Identification of the Maltose Transport Protein of Saccharomyces cerevisiae

Broek, Peter J.A. Van den; Leeuwen, C. C. M. Van; Weusthuis, Ruud A.; Postma, Erik; Dijken, Johannes P. van; Karssies, R.; Amons, Reinout

doi:10.1006/bbrc.1994.1411

Cited by 14 publications

(12 citation statements)

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“…It has been shown before that the antibodies can react with three or more bands in the 66-kDa region (29). These bands may reflect different degradation products and/or phosphorylation states of the protein (47). Our current research is aimed at isolating Mal61p mutants and S. cerevisiae strains in which this heterogeneity in the maltose transport protein(s) is absent.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes

et al. 1995

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For maltose uptake in Saccharomyces cerevisiae, multiple kinetic forms of transport as well as inhibition of transport by high concentrations of maltose at the trans side of the plasma membrane have been described. Most of these studies were hampered by a lack of genetically well-defined mutants and/or the lack of an artificial membrane system to study translocation catalysis in vitro. A genetically well-defined S. cerevisiae strain lacking the various MAL loci was constructed by gene disruption. Expression of the maltose transport protein (Mal61p) was studied by using various plasmid vectors that differed in copy number and/or type of promoter. The expression levels were quantitated by immunoblotting with antibodies generated against the N-terminal half of Mal61p. The levels of expression as well as the initial uptake rates were increased 20-fold compared with those in a yeast strain carrying only one chromosomal MAL locus. Similar results were obtained when the transport activities were compared in hybrid membranes of the corresponding strains. To generate a proton motive force, isolated membranes were fused with liposomes containing cytochrome c oxidase as a proton pump. Fusion was achieved by a cycle of freeze-thawing, after which the hybrid membranes were passed through a filter with a defined pore size to obtain unilamellar membrane vesicles. Proton motive force-driven maltose uptake, maltose efflux down the concentration gradient, and equilibrium exchange of maltose in the hybrid membranes vesicles have been analyzed. The data indicate that maltose transport by the maltose transporter is kinetically monophasic and fully reversible under all conditions tested.

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Section: Discussionmentioning

confidence: 99%

Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes

et al. 1995

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“…However based on the sequence of the FCY2 gene, the permease is assumed to be a 58-kDa hydrophobic protein. Such a discrepancy between the molecular mass calculated from sequence data and that observed on SDS/PAGE is typical of integral membrane proteins such as plasma-membrane permeases (16,29,301. These hydrophobic proteins bind excessive amounts of SDS, which results in an abnormally high electrophoretic mobility [31].…”

Section: -mentioning

confidence: 99%

In vivo Phosphorylation of the Purine/Cytosine Permease from the Plasma Membrane of the Yeast Saccharomyces cerevisiae

Pinson

Pillois

Brèthes

et al. 1996

European Journal of Biochemistry

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The purinekytosine permease, encoded by the FCY2 gene, is a carrier located in the plasma membrane of the yeast Saccharomyces cerevisiae. Polyclonal antibodies were raised against two peptides that corresponded to the sub-N-terminal and C-terminal sequences of the putative protein deduced from the FCY2 gene. Immunoprecipitation experiments performed with protein extracts labelled in vivo with -3 showed that purinekytosine permease is specifically detected as a broad and diffuse band. The apparent molecular mass of this protein was 45-50 kDa. By means of in vivo pulsekhase '3-labelling experiments, we observed a slight increase in the apparent molecular mass of purinekytosine permease during the chase. This shift in electrophoretic mobility of the protein suggested a post-translational modification. This molecular mass increase was eliminated by alkaline phosphatase treatment of the immunoprecipitate, which strongly suggested phosphorylation of the carrier. This proposal was confirmed by in vivo [""pP, labelling and immunoprecipitation of purinekytosine permease with purified anti-(sub-N-terminal peptide) IgG or anti-(C-terminal peptide) IgG. Phosphoamino acid analysis indicated that phosphorylation occurred on seryl residues of purine/cytosine permease. By means of thermosensitive secretory-pathwaymutant strains, we demonstrated that purinekytosine permease phosphorylation occurred either between the Golgi apparatus and the plasma membrane or in the plasma membrane itself.Keywords: Saccharomyces cerevisiae ; yeast plasma membrane ; purine/cytosine permease; phosphorylation.In the yeast Saccharomyces cerevisiae, active metabolite uptake across the plasma membrane has been investigated by means of physiological and genetic studies. An increasing number of genes that encode specific active-transport systems have been cloned and sequenced [l -41. A transmembrane electrochemical-potential difference created by the proton-translocating ATPase of the plasma membrane [5, 61 leads to the accumulation of various metabolites in the cytoplasm.Purinekytosine permease mediates symport through the plasma membrane of protons and either purine bases (adenine, hypoxanthine and guanine) or a pyrimidine base (cytosine) 17-101. Purinekytosine permease is encoded by the FCY2 gene, which has been cloned 1111 and sequenced 1121. The amino acid sequence that was deduced from the FCY2 gene corresponds to a protein of 533 amino acid residues with a molecular mass of 58.2 kDa. Based on this sequence, predictive structural analyses suggested that purinekytosine permease is a polytopic protein with nine a-helical transmembrane segments (Chevallier, M. R., personal communication).The FCY2 gene was cloned into a multicopy plasmid, which led to the amplification of gene expression and an increase in the amount of active purinekytosine permease in the plasma membrane [111. Such a permease-proficient strain is a valuable tool for identification of the protein and to study its transport mechanism. The maximal rate of uptake of the bases and the m...

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“…In industrial strains, there is evidence for the existence of a low-affinity maltose transporter [39] and this is not unusual, since industrial strains clearly differ from laboratory strains with regard to maltose uptake characteristics. The high-affinity maltose transporter in S. cerevisiae is induced by maltose, repressed by high (>0.4%, w/v) glucose and inactivated following growth on glucose or by nitrogen exhaustion [12,13,40,42]. This transporter has been characterized both genetically and biochemically.…”

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confidence: 99%

Substrate inhibition kinetics of Saccharomyces cerevisiae in fed-batch cultures operated at constant glucose and maltose concentration levels

Papagianni

Boonpooh

Mattey

et al. 2007

J Ind Microbiol Biotechnol

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Fed-batch culture is the mode of operation of choice in industrial baker's yeast fermentation. The particular mode of culture, operated at stable glucose and maltose concentration levels, was employed in this work in order to estimate important kinetic parameters in a process mostly described in the literature as batch or continuous culture. This way, the effects of a continuously falling sugar level during a batch process were avoided and therefore the effects of various (stable) sugar levels on growth kinetics were evaluated. Comparing the kinetics of growth and the inhibition by the substrate in cultures grown on glucose, which is the preferential sugar source for Saccharomyces cerevisiae, and maltose, the most common sugar source in industrial media for baker's yeast production, a milder inhibition effect by the substrate in maltose-grown cells was observed, as well as a higher yield coefficient. The observed sugar inhibition effect in glucostat cultures was taken into account in modeling substrate inhibition kinetics. The inhibition coefficient Ki increased with increasing sugar concentration levels, but it appeared to be unaffected by the type of substrate and almost equal for both substrates at elevated concentration levels.

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Identification of the Maltose Transport Protein of Saccharomyces cerevisiae

Cited by 14 publications

References 0 publications

Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes

Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes

In vivo Phosphorylation of the Purine/Cytosine Permease from the Plasma Membrane of the Yeast Saccharomyces cerevisiae

Substrate inhibition kinetics of Saccharomyces cerevisiae in fed-batch cultures operated at constant glucose and maltose concentration levels

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