Abstract
To characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and –E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose.
Highlights
Glucose represents the main source of carbon and energy for most heterotrophic organisms, in turn influencing the regulation of cell growth, metabolism and development [1]
The transcription of the four hxt genes when A. nidulans is grown in the presence of either 1 or 0.1 % glucose was confirmed via RT-qPCR (Figure 2)
A putative high-affinity glucose transporter, hxtA, which has increased mRNA accumulation when A. nidulans is grown in the presence of low glucose concentrations or during carbon starvation was used as a control [42]
Summary
Glucose represents the main source of carbon and energy for most heterotrophic organisms, in turn influencing the regulation of cell growth, metabolism and development [1]. In S. cerevisiae, extracellular glucose is sensed by two specific transmembrane proteins that act as sensors, Rgt and Snf, which demonstrate similarity to hexose transporters (Hxt proteins). These sensor proteins are unable to transport glucose and have unusually long C-terminal tails (around 200 amino acids) that are predicted to reside in the cytoplasm [13] and are necessary for the sensing mechanisms [14,15,16]. In the absence of extracellular glucose, a transcriptional repressor complex, comprised of Rgt, Std and Mth, is bound to the promoter regions of HXT genes inhibiting transcription [17]. When Snf and Rgt detect extracellular glucose, the Std and Mth co-repressors are phosphorylated by the Yck and Yck kinases [18] and targeted to the SCFGrr E2/E3 ubiquitin complex for
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