OVEREXPRESSION OF MAL61P IN SACCHAROMYCES-CEREVISIAE AND CHARACTERIZATION OF MALTOSE TRANSPORT IN ARTIFICIAL MEMBRANES

For maltose uptake in Saccharomyces cerevisiae, multiple kinetic forms of transport as well as inhibition of transport by high concentration s of maltose at the trans side of the plasma membrane have been descri bed. Most of these studies were hampered by a lack of genetically well -defined mutants and/or the lack of an artificial membrane system to s tudy translocation catalysis in vitro. A genetically well defined S. c erevisiae strain lacking the various MAL loci was constructed by gene disruption. Expression of the maltose transport protein (Mal61p) was s tudied by using various plasmid vectors that differed in copy number a nd/or type of promoter. The expression levels were quantitated by immu noblotting with antibodies generated against the N-terminal half of Ma l61p. The levels df expression as well as the initial uptake rates wer e increased 20-fold compared with those in a yeast strain carrying onl y one chromosomal MAL locus. Similar results were obtained when the tr ansport activities were compared in hybrid membranes of the correspond ing strains. To generate a proton motive force, isolated membranes wer e fused with liposomes containing cytochrome c oxidase as a proton pum p. Fusion was achieved by a cycle of freeze-thawing, after which the h ybrid membranes were passed through a filter with a defined pore size to obtain unilamellar membrane vesicles. Proton motive force driven ma ltose uptake, maltose efflux down the concentration gradient, and equi librium exchange of maltose in the hybrid membranes vesicles have been analyzed. The data indicate that maltose transport by the maltose tra nsporter is kinetically monophasic and fully reversible under all cond itions tested.