Identification of Glucose Transporters in Aspergillus nidulans

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.

Comparison of the oxidative stress response of two Antarctic fungi to different growth temperatures

“…Under temperature induced oxidative stress, fungi reduce the glucose and ammonia uptake (Li et al 2008). In this way, the low temperature-induced stress can cause a dynamic rearrangement of the metabolic flux to the pentose phosphate pathway leading to the generation of the reduced electron carrier NADPH (Dos Reis et al 2013). Fungal cells accelerated the production of glucose by gluconeogenesis and the production of amino acids important to compose the repertoire of molecules to the oxidative stress response (Dos Reis et al 2013).…”

Organic acid production in Aspergillus niger and other filamentous fungi

“…This fact, plus additional insights observed at genomic, transcriptomic and proteomic level gave strong indications that RhtA acts as a specific transporter for L-rhamnose, but did not provide direct evidence. To assay transport capacity and quantify uptake kinetics, radiolabeled sugar uptake experiments can be performed with the mentioned yeast strain (Colabardini et al, 2014;dos Reis et al, 2013;Leandro et al, 2013;Sloothaak et al, 2015;Subtil and Boles, 2011;Wahl et al, 2010). This approach was followed in the present study as a final step to prove the ability of RhtA to transport L-rhamnose.…”

“…MstA also showed a preference for glucose, while being able to transport other monosaccharides as well (Vankuyk PA et al, 2004). Similarly, substrate specificity of Aspergillus nidulans hexose transporters HxtB and HxtC has been assayed (dos Reis et al, 2013), and a reduction of glucose uptake of 70 to 80 % was observed for galactose, fructose and mannose. In accordance to the functioning of many glucose transporters from other filamentous fungi, the function of MstG and…”

“…These yeast mutant strains, unable to grow on glucose, fructose, mannose and galactose as a single carbon source, have also subsequently been used as tools for the functional characterisation of sugar transporters from other fungal species (Colabardini et al, 2014;dos Reis et al, 2013;Du et al, 2010;Leandro et al, 2013;Polidori et al, 2007;Saloheimo et al, 2007;Vankuyk PA et al, 2004;Wahl et al, 2010) In contrast to S. cerevisiae, the only functionally validated sugar transporters in A. niger are the recently identified D-galacturonic acid transporter GatA (Martens-Uzunova and Schaap, 2008;Sloothaak et al, 2014), two fructose transporters (Coelho et al, 2013) and the high-affinity sugar/H + symporter MstA (Vankuyk PA et al, 2004). Furthermore, transcriptional data for the A. niger mstC gene suggests that it encodes a low-affinity glucose transporter (Jørgensen et al, 2007), but no experimental data supporting its role as a functional sugar transporter is publicly available.…”

“…Yeast transporter null mutants were subsequently also used for the functional characterization of sugar porters from other fungal species (dos Reis et al, 2013;Du et al, 2010;Leandro et al, 2013;Polidori et al, 2007;Saloheimo et al, 2007;Vankuyk PA et al, 2004).…”