Maltose Transport Research Articles

The Transcription Factors Atf1 and Pcr1 Are Essential for Transcriptional Induction of the Extracellular Maltase Agl1 in Fission Yeast

The fission yeast Schizosaccharomyces pombe secretes the extracellular maltase Agl1, which hydrolyzes maltose into glucose, thereby utilizing maltose as a carbon source. Whether other maltases contribute to efficient utilization of maltose and how Agl1 expression is regulated in response to switching of carbon sources are unknown. In this study, we show that three other possible maltases and the maltose transporter Sut1 are not required for efficient utilization of maltose. Transcription of agl1 was induced when the carbon source was changed from glucose to maltose. This was dependent on Atf1 and Pcr1, which are highly conserved transcription factors that regulate stress-responsive genes in various stress conditions. Atf1 and Pcr1 generally bind the TGACGT motif as a heterodimer. The agl1 gene lacks the exact motif, but has many degenerate TGACGT motifs in its promoter and coding region. When the carbon source was switched from glucose to maltose, Atf1 and Pcr1 associated with the promoters and coding regions of agl1, fbp1, and gpx1, indicating that the Atf1-Pcr1 heteromer binds a variety of regions in its target genes to induce their transcription. In addition, the association of Mediator with these genes was dependent on Atf1 and Pcr1. These data indicate that Atf1 and Pcr1 induce the transcription of agl1, which allows efficient utilization of extracellular maltose.

Leaves of the Arabidopsis maltose exporter1 mutant exhibit a metabolic profile with features of cold acclimation in the warm.

BackgroundArabidopsis plants accumulate maltose from starch breakdown during cold acclimation. The Arabidopsis mutant, maltose excess1-1, accumulates large amounts of maltose in the plastid even in the warm, due to a deficient plastid envelope maltose transporter. We therefore investigated whether the elevated maltose level in mex1-1 in the warm could result in changes in metabolism and physiology typical of WT plants grown in the cold.Principal FindingsGrown at 21 °C, mex1-1 plants were much smaller, with fewer leaves, and elevated carbohydrates and amino acids compared to WT. However, after transfer to 4 °C the total soluble sugar pool and amino acid concentration was in equal abundance in both genotypes, although the most abundant sugar in mex1-1 was still maltose whereas sucrose was in greatest abundance in WT. The chlorophyll a/b ratio in WT was much lower in the cold than in the warm, but in mex1-1 it was low in both warm and cold. After prolonged growth at 4 °C, the shoot biomass, rosette diameter and number of leaves at bolting were similar in mex1-1 and WT.ConclusionsThe mex1-1 mutation in warm-grown plants confers aspects of cold acclimation, including elevated levels of sugars and amino acids and low chlorophyll a/b ratio. This may in turn compromise growth of mex1-1 in the warm relative to WT. We suggest that elevated maltose in the plastid could be responsible for key aspects of cold acclimation.

Phosphatidylglycerol Directs Binding and Inhibitory Action of EIIAGlc Protein on the Maltose Transporter

The signal-transducing protein EIIA(Glc) belongs to the phosphoenolpyruvate carbohydrate phosphotransferase system. In its dephosphorylated state, EIIA(Glc) is a negative regulator for several permeases, including the maltose transporter MalFGK2. How EIIA(Glc) is targeted to the membrane, how it interacts with the transporter, and how it inhibits sugar uptake remain obscure. We show here that acidic phospholipids together with the N-terminal tail of EIIA(Glc) are essential for the high affinity binding of the protein to the transporter. Using protein docking prediction and chemical cross-linking, we demonstrate that EIIA(Glc) binds to the MalK dimer, interacting with both the nucleotide-binding and the C-terminal regulatory domains. Dissection of the ATPase cycle reveals that EIIA(Glc) does not affect the binding of ATP but rather inhibits the capacity of MalK to cleave ATP. We propose a mechanism of maltose transport inhibition by this central amphitropic regulatory protein.

Carbon catabolite repression of the maltose transporter revealed by X-ray crystallography

SummaryEfficient carbon utilization is critical to the survival of microorganisms in competitive environments. To optimize energy usage, bacteria have developed an integrated control system to preferentially uptake carbohydrates that support rapid growth. The availability of a preferred carbon source such as glucose represses the synthesis and activities of proteins necessary for the transport and metabolism of secondary carbon sources. This regulatory phenomenon is defined as carbon catabolite repression (CCR)1. In enteric bacteria, the key player of CCR is a component of the glucose-specific phosphotransferase system, enzyme IIA (EIIAGlc)1,2. It is known that unphosphorylated EIIAGlc binds and inhibits a variety of transporters when glucose is available1,2. However, understanding the underlying molecular mechanism has been hindered by the complete absence of structures for any EIIAGlc-transporter complexes. Here, we present the 3.9 Å crystal structure of EIIAGlc in complex with the maltose transporter, an ATP-binding cassette (ABC) transporter. The structure shows that two EIIAGlc molecules bind to the cytoplasmic ATPase subunits, stabilizing the transporter in an inward-facing conformation and preventing the structural rearrangements necessary for ATP hydrolysis. We also show that the half maximal inhibitory concentrations of the full-length EIIAGlc and an N-terminal truncation mutant differ by 60 fold, consistent with the hypothesis that the N-terminal region, disordered in the crystal structure, functions as a membrane-anchor to increase the effective EIIAGlc concentration at the membrane3,4. Together these data suggest a model of how the central regulatory protein EIIAGlc allosterically inhibits maltose uptake in E. coli.

Reinterpreting the Action of ATP Analogs on KATP Channels

Neuroendocrine-type K(ATP) channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K(+), and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting β-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.

Amy L. Davidson 1958–2013

On 2 April 2013, Amy L. Davidson lost her battle against thyroid cancer. The membrane transport field has lost one of its best biochemists and a superb mechanistic thinker. Amy devoted her career to uncovering the secrets of the maltose transporter, a prototype of a large protein family called ATP-binding cassette (ABC) transporters.

Molecular mechanism of the Escherichia coli maltose transporter

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins that import and export a large variety of materials across the lipid bilayer. A key question that drives ABC transporter research is how ATP hydrolysis is coupled to substrate translocation. This review uses the maltose transporter of Escherichia coli as a model system to understand the molecular mechanism of ABC importers. X-ray crystallography was used to capture the structures of the maltose transporter in multiple conformations. These structures, interpreted in the light of functional data, are discussed to address the following questions: first, what is the nature of conformational changes in a transport cycle? Second, how does substrate activate ATPase activity? Third, how does ATP hydrolysis enable substrate transport?

The maltose ABC transporter: Action of membrane lipids on the transporter stability, coupling and ATPase activity

The coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency.

Expression and functional analysis of rice plastidic maltose transporter, OsMEX1

In Arabidopsis, maltose is a major product of the transitory starch degradation pathway at night, and its mobilization from the chloroplasts to the cytosol in leaf tissues via a plastidic maltose transporter, AtMEX1, is essential for normal plant growth. However, such a starch utilization pathway has not yet been characterized in rice (Oryza sativa), a monocot model plant. Examination of expression profiles of a rice plastidic maltose transporter, OsMEX1, by real-time polymerase chain reaction showed that it is abundant in the pollen grain-containing stamens of mature flowers. Consistently, high performance liquid chromatography analysis revealed a relatively high maltose content in mature flowers, suggesting that OsMEX1 mainly functions in the tissues. OsMEX1-green fluorescent protein fusion experiment confirmed that OsMEX1 localizes at the chloroplast envelope in both rice and Arabidopsis. Arabidopsis maltose excess1 (mex1) mutant was transformed with OsMEX1 fused to the cauliflower mosaic virus 35S (CaMV35S) promoter. In the resulting transgenic plants, the typical mutant phenotypes of Arabidopsis mex1, such as chlorosis, stunted growth, and maltose and starch deposition at the end of the night, are clearly rescued. This result demonstrates that OsMEX1 functions as a plastidic maltose transporter in Arabidopsis. Our present findings thus suggest that whereas the Arabidopsis MEX1 gene essentially functions in source leaf tissues, its rice counterpart likely has a role in the pollens of mature flowers.

ATP Alone Triggers the Outward Facing Conformation of the Maltose ATP-binding Cassette Transporter

The maltose transporter MalFGK(2) is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction.

Structural Mechanism of Action of Binding Protein Independent Mutant MalG511 of Escherichia Coli Maltose Transporter

ATP-binding cassette (ABC) transporters couple the uphill transport of substances to the hydrolysis of ATP. Mutations in the E. coli maltose ABC transporter have been isolated that allow transport in the absence of periplasmic maltose binding protein (MBP), a protein that delivers maltose to the transporter with high affinity and stimulates the ATPase activity of the transporter. One such mutant, MalG511, shows an interesting biphasic behavior with low levels of MBP stimulating transport activity while higher levels (>50µM) inhibit transport activity. Remarkably, the rescuing effect of MBP suppressor mutants isolated at high, inhibitory MBP concentrations provided insight into regulatory mechanisms in the ABC transporter superfamily. Site directed spin labeling EPR spectroscopy was used to follow the catalytic cycle of MalG511 without any binding protein, with MBP and with suppressor MBP by attaching spin label at two positions in the MalK ATPase subunit (V16 and R129) that can be used to detect the closure of nucleotide-binding interface. MalG511 responded differently to the addition of ligands, including nucleotide and MBP, than wild type transporter. Notably, MalG511 can sample all conformational states (open, semi-open and closed interface) in the absence of MBP, as expected for a MBP-independent transporter. However, the closed state could be stabilized only in the presence of a transition state analogue (vanadate) suggesting that the mutations in MalG511 altered the conformational equilibrium of the transporter, favoring a semi-open over a closed conformation, even in the presence of ATP, plus or minus MBP. The addition of MBP, but not suppressor MBP, also resulted in a failure of the transporter to return to the fully open state at the end of a catalytic cycle, suggesting a mechanism for inhibition of transport at high MBP concentration.

Mapping the Conformational Stability of Maltose Binding Protein at the Residue Scale Using Nuclear Magnetic Resonance Hydrogen Exchange Experiments

Being able to differentiate local fluctuations from global folding-unfolding dynamics of a protein is of major interest for improving our understanding of structure-function determinants. The maltose binding protein (MBP), a protein that belongs to the maltose transport system, has a structure composed of two globular domains separated by a rigid-body "hinge bending". Here we determined, by using hydrogen exchange (HX) nuclear magnetic resonance experiments, the apparent stabilization free energies of 101 residues of MBP bound to β-cyclodextrin (MBP-βCD) under native conditions. We observed that the last helix of MBP (helix α14) has a lower protection factor than the rest of the protein. Further, HX experiments were performed using guanidine hydrochloride under subdenaturing conditions to discriminate between local fluctuations and global unfolding events and to determine the MBP-βCD energy landscape. The results show that helix α4 and a part of helices α5 and α6 are clearly grouped into a subdenaturing folding unit and represent a partially folded intermediate under native conditions. In addition, we observed that amide protons located in the hinge between the two globular domains share similar ΔG(gu)(app) and m values and should unfold simultaneously. These observations provide new points of view for improving our understanding of the thermodynamic stability and the mechanisms that drive folding-unfolding dynamics of proteins.

Proteome analysis of chlorotic leaves of the Arabidopsis mex1 mutant defective in the mobilization of starch degradation products

Leaf starch synthesized during the day for transient storage of photoassimilated carbon is degraded the following night to support respiration and growth in plants. Maltose is a major product of starch degradation, and is exported to the cytosol through the maltose transporter (MEX1). The Arabidopsis mex1 mutant displays growth retardation and an exceptional chlorotic phenotype that is not observed in other mutants demonstrating defective starch synthesis or degradation. Consistent with the chlorotic phenotype, proteomic analysis revealed degeneration of the photosynthetic machinery in mex1, and the down-regulation of essential components for photosynthesis was also observed. The chlorosis observed in mex1 occurs during vegetative growth period under normal growth conditions, which is distinct from general senescence-induced chlorosis. No up-regulation of senescence-related genes was found in the proteomic analysis of mex1, suggesting that the chlorotic process occurring in mex1 is likely distinct from senescence-dependent processes. On the other hand, cellular processes needed to survive stress situations caused by the blocking of maltose export are induced in mex1 by up-regulation of stress-related proteins, such as a germin-like protein and glutathione S-transferase. The increased abundance of heat shock protein 93-V participating in chloroplast biogenesis and rubisco activase, a regulatory protein of photosynthesis, likely reflects an attempt by the mex1 mutant to maintain chloroplast function to survive stress conditions.

Glucose Transport in Escherichia coli Mutant Strains with Defects in Sugar Transport Systems

In Escherichia coli, several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase system (glucose PTS), but the mannose PTS and the galactose and maltose transporters also can translocate glucose. Mutant strains which lack the enzyme IIBC (EIIBC) protein of the glucose PTS have been investigated previously because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit have not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as glucose-limited continuous cultivations. Deletion of EIIBC disturbs glucose transport severely in batch cultures; cyclic AMP (cAMP)-cAMP receptor protein (CRP) levels rise, and induction of the mgl operon occurs. Nevertheless, Mgl activity is not essential for growth of these mutants, since deletion of this transporter did not affect the growth rate; the activities of the remaining transporters seem to be sufficient. Under conditions of glucose limitation, mgl is upregulated 23-fold compared to levels for growth under glucose excess. Despite the strong induction of mgl upon glucose limitation, deletion of this transport system did not lead to further changes. Although the galactose transporters are often regarded as important for glucose uptake at micromolar concentrations, the glucose as well as mannose PTS might be sufficient for growth at this relatively low dilution rate.

Obacunone Represses Salmonella Pathogenicity Islands 1 and 2 in anenvZ-Dependent Fashion

Obacunone belongs to a class of unique triterpenoids called limonoids, present in Citrus species. Previous studies from our laboratory suggested that obacunone possesses antivirulence activity and demonstrates inhibition of cell-cell signaling in Vibrio harveyi and Escherichia coli O157:H7. The present work sought to determine the effect of obacunone on the food-borne pathogen Salmonella enterica serovar Typhimurium LT2 by using a cDNA microarray. Transcriptomic studies indicated that obacunone represses Salmonella pathogenicity island 1 (SPI1), the maltose transporter, and the hydrogenase operon. Furthermore, phenotypic data for the Caco-2 infection assay and maltose utilization were in agreement with microarray data suggesting repression of SPI1 and maltose transport. Further studies demonstrated that repression of SPI1 was plausibly mediated through hilA. Additionally, obacunone seems to repress SPI2 under SPI2-inducing conditions as well as in Caco-2 infection models. Furthermore, obacunone seems to repress hilA in an EnvZ-dependent fashion. Altogether, the results of the study seems to suggest that obacunone exerts an antivirulence effect on S. Typhimurium and may serve as a lead compound for development of antivirulence strategies for S. Typhimurium.

Characterization of the Saccharomyces bayanus-type AGT1 transporter of lager yeast

Transport of maltose and maltotriose into the yeast cell is thought to be rate-limiting in the utilization of these sugars. The maltose and maltotriose transporters Malx1, Agt1, Mtt1 and Mphx are present in different combinations in brewer's yeast strains, conferring different maltose and maltotriose transport characteristics on the strains. A new putative maltose/maltotriose transporter ORF was identified during whole genome sequencing of the lager strain WS34/70 (Y. Nakao et al., DNA Res., 2009, 16, 115–129). Sequence comparisons suggested that this putative α-glucoside transporter might be a Saccharomyces bayanus counterpart of the Agt1 (Saccharomyces cerevisiae type) transporter. In the present work, the transporter coded by a SbAGT1 gene from a lager strain, A15 (and with the same sequence as the corresponding gene in WS34/70) was characterized. It is shown that this SbAGT1 encodes a functional α-glucoside transporter with a wide-substrate range, including maltose and maltotriose. Trehalose, α-methylglucoside and sucrose were inhibitors, suggesting they are also substrates. The SbAgt1 transporter had similar affinities for maltose and maltotriose (17 ± 7 and 22 ± 2 m m, respectively) and a higher Vmax for maltose than maltotriose (21 ± 7 and 12 ± 2 µmol min−1 g dry yeast−1, respectively). Copyright © 2012 The Institute of Brewing & Distilling

The ABC transporter MalFGK2 sequesters the MalT transcription factor at the membrane in the absence of cognate substrate

MalK, the cytoplasmic component of the maltose ABC transporter from Escherichia coli is known to control negatively the activity of MalT, the activator of the maltose regulon, through complex formation. Here we further investigate this regulatory process by monitoring MalT activity and performing fluorescence microscopy analyses under various conditions. We establish that, under physiological conditions, the molecular entity that interacts with MalT is not free MalK, but the maltose transporter, MalFGK(2) , which sequesters MalT to the membrane. Furthermore, we provide compelling evidence that the transporter's ability to bind MalT is not constitutive, but strongly diminished when MalFGK(2) is engaged in sugar transport. Notably, the outward-facing transporter, i.e. the catalytic intermediate, is ineffective in inhibiting MalT compared to the inward-facing state, i.e. the resting form. Analyses of available genetic and structural data suggest how the interaction between one inactive MalT molecule and MalFGK(2) would be sensitive to the transporter state, thereby allowing MalT release upon maltose entrance. A related mechanism may underpin signalling by other ABC transporters.

Killing two birds with one stone: an ABC transporter regulates gene expression through sequestration of a transcriptional regulator at the membrane

Transcriptional regulators are controlled through various, mostly well-understood, principles. In the study of Richet et al., published in this issue of Molecular Microbiology, fluorescence microscopy was used to uncover an unorthodox mechanism that relies on the dynamic shuttling of a gene regulator between the membrane and the chromosome. When not occupied with transport, the maltose-specific ABC transporter sequesters and thereby inactivates its cognate transcriptional regulator MalT. Upon maltose transport, MalT is released from the membrane and activates the maltose utilization and transport genes. This mechanism prevents induction of MalT by endogenously produced maltotriose, which is the inducer. Thus, the maltose uptake system is a trigger transporter with a bi-functional role in transport and regulation.

Substrate Transport Activation Is Mediated through Second Periplasmic Loop of Transmembrane Protein MalF in Maltose Transport Complex of Escherichia coli

In a recent study we described the second periplasmic loop P2 of the transmembrane protein MalF (MalF-P2) of the maltose ATP-binding cassette transporter (MalFGK(2)-E) as an important element in the recognition of substrate by the maltose-binding protein MalE. In this study, we focus on MalE and find that MalE undergoes a structural rearrangement after addition of MalF-P2. Analysis of residual dipolar couplings (RDCs) shows that binding of MalF-P2 induces a semiopen state of MalE in the presence and absence of maltose, whereas maltose is retained in the binding pocket. These data are in agreement with paramagnetic relaxation enhancement experiments. After addition of MalF-P2, an increased solvent accessibility for residues in the vicinity of the maltose-binding site of MalE is observed. MalF-P2 is thus not only responsible for substrate recognition, but also directly involved in activation of substrate transport. The observation that substrate-bound and substrate-free MalE in the presence of MalF-P2 adopts a similar semiopen state hints at the origin of the futile ATP hydrolysis of MalFGK(2)-E.

Discovery of an Auto-Regulation Mechanism for the Maltose ABC Transporter MalFGK2

The maltose transporter MalFGK2, 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.