW6, CCW7 and CCW8, were disrupted and the phenotype of the mutants obtained was examined. They grew somewhat more slowly, were larger and irregularly shaped, and showed pronounced susceptibility to cell wall synthesis inhibitors like Calcofluor white and Congo red. In addition, the triple and the quadruple deletants had a decreased mating ability. All these properties were more obvious the more of these genes were disrupted, indicating that probably all members of this protein family are at least functionally equivalent in the cell wall.

Base Sequence↗

PubMed1999

Deletion of new covalently linked cell wall glycoproteins alters the electrophoretic mobility of phosphorylated wall components of Saccharomyces cerevisiae.

The incorporation of radioactive orthophosphate into the cell walls of Saccharomyces cerevisiae was studied. 33P-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase. This radioactive material stayed in the stacking gel during SDS-polyacrylamide gel electrophoresis but entered the separating gel upon treatment with N-glycosidase F, indicating that phosphate was linked directly or indirectly to N-mannosylated glycoproteins. The phosphate was bound to covalently linked cell wall proteins as mannose-6-phosphate, the same type of linkage shown previously for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc. Natl. Acad. Sci. USA 87:3368-3372, 1990). From the phosphate-labeled glycoprotein fraction released by laminarinase, three cell wall mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified by N-terminal sequencing. For Ccw13p (encoded by DAN1 [also called TIR3]) and Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A. Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154-2162, 1997). In ccw12, ccw13, or ccw14 single or double mutants neither the amount of radioactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a shift of the 33P-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of CCW12 results in a pronounced sensitivity of the cells to calcofluor white and Congo red. In addition, the ccw12 mutant shows a decrease in mating efficiency and a defect in agglutination.

Amino Acid Sequence↗

PubMed1998

Alteration of substrate affinities and specificities of the Chlorella Hexose/H+ symporters by mutations and construction of chimeras.

The cDNAs HUP1 and HUP2 of Chlorella kessleri code for monosaccharide/H+ symporters that can be functionally expressed in Schizosaccharomyces pombe. By random mutagenesis three HUP1 mutants with an increased Km value for D-glucose were isolated. The 40-fold increase in Km of the first mutant is due to the amino acid exchange N436I in putative transmembrane helix XI. Two substitutions were found in a second (G97C/I303N) and third mutant (G120D/F292L), which show a 270-fold and 50-fold increase in Km for D-glucose, respectively. An investigation of the individual mutations revealed that the substitutions I303N and F292L (both in helix VII) cause the Km shifts seen in the corresponding double mutants. These mutations together with those previously found support the hypothesis that helices V, VII, and XI participate in the transmembrane sugar pathway. Whereas for most mutants obtained so far the Km change for D-glucose is paralleled by a corresponding change for other hexoses tested, the exchange D44E exclusively alters the Km for D-glucose. Moreover the pH profile of this mutant is shifted by more than 2 pH units to alkaline values, indicating that the activity of the transporter may require deprotonation of the corresponding carboxyl group. Chimeric transporters were constructed to study the 100-fold lower affinity for D-galactose of the HUP1 symporter as compared with that of the HUP2 protein. A crucial determinant for the differential D-galactose recognition was shown to be associated with the first external loop. The effect could be pinpointed to a single amino acid change: replacement of Asn-45 of HUP1 with isoleucine, the corresponding amino acid of HUP2, yields a transporter with a 20 times higher affinity for D-galactose. The reverse substitution (I47N) decreases the affinity of HUP2 for D-galactose 20-fold.

Amino Acid Sequence↗

PubMed1998

Post-translational fate of CAN1 permease of Saccharomyces cerevisiae.

To study the post-translational fate of arginine permease (Can1p), the gene coding for this transport protein was placed behind a constitutive promoter of plasma membrane ATPase (PMA1) and furnished with a Myc tag. In exponential-phase cells the amount of Can1p is constant, although turnover can be demonstrated. A rapid decrease in transport activity during the early stationary phase is paralleled by a corresponding net degradation of the protein. The amount of Can1p present in exponential cells grown on various nitrogen sources is the same, except in arginine-grown cells, in which the amount of the protein is markedly lower. This occurs solely when arginine serves as nitrogen source but not as an immediate consequence of, for example, arginine addition to cells growing on other nitrogen sources. it was demonstrated that Can1p is phosphorylated. Since Can1p expression under the PMA1 promoter is glucose-dependent, the amount of the permease expressed in high-glucose-grown cells is higher than in low-glucose-grown ones. Only a part of the Can1p overexpressed in high-glucose-grown cells is phosphorylated, while in low-glucose-grown cells the phosphorylated form probably represents the majority of Can1p. The permease phosphorylation or dephosphorylation is not related to transinhibition.

Blotting, Western↗

PubMed1998

New potential cell wall glucanases of Saccharomyces cerevisiae and their involvement in mating.

Biotinylation of intact Saccharomyces cerevisiae cells with a nonpermeant reagent (Sulfo-NHS-LC-Biotin) allowed the identification of seven cell wall proteins that were released from intact cells by dithiothreitol (DTT). By N-terminal sequencing, three of these proteins were identified as the known proteins beta-exoglucanase 1 (Exg1p), beta-endoglucanase (Bgl2p), and chitinase (Cts1p). One protein was related to the PIR protein family, whereas the remaining three (Scw3p, Scw4p, and Scw10p [for soluble cell wall proteins]) were found to be related to glucanases. Single knockouts of these three potential glucanases did not result in dramatic phenotypes. The double knockout of SCW4 and the homologous gene SCW10 resulted in slower growth, significantly increased release of proteins from intact cells by DTT, and highly decreased mating efficiency when these two genes were disrupted in both mating types. The synergistic behavior of the disruption of SCW4 and SCW10 was partly antagonized by the disruption of BGL2. The data are discussed in terms of a possible counterplay of transglucosidase and glucosidase activities.

Amino Acid Sequence↗

PubMed1997

Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae.

Intact Saccharomyces cerevisiae cells were biotinylated with the non-permeable sulfosuccinimidyl-6-(biotinamido) hexanoate reagent. Twenty specifically labelled cell wall proteins would be extracted and visualized on SDS gels via streptavidin/horseradish peroxidase. Nine cell wall proteins were released by SDS extraction under reducing conditions and were designated Scw1-9p for (soluble cell wall proteins); five proteins were released from SDS-extracted cell walls by laminarinase (Ccw1-5p for covalently linked cell wall proteins) and six with mild (30 mM-NaOH, 4 degrees C, 14 h) alkali treatment (Ccw6-11p). N-terminal sequences of the Ccw proteins 6, 7, 8 and 11 showed that these cell wall proteins are members of the PIR gene family (predicted proteins with internal repeats), CCW6 being identical to PIR1 and CCW8 to PIR3. Single gene disruptions of all four genes did not yield a phenotype. In the CCW11 disruption the Ccw11p as well as the laminarinase-extracted Ccw5 protein was missing. The new cell wall proteins are O-mannosylated, contain a Kex2 processing site, but no C-terminal GPI anchor sequence.

Amino Acid Sequence↗

PubMed1997

Characterization of two new genes down-regulated by alpha-factor.

To detect genes directly down-regulated by alpha-factor, 55,000 plaque-forming units of a Saccharomyces cerevisiae lambda gt10 gene bank were differentially screened with cDNA of cells treated with alpha-factor for 20 min. Two new genes were detected in this way, called alpha0.5 and alpha0.6. The former is transiently down-regulated by alpha-factor; it is very highly transcribed in late exponential-phase cells. The gene, located on the right arm of chromosome XIII, codes for a 59 amino-acid protein with a signal peptide. The protein has been shown with an antibody to be present in the membrane fraction. The gene has also been cloned as HOR7 (hyperosmolarity-responsive protein; Hirayama et al., 1995). No other homologous sequences have been detected in the yeast genome. alpha0.6, located on the right arm of chromosome XII, corresponds to the open reading frame YLR110c; it codes for a 133 amino-acid protein containing a signal peptide. Its derived amino-acid sequence is homologous to the N-terminal half of the SED1 gene product. SED1, when overexpressed, is able to suppress a defect in the HDEL receptor coded for by the ERD2 gene (Hardwick and Pelham, 1994); however, alpha0.6 is not able to do so. The disruption of alpha0.5 or alpha0.6 does not lead to a special phenotype.

Amino Acid Sequence↗

PubMed1997

Protein-O-glycosylation in yeast: protein-specific mannosyltransferases.

S. cerevisiae contains at least six genes (PMT1-6) for dolicholphosphate-D-mannose: protein-O-D-mannosyltransferases. The in vivo mannosylation of seven O-mannosylated yeast proteins has been analyzed in a number of pmt mutants. The results clearly indicate that the various protein O-mannosyltransferases have different specificities for protein substrates. Five of the proteins tested (chitinase, a-agglutinin, Kre9p, Bar1p, Pir2p/hsp 150) are mainly underglycosylated in pmt1 and pmt2 mutants, whereby qualitative differences exist among the various proteins. Two of the O-mannosylated proteins (Ggp1p and Kex2p) are not at all affected in pmt1 and pmt2 mutants but are clearly underglycosylated when PMT4 is mutated. Although the PMT4 gene product is shown to be responsible for O-mannosylating a Ser-rich region of Ggp1p in vivo, a penta-seryl-peptide is not an in vitro substrate for this transferase. A PMT3 mutation does affect O-mannosylation of chitinase only in the genetic background of a pmt1pmt2 double mutation, indicating that PMT1 and PMT2 can compensate for a deleted PMT3 gene.

Chitinases↗

PubMed1996

The PMT gene family: protein O-glycosylation in Saccharomyces cerevisiae is vital.

The transfer of mannose to seryl and threonyl residues of secretory proteins is catalyzed by a family of protein mannosyltransferases coded for by seven genes (PMT1-7). Mannose dolichylphosphate is the sugar donor of the reaction, which is localized at the endoplasmic reticulum. By gene disruption and crosses all single, double and triple mutants of genes PMT1-4 were constructed. Two of the double and three of the triple mutants were not able to grow under normal conditions; three of these mutants could grow, however, when osmotically stabilized. The various mutants were extensively characterized concerning growth, morphology and their sensitivity to killer toxin K1, caffeine and calcofluor white. O-Mannosylation of gp115/Gas1p was affected only in pmt4 mutants, whereas glycosylation of chitinase was mainly affected in pmt1 and pmt2 mutants. The results show that protein O-glycosylation is essential for cell wall rigidity and cell integrity and that this protein modification, therefore, is vital for Saccharomyces cerevisiae.

Fungal Proteins↗