MAL11 and MAL61 encode the inducible high-affinity maltose transporter of Saccharomyces cerevisiae.

We have investigated the transport of maltose in a genetically defined maltose-fermenting strain of Saccharomyces cerevisiae carrying the MAL1 locus. Two kinetically different systems were identified: a high-affinity transporter with a Km of 4 mM and a low-affinity transporter with a Km of 70 to 80 mM. The high-affinity maltose transporter is maltose inducible and is encoded by the MAL11 (and/or MAL61) gene of the MAL1 (and/or MAL6) locus. The low-affinity maltose transporter is expressed constitutively and is not related to MAL11 and/or MAL61. Both maltose transporters are subject to glucose-induced inactivation.     

Kinetics of active alpha-glucoside transport in Saccharomyces cerevisiae

alpha-Glucosides are the most abundant fermentable sugars in the industrial applications of Saccharomyces cerevisiae, and the active transport across the plasma membrane is the rate-limiting step for their metabolism. In this report we performed a detailed kinetic analysis of the active alpha-glucoside transport system(s) present in a wild-type strain, and in strains with defined alpha-glucoside permeases. Our results indicate that the wild-type strain harbors active transporters with high and low affinity for maltose and trehalose, and low-affinity transport systems for maltotriose and alpha-methylglucoside. The maltose permease encoded by the MAL21 gene showed a high affinity (K(m) approximately 5 mM) for maltose, and a low affinity (K(m) approximately 90 mM) for trehalose. On the other hand, the alpha-glucoside permease encoded by the AGT1 gene had a high affinity (K(m) approximately 7 mM) for trehalose, a low affinity (K(m) approximately 18 mM) for maltose and maltotriose, and a very low affinity (K(m) approximately 35 mM) for alpha-methylglucoside.     

Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli

Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system. We observed a 10-fold increase in the level of transport activity in assays with membrane vesicles when the three membrane-associated components of the transport system (the MalF, MalG, and MalK proteins) were overproduced. In addition, we have successfully reconstituted maltose transport activity in proteoliposome vesicles from solubilized proteins using a detergent dilution procedure. The addition of ATP as an energy source was sufficient to obtain transport, and this activity was dependent on the presence of maltose binding protein and was not seen in proteoliposomes prepared from a strain with a deletion of the maltose genes. We determined that hydrolysis of ATP was directly coupled to maltose uptake. In the majority of these experiments, an average of 1.4 mol of ATP was hydrolyzed for each mole of maltose accumulated. However, in the remaining experiments, ATP hydrolysis was observed to be much higher and averaged 17 mol of ATP hydrolyzed per mol of maltose transported. Possible explanations for a variable stoichiometry are discussed. These results provide strong evidence that it is the hydrolysis of ATP by a component of the transport complex that provides the energy required for active maltose transport.     

Identification and characterization of the maltose permease in genetically defined Saccharomyces strain

Saccharomyces yeasts ferment several alpha-glucosides including maltose, maltotriose, turanose, alpha-methylglucoside, and melezitose. In the utilization of these sugars transport is the rate-limiting step. Several groups of investigators have described the characteristics of the maltose permease (D. E. Kroon and V. V. Koningsberger, Biochim. Biophys. Acta 204:590-609, 1970; R. Serrano, Eur. J. Biochem. 80:97-102, 1977). However, Saccharomyces contains multiple alpha-glucoside transport systems, and these studies have never been performed on a genetically defined strain shown to have only a single permease gene. In this study we isolated maltose-negative mutants in a MAL6 strain and, using a high-resolution mapping technique, we showed that one class of these mutants, the group A mutants, mapped to the MAL61 gene (a member of the MAL6 gene complex). An insertion into the N-terminal-coding region of MAL61 resulted in the constitutive production of MAL61 mRNA and rendered the maltose permease similarly constitutive. Transformation by high-copy-number plasmids containing the MAL61 gene also led to an increase in the maltose permease. A deletion-disruption of MAL61 completely abolished maltose transport activity. Taken together, these results prove that this strain has only a single maltose permease and that this permease is the product of the MAL61 gene. This permease is able to transport maltose and turanose but cannot transport maltotriose, alpha-methylglucoside, or melezitose. The construction of strains with only a single permease will allow us to identify other maltose-inducible transport systems by simple genetic tests and should lead to the identification and characterization of the multiple genes and gene products involved in alpha-glucoside transport in Saccharomyces yeasts.     

Maltose transport in Escherichia coli K-12: involvement of the bacteriophage lambda receptor.

Mutants affected in lamB, the structural gene for phage lambda receptor, are unable to utilize maltose when it is present at low concentrations (less than or equal 10 muM). During growth in a chemostat at limiting maltose concentrations, the lamB mutants tested were selected against in the presence of the wild-type strain. Transport studies demonstrate that most lamB mutants have deficient maltose transport capacities at low maltose concentrations. When antibodies against purified phage lambda receptor are added to a wild-type strain, transport of maltose at low concentrations is significantly reduced. These results strongly suggest that the phage lambda receptor molecule is involved in maltose transport.     

Evidence for the Transport of Maltose by the Sucrose Permease,CscB, of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The purpose of this study was to examine the sugar recognition and transport properties of the sucrose permease (CscB), a secondary active transporter from Escherichia coli. We tested the hypothesis that maltose transport is conferred by the wild-type CscB transporter. Cells of E. coli HS4006 harboring pSP72/cscB were red on maltose MacConkey agar indicator plates. We were able to measure “downhill” maltose transport and establish definitive kinetic behavior for maltose entry in such cells. Maltose was an effective competitor of sucrose transport in cells with CscB, suggesting that the respective maltose and sucrose binding sites and translocation pathways through the CscB channel overlap. Accumulation (“uphill” transport) of maltose by cells with CscB was profound, demonstrating active transport of maltose by CscB. Sequencing of cscB encoded on plasmid pSP72/cscB used in cells for transport studies indicate an unaltered primary CscB structure, ruling out the possibility that mutation conferred maltose transport by CscB. We conclude that maltose is a bona fide substrate for the sucrose permease of E. coli. Thus, future studies of sugar binding, transport, and permease structure should consider maltose, as well as sucrose. Yang Peng and Sanath Kumar contributed equally to this paper.     

Residues in the alpha helix 7 of the bacterial maltose binding protein which are important in interactions with the Mal FGK2 complex.

The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose transport, most probably in a productive interaction between the MalE protein and the membrane bound MalFGK2 complex.     

Escherichia coli K-12 mutants that allow transport of maltose via the beta-galactoside transport system.

We have isolated mutants of Escherichia coli that have an altered beta-galactoside transport system. This altered transport system is able to transport a sugar, maltose, that the wild-type beta-galactoside transport system is unable to transport. The mutation that alters the specificity of the transport system is in the lacY gene, and we refer to the allele as lacYmal. The lacYmal allele was detected originally in strains in which the lac genes were fused to the malF gene. Thus, as a result of gene fusion and isolation of the lacYmal mutation, a new transport system was evolved with regulatory properties and specificity similar to those of the original maltose transport system. Maltose transport via the lacYmal gene product is independent of all of the normal maltose transport system components. The altered transport system shows a higher affinity than the wild-type transport system for two normal substrates of the beta-galactoside transport system, thiomethyl-beta-D-galactoside and o-nitrophenyl-beta-D-galactoside.     

Molecular Analysis of Maltotriose Active Transport and Fermentation by Saccharomyces cerevisiae Reveals a Determinant Role for the AGT1 Permease

Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known α-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and MAL inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high- and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (K_m, 36 ± 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.     

Maltose chemoreceptor of Escherichia coli.

Strains carrying mutations in the maltose system of Escherichia coli were assayed for maltose taxis, maltose uptake at 1 and 10 muM maltose, and maltose-binding activity released by osmotic shock. An earlier conclusion that the metabolism of maltose is not necessary for chemoreception is extended to include the functioning of maltodextrin phosphorylase, the product of malP, and the genetic control of the maltose receptor by the product of malT is confirmed. Mutants in malF and malK are defective in maltose transport at low concentrations as well as high concentrations, as previously shown, but are essentially normal in maltose taxis. The product of malE has been previously shown to be the maltose-binding protein and was implicated in maltose transport. Most malE mutants are defective in maltose taxis, and all those tested are defective in maltose transport at low concentrations. Thus, as previously suggested, the maltose-binding protein probably serves as the recognition component of the maltose receptor, as well as a component of the transport system. tsome malE mutants release maltose-binding activity and are tactic toward maltose, although defective in maltose transport, implying that the binding protein has separate sites for interaction with the chemotaxis and transport systems. Some mutations in lamB, whose product is the receptor for the bacteriophage lamba, cause defects in maltose taxis, indicating some involvement of that product in maltose reception.     

Crystal structure of a defective folding protein

Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32-->Asp and Ile 33-->Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose-binding activity comparable with that of the wild-type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose-bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 A resolution. The conformation of MalE31 closely resembles that of wild-type MalE, with very small displacements of the mutated residues located in the loop connecting the first alpha-helix to the first beta-strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild-type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system.     

Bacterial chemotaxis to saccharides is governed by a trade-off between sensing and uptake

To swim up gradients of nutrients, E. coli senses nutrient concentrations within its periplasm. For small nutrient molecules, periplasmic concentrations typically match extracellular concentrations. However, this is not necessarily the case for saccharides, such as maltose, which are transported into the periplasm via a specific porin. Previous observations have shown that, under various conditions, E. coli limits maltoporin abundance so that, for extracellular micromolar concentrations of maltose, there are predicted to be only nanomolar concentrations of free maltose in the periplasm. Thus, in the micromolar regime, the total uptake of maltose from the external environment into the cytoplasm is limited not by the abundance of cytoplasmic transport proteins but by the abundance of maltoporins. Here, we present results from experiments and modeling suggesting that this porin-limited transport enables E. coli to sense micromolar gradients of maltose despite having a high-affinity ABC transport system that is saturated at these micromolar levels. We used microfluidic assays to study chemotaxis of E. coli in various gradients of maltose and methyl-aspartate and leveraged our experimental observations to develop a mechanistic transport-and-sensing chemotaxis model. Incorporating this model into agent-based simulations, we discover a trade-off between uptake and sensing: although high-affinity transport enables higher uptake rates at low nutrient concentrations, it severely limits the range of dynamic sensing. We thus propose that E. coli may limit periplasmic uptake to increase its chemotactic sensitivity, enabling it to use maltose as an environmental cue.     

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 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.     

Discovery of an auto-regulation mechanism for the maltose ABC transporter MalFGK2

The maltose transporter MalFGK(2), together with the substrate-binding protein MalE, is one of the best-characterized ABC transporters. In the conventional model, MalE captures maltose in the periplasm and delivers the sugar to the transporter. Here, using nanodiscs and proteoliposomes, we instead find that MalE is bound with high-affinity to MalFGK2 to facilitate the acquisition of the sugar. When the maltose concentration exceeds the transport capacity, MalE captures maltose and dissociates from the transporter. This mechanism explains why the transport rate is high when MalE has low affinity for maltose, and low when MalE has high affinity for maltose. Transporter-bound MalE facilitates the acquisition of the sugar at low concentrations, but also captures and dissociates from the transporter past a threshold maltose concentration. In vivo, this maltose-forced dissociation limits the rate of transport. Given the conservation of the substrate-binding proteins, this mode of allosteric regulation may be universal to ABC importers.     

MBP-MalFGK2相互作用动力学:ABC转运蛋白的模型研究

本文主要描述了麦芽糖结合蛋白(MBP)和属于ATP结合盒式蛋白(ABC)家族的麦芽糖转运蛋白复合物MalFGK2的相互作用。通过基因、结构和生化分析可知,MBP和MalFGK2以不同构象进行相互作用。在这个转运系统中,MBP与麦芽糖结合,并与MalFGK2发生相互作用,从而将麦芽糖从胞外转运至胞内,但由于MBP和MalFGK2都有多种构象,所以它们的相互作用很复杂。相互作用机理模型最重要的特点是结合配体的MBP,通过稳定MalFGK2的高能量构象来启动依赖ATP的麦芽糖转运过程。麦芽糖转运蛋白机理模型表明,ABC型转运系统利用外周结合蛋白,其转运过程基本上是不可逆的。     

Transport kinetics of maltotriose in strains ofSaccharomyces

Summary Maltotriose transport was studied in two brewer's yeast strains, an ale strain 3001 and a lager strain 3021, using laboratory-synthesized¹⁴C-maltotriose. The maltotriose transport systems preferred a lower pH (pH 4.3) to a higher pH (pH 6.6). Two maltotriose transport affinity systems have been indentified. The high affinity system hasK _m values of 1.3 mM for strain 3021 and 1.4 mM for strain 3001. The low affinity competitively inhibited by maltose and glucose withK _i values of 58 mM and 177 mM. respectively, for strain 3021, and 55 mM and 147 mM, respectively, for strain 3001. Cells grown in maltotriose and maltose had higher maltotriose and maltose transport rates, and cells grown in glucose had lower maltortriose and maltose transport rates. Early-logarithmic phase cells transported glucose faster than either maltose or maltotriose. Cells harvested later in the growth phase had increased maltotriose and maltose transport activity. Neither strain exhibited significant differences with respect to maltose and maltotriose transport activity.     

Energetics and kinetics of maltose transport in Saccharomyces cerevisiae: a continuous culture study.

In Saccharomyces cerevisiae, maltose is transported by a proton symport mechanism, whereas glucose transport occurs via facilitated diffusion. The energy requirement for maltose transport was evaluated with a metabolic model based on an experimental value of YATP for growth on glucose and an ATP requirement for maltose transport of 1 mol.mol-1. The predictions of the model were verified experimentally with anaerobic, sugar-limited chemostat cultures growing on a range of maltose-glucose mixtures at a fixed dilution rate of 0.1 h-1. The biomass yield (grams of cells.gram of sugar-1) decreased linearly with increasing amounts of maltose in the mixture. The yield was 25% lower during growth on maltose than during that on glucose, in agreement with the model predictions. During sugar-limited growth, the residual concentrations of maltose and glucose in the culture increased in proportion to their relative concentrations in the medium feed. From the residual maltose concentration, the in situ rates of maltose consumption by cultures, and the Km of the maltose carrier for maltose, it was calculated that the amount of this carrier was proportional to the in situ maltose consumption rate. This was also found for the amount of intracellular maltose. These two maltose-specific enzymes therefore exert high control over the maltose flux in S. cerevisiae in anaerobic, sugar-limited, steady-state cultures.     

Regulation of the maltose transport system of Escherichia coli by the glucose-specific enzyme III of the phosphoenolpyruvate-sugar phosphotransferase system. Characterization of inducer exclusion-resistant mutants and reconstitution of inducer exclusion in proteoliposomes

Maltose transport in Escherichia coli is regulated at the protein level by the glucose-specific enzyme III (IIIglc) of the phosphoenolpyruvate-sugar phosphotransferase system, by a mechanism known as inducer exclusion. We have isolated and characterized four mutants in the maltose transport system, all of which are in malK, which are resistant to inducer exclusion. The mutations in three of these mutants fall within the COOH-terminal domain of MalK and suggest the first reported function for this domain. Two of these are in a region which shows sequence similarity to lacY and melB, both of which are also regulated by IIIglc, and thus may define a IIIglc-binding domain. We have also reconstituted inducer exclusion in proteoliposomes made from membranes overexpressing the maltose permease. Maltose transport is inhibited by 50-60% when IIIglc is included in the intravesicular space. The inhibition is due to a decrease in the Vmax of transport by a factor of 2. IIIglc does not affect the coupling of ATP hydrolysis to maltose transport, since the ratio of ATP hydrolyzed/maltose transported remained constant in the presence and absence of IIIglc. Finally, the Ki for IIIglc was 40 microM, roughly the same as the in vivo concentration of IIIglc.