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

Abstract

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.

Alkaline Response Genes of Saccharomyces cerevisiaeand Their Relationship to the RIM101 Pathway

“…One effect of external alkalinity is the disruption of membrane proton gradients, which normally supply energy for nutrient and ion transport (10). Prior studies indicated that yeast responds by expressing ENA1, a plasma membrane Na ϩ -ATPase gene (14,18).…”

“…4). The rim101⌬ strain grew poorly in the presence of NaCl and LiCl, as do ena1 mutants (10,42). This growth defect did not arise from general osmotic sensitivity because RIM101 and rim101⌬ strains grew equally well on medium 3-6 and 7-10, respectively).…”

“…They neutralize the cytoplasm through activity of Pma1p, the plasma membrane H ϩ -ATPase, which hydrolyzes ATP to pump protons out of the cell (10). The proton gradient is used for transport of amino acids, nucleotide bases, phosphate, and many other molecules in symport reactions (10).…”

“…It also provides an electrochemical gradient that favors uptake of cations (11,12). Thus, the plasma membrane proton gradient is required for accumulation of solutes from the medium, and PMA1 is an essential gene (10,13).…”

Regulation of maltose transport in Saccharomyces cerevisiae

“…This so called trans-inhibition involves a decrease in transport activity as the intracellular substrate concentration increases and has been well-described for bacterial transport systems involved in osmoregulation (Poolman and Glaasker 1998). Studies in hybrid membranes with overexpressed Mal61p indicate that maltose transport is reversible, because influx as well as efflux transport can be observed (Van der Rest et al 1995). Since the orientation of the maltose transporter in the hybrid membranes is most likely random, these experiments do not rule out the possibility that the maltose transport system is trans-inhibited or, for some other reason, kinetically different for uptake and efflux transport, as both modes of transport may be facilitated by oppositely oriented Mal61p molecules.…”