Passive plasma membrane transporters play a critical role in perception of carbon availability in yeast

Recently, our lab found that the canonical glucose/galactose regulation pathway in yeast makes the decision to metabolize galactose based on the ratio of glucose to galactose concentrations in the external medium. This led to the question of where and how the ratio-sensing is achieved. Here, we consider the possibilities of an intracellular, extracellular, or membrane bound ratio sensing mechanisms. We show that hexose transporters in the plasma membrane are mainly responsible for glucose/galactose ratio-sensing in yeast. Further, while the glucose sensors Gpr1, Snf3, and Rgt2 are not required for ratio sensing, they help modulate the ratio sensing phenotype by regulating the expression of individual transporters in different environments. Our study provides an example of an unexpected, but potentially widespread, mechanism for making essential decisions.

The Role of Hexokinase and Hexose Transporters in Preferential Use of Glucose over Fructose and Downstream Metabolic Pathways in the Yeast Yarrowia lipolytica

The development of efficient bioprocesses requires inexpensive and renewable substrates. Molasses, a by-product of the sugar industry, contains mostly sucrose, a disaccharide composed of glucose and fructose, both easily absorbed by microorganisms. Yarrowia lipolytica, a platform for the production of various chemicals, can be engineered for sucrose utilization by heterologous invertase expression, yet the problem of preferential use of glucose over fructose remains, as fructose consumption begins only after glucose depletion what significantly extends the bioprocesses. We investigated the role of hexose transporters and hexokinase (native and fructophilic) in this preference. Analysis of growth profiles and kinetics of monosaccharide utilization has proven that the glucose preference in Y. lipolytica depends primarily on the affinity of native hexokinase for glucose. Interestingly, combined overexpression of either hexokinase with hexose transporters significantly accelerated citric acid biosynthesis and enhanced pentose phosphate pathway leading to secretion of polyols (31.5 g/L vs. no polyols in the control strain). So far, polyol biosynthesis was efficient in glycerol-containing media. Moreover, overexpression of fructophilic hexokinase in combination with hexose transporters not only shortened this process to 48 h (84 h for the medium with glycerol) but also allowed to obtain 23% more polyols (40 g/L) compared to the glycerol medium (32.5 g/L).

A Novel Hexose Transporter ChHxt6 Is Required for Hexose Uptake and Virulence in Colletotrichum higginsianum

Colletotrichum higginsianum is an important hemibiotrophic plant pathogen that causes crucifer anthracnose worldwide. To date, some hexose transporters have been identified in fungi. However, the functions of hexose transporters in virulence are not clear in hemibiotrophic phytopathogens. In this study, we identified and characterized a new hexose transporter gene named ChHxt6 from a T-DNA insertion pathogenicity-deficient mutant G256 in C. higginsianum. Expression profiling analysis revealed that six ChHxt genes, ChHxt1 to ChHxt6, exhibited specific expression patterns in different infection phases of C. higginsianum. The ChHxt1 to ChHxt6 were separately deleted using the principle of homologous recombination. ChHxt1 to ChHxt6 deletion mutants grew normally on PDA plates, but only the virulence of ChHxt4 and ChHxt6 deletion mutants was reduced. ChHxt4 was required for fungal infection in both biotrophic and necrotrophic stages, while ChHxt6 was important for formation of necrotrophic hyphae during infection. In addition, ChHxts were functional in uptake of different hexoses, but only ChHxt6-expressing cells could grow on all five hexoses, indicating that the ChHxt6 was a central hexose transporter and crucial for hexose uptake. Site-directed mutation of T169S and P221L positions revealed that these two positions were necessary for hexose transport, whereas only the mutation Thr169 caused reduced virulence and defect in formation of necrotrophic hyphae. Taken together, ChHxt6 might regulate fungal virulence by modulating the utilization of hexose.

TORC2 inhibition of α-arrestin Aly3 effects cell surface persistence of S. pombe Ght5 glucose transporter in low-glucose

In the fission yeast, Schizosaccharomyces pombe, the high-affinity hexose transporter, Ght5, must be transcriptionally upregulated and localized to the cell surface for cell division under limited glucose. While cell-surface localization of Ght5 depends on Target Of Rapamycin Complex 2 (TORC2), the molecular mechanisms by which TORC2 ensures proper localization of Ght5 remain unknown. We performed genetic screening for gene mutations that restore Ght5 localization on the cell surface in TORC2-deficient mutant cells, and identified a gene encoding an uncharacterized α-arrestin-like protein, Aly3/SPCC584.15c. α-arrestins are thought to recruit a ubiquitin ligase to membrane-associated proteins. Consistently, Ght5 is ubiquitinated in TORC2-deficient cells, and this ubiquitination is dependent on Aly3. TORC2 supposedly enables cell-surface localization of Ght5 by preventing Aly3-dependent ubiquitination and subsequent ubiquitination-dependent translocation of Ght5 to vacuoles. Surprisingly, nitrogen starvation, but not glucose depletion, triggers Aly3-dependent transport of Ght5 to vacuoles in S. pombe, unlike budding yeast hexose transporters, vacuolar transport of which is initiated upon changes in the hexose concentration. This study provides new insights into molecular mechanisms controlling subcellular localization of hexose transporters in response to extracellular stimuli.

A push-pull system of repressors matches levels of glucose transporters to extracellular glucose in budding yeast

SummaryA common cellular task is to match gene expression dynamically to a range of concentrations of a regulatory molecule. Studying glucose transport in budding yeast, we determine mechanistically how such matching occurs for seven hexose transporters. By combining time-lapse microscopy with mathematical modelling, we find that levels of transporters are history-dependent and are regulated by a push-pull system comprising two types of repressors. Repression by these two types varies with glucose in opposite ways, and not only matches the expression of transporters by their affinity to a range of glucose concentrations, but also the expression of some to how glucose is changing. We argue that matching is favoured by a rate-affinity trade-off and that the regulatory system allows yeast to import glucose rapidly enough to starve competitors. Matching expression to a pattern of input is fundamental, and we believe that push-pull repression is widespread.

The Sugar Porter gene family of Piriformospora indica: Nomenclature, Transcript Profiling and Characterization

Piriformospora indica is one of the prominent mutualistic root endophyte known to overcome phosphate and nitrogen limitation in a wide variety of plant species, reciprocally takes up carbohydrates for its survival and growth. A total of nineteen potential hexose transporters have been identified from P. indica genome, that may contributes to its potential of carbohydrate assimilation from host plant. Phylogenetic analysis assembles it in 10 groups within 3 clusters. To ease the study, systematic nomenclature were provided to 19 putative hexose transporters as “PiST1-PiST19” in accordance to their appearance on the supercontigs genome sequence of P. indica. The protein length ranges from 487 to 608 amino acids. Out of 19 putative hexose transporters, 9 have been predicted to contain 12 transmembrane domains (PiST1, PiST2, PiST5, PiST6, PiST9, PiST10, PiST11, PiST12 and PiST17), along with MFS family and Sugar porter subfamily motif. Therefore, transcripts were detected for these 9 genes. During colonization, three P. indica genes PiST1, PiST5 and PiST9 have shown induction as compared to axenic culture. Similarly during phosphate starvation, revealed PiST12 to be strongly enhanced. Carbon starvation study in liquid axenic culture resulted in induction of 4 genes, PiST6, PiST9, PiST12 and PiST17. We found co-relation in the expression pattern of PiPT and PiST12 during phosphate starvation. In silico analysis revealed the presence of functional conserved fucose permease (FucP) domain, involved in fructose transport. Phylogenetic analysis revealed that PiST12 groups closely with basidiomycetes hexose transporters. Further, functional complementation of Δhxt null mutant revealed, PiST12 is able to complement growth on fructose and galactose but negligible on glucose.

Computational analysis of GAL pathway pinpoints mechanisms underlying natural variation

Quantitative traits such as human height are measurable phenotypes that show continuous variation over a wide phenotypic range. Enormous effort has recently been put into determining the genetic influences on a variety of quantitative traits, including human genetic diseases, with mixed success. We identified a quantitative trait in a tractable model system, the GAL pathway in yeast, which controls the uptake and metabolism of the sugar galactose. GAL pathway activation depends both on galactose concentration and on the concentrations of competing, preferred sugars such as glucose. Natural yeast isolates show substantial variation in the behavior of the pathway. All studied yeast strains exhibit bimodal responses relative to external galactose concentration, i.e. a set of galactose concentrations existed at which both GAL-induced and GAL-repressed subpopulations were observed. However, these concentrations differed in different strains. We built a mechanistic model of the GAL pathway and identified parameters that are plausible candidates for capturing the phenotypic features of a set of strains including standard lab strains, natural variants, and mutants. In silico perturbation of these parameters identified variation in the intracellular galactose sensor, Gal3p, the negative feedback node within the GAL regulatory network, Gal80p, and the hexose transporters, HXT, as the main sources of the bimodal range variation. We were able to switch the phenotype of individual yeast strains in silico by tuning parameters related to these three elements. Determining the basis for these behavioral differences may give insight into how the GAL pathway processes information, and into the evolution of nutrient metabolism preferences in different strains. More generally, our method of identifying the key parameters that explain phenotypic variation in this system should be generally applicable to other quantitative traits.Author summaryMicrobes adopt elaborate strategies for the preferred uptake and use of nutrients to cope with complex and fluctuating environments. As a result, yeast strains originating from different ecological niches show significant variation in the way they induce genes in the galactose metabolism (GAL) pathway in response to nutrient signals. To identify the mechanistic sources of this variation, we built a mathematical model to simulate the dynamics of the galactose metabolic regulation network, and studied how parameters with different biological implications contributed to the natural variation. We found that variations in the behavior of the galactose sensor Gal3p, the negative feedback node Gal80p, and the hexose transporters HXT were critical elements in the GAL pathway response. Tuning single parameters in silico was sufficient to achieve phenotype switching between different yeast strains. Our computational approach should be generally useful to help pinpoint the genetic and molecular bases of natural variation in other systems.

A label-free real-time method for measuring glucose uptake kinetics in yeast

Glucose uptake assays commonly rely on the isotope-labeled sugar, which is associated with radioactive waste and exposure of the experimenter to radiation. Here, we show that the rapid decrease of the cytosolic pH after a glucose pulse to starved Saccharomyces cerevisiae cells is dependent on the rate of sugar uptake and can be used to determine the kinetic parameters of sugar transporters. The pH-sensitive GFP variant pHluorin is employed as a genetically encoded biosensor to measure the rate of acidification as a proxy of transport velocity in real time. The measurements are performed in the hexose transporter-deficient (hxt0) strain EBY.VW4000 that has been previously used to characterize a plethora of sugar transporters from various organisms. Therefore, this method provides an isotope-free, fluorometric approach for kinetic characterization of hexose transporters in a well-established yeast expression system.

Sugar transport for enhanced xylose utilization in Ashbya gossypii

The co-utilization of mixed (pentose/hexose) sugars constitutes a challenge for microbial fermentations. The fungus Ashbya gossypii, which is currently exploited for the industrial production of riboflavin, has been presented as an efficient biocatalyst for the production of biolipids using xylose-rich substrates. However, the utilization of xylose in A. gossypii is hindered by hexose sugars. Three A. gossypii homologs (AFL204C, AFL205C and AFL207C) of the yeast HXT genes that code for hexose transporters have been identified and characterized by gene-targeting approaches. Significant differences in the expression profile of the HXT homologs were found in response to different concentrations of sugars. More importantly, an amino acid replacement (N355V) in AFL205Cp, introduced by CRISPR/Cas9-mediated genomic edition, notably enhanced the utilization of xylose in the presence of glucose. Hence, the introduction of the afl205c-N355V allele in engineered strains of A. gossypii will further benefit the utilization of mixed sugars in this fungus.