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.

Functional Expression of the Human Glucose Transporters GLUT2 and GLUT3 in Yeast Offers Novel Screening Systems for GLUT-Targeting Drugs

Human GLUT2 and GLUT3, members of the GLUT/SLC2 gene family, facilitate glucose transport in specific tissues. Their malfunction or misregulation is associated with serious diseases, including diabetes, metabolic syndrome, and cancer. Despite being promising drug targets, GLUTs have only a few specific inhibitors. To identify and characterize potential GLUT2 and GLUT3 ligands, we developed a whole-cell system based on a yeast strain deficient in hexose uptake, whose growth defect on glucose can be rescued by the functional expression of human transporters. The simplicity of handling yeast cells makes this platform convenient for screening potential GLUT2 and GLUT3 inhibitors in a growth-based manner, amenable to high-throughput approaches. Moreover, our expression system is less laborious for detailed kinetic characterization of inhibitors than alternative methods such as the preparation of proteoliposomes or uptake assays in Xenopus oocytes. We show that functional expression of GLUT2 in yeast requires the deletion of the extended extracellular loop connecting transmembrane domains TM1 and TM2, which appears to negatively affect the trafficking of the transporter in the heterologous expression system. Furthermore, single amino acid substitutions at specific positions of the transporter sequence appear to positively affect the functionality of both GLUT2 and GLUT3 in yeast. We show that these variants are sensitive to known inhibitors phloretin and quercetin, demonstrating the potential of our expression systems to significantly accelerate the discovery of compounds that modulate the hexose transport activity of GLUT2 and GLUT3.

Extra Fructose in the Growth Medium Fuels Lipogenesis of Adipocytes

Fructose in excessive amounts exerts negative effects on insulin sensitivity, blood pressure, and liver metabolism. These adverse outcomes were attributed to its disturbances of key metabolic pathways in the liver. Recently, possible consequences of high fructose levels directly on adipocytesin vivohave been considered. We have cultured adipocytes in growth media containing 1 g/L fructose additionally to glucose and monitored the cells fate. Cells developed lipid vesicles much earlier with fructose and showed altered kinetics of the expression of mRNAs involved in lipogenesis and hexose uptake. Adiponectin secretion, too, peaked earlier in fructose containing media than in media with glucose only. From these data it can be speculated that similar effects of fructose containing diets happenin vivoalso. Apart from toxic action on liver cells, adipocytes might be stimulated to take up extra fructose and generate new lipid vesicles, further dysregulating energy homeostasis.

Identification of Hexose Transporter-Like Sensor HXS1 and Functional Hexose Transporter HXT1 in the Methylotrophic Yeast Hansenula polymorpha

ABSTRACT We identified in the methylotrophic yeast Hansenula polymorpha (syn. Pichia angusta) a novel hexose transporter homologue gene, HXS1 (hexose sensor), involved in transcriptional regulation in response to hexoses, and a regular hexose carrier gene, HXT1 (hexose transporter). The Hxs1 protein exhibits the highest degree of primary sequence similarity to the Saccharomyces cerevisiae transporter-like glucose sensors, Snf3 and Rgt2. When heterologously overexpressed in an S. cerevisiae hexose transporter-less mutant, Hxt1, but not Hxs1, restores growth on glucose or fructose, suggesting that Hxs1 is nonfunctional as a carrier. In its native host, HXS1 is expressed at moderately low level and is required for glucose induction of the H. polymorpha functional low-affinity glucose transporter Hxt1. Similarly to other yeast sensors, one conserved amino acid substitution in the Hxs1 sequence (R203K) converts the protein into a constitutively signaling form and the C-terminal region of Hxs1 is essential for its function in hexose sensing. Hxs1 is not required for glucose repression or catabolite inactivation that involves autophagic degradation of peroxisomes. However, HXS1 deficiency leads to significantly impaired transient transcriptional repression in response to fructose, probably due to the stronger defect in transport of this hexose in the hxs1Δ deletion strain. Our combined results suggest that in the Crabtree-negative yeast H. polymorpha, the single transporter-like sensor Hxs1 mediates signaling in the hexose induction pathway, whereas the rate of hexose uptake affects the strength of catabolite repression.

Hexose uptake by developing cotyledons of Vicia faba: physiological evidence for transporters of differing affinities and specificities

Cotyledons of broad bean (Vicia faba L.) develop in an apoplasmic environment that shifts in composition from one dominated by hexoses to one dominated by sucrose. During the latter phase of development, sucrose / H+ symporter activity and expression is restricted to cotyledon epidermal transfer cell complexes that support sucrose fluxes that are 8.5-fold higher than those exhibited by the storage parenchyma. In contrast, the flux difference between these cotyledon tissues is only 1.7-fold for hexoses. Glucose and fructose uptake was shown to be sensitive to PCMBS and phloridzin, both of which slow H+-sugar transport. A low Km (or high affinity transporter, HAT) mechanism transports glucose and glucose-analogues exclusively. No HAT system for fructose could be found. A high Km (low affinity transporter, LAT) mechanism transports a broader range of hexoses, including glucose and fructose. Consistent with glucose and fructose transport being H+-coupled, their uptake was inhibited by dissipating the proton motive force (pmf) by treating cotyledons with carbonyl cyanide m-chlorophenol hydrazone, propionic acid or tetraphenylphosphonium ion. Erythrosin B inhibited hexose uptake, indicating a role for the P-type H+-ATPase in establishing the pmf. It is concluded that H+-coupled glucose and fructose transport mechanisms occur at plasma membranes of dermal transfer cell complexes and storage parenchyma cells. These transport mechanisms are active during pre- and storage phases of cotyledon development. However, hexose symport only makes a quantitative contribution to cotyledon biomass gain during the pre-storage stage of development.