Functional analysis of ght5 hexose transporter under iron stress in Schizosaccharomyces pombe

All eukaryotes need the element iron for metabolic functions. While iron is essential for electron transport and many enzymatic activities, it exerts a cytotoxic effect under certain conditions. On the other hand, glucose is the preferred energy and carbon source by most organisms and is an important signaling molecule in the regulation of biological processes. The uptake of glucose into the cell is provided by hexose carrier proteins. Schizosaccharomyces pombe has eight different hexose transporter proteins (Ght1-8). Among them, Ght5 is essential and sufficient for cell proliferation under low-glucose conditions. In this study, the function of the ght5 gene in S. pombe in sensing and signaling iron stress was investigated. For this purpose, the ght5 gene was cloned into pSLF172 and pSLF173 vectors and the resulting recombinant plasmids (SA01, SA02, SA03) were transformed into S. pombe EDD666 strain. The resulting SA01 and SA02 recombinant plasmids were used to investigate whether the HA epitope made any difference at the carboxy or amino terminus of the Ght5 protein. On the other hand, SA03 transformant S. pombe strain was produced under high (2%) and low (0.5%) glucose conditions and by applying iron at different concentrations (0, 0.5, 1 and 2 mM). The effect of iron stress on the expression profile of the ght5 gene was analyzed at the protein level by western blot method.It was observed that binding the HA epitope to the amino or carboxy terminus of the Ght5 protein did not make any difference, and the epitope worked in both cases. For SA03 strain; The observation of a darker and thicker band in the medium containing 0.5% glucose than that produced in the medium containing 2% glucose confirms the expression profile of the ght5 gene, which is known to have high glucose affinity. While a very dark and thick band was observed in the presence of 1 mM iron in the medium containing 0.5% glucose, the observation of lighter bands at 0.5 mM and 2 mM iron stress indicates that iron may be a factor on ght5 expression. In addition, the bands obtained from SA03 strain grown in 2% glucose media were lighter than those in 0.5% glucose media, but the presence of Ght5-HA protein bands that became evident due to increased iron stress ,further strengthens our thesis. In conclusion, the difference in the Ght5-HA protein bands observed in the SA03 strain against changing iron stress in repressed and depressed conditions suggests that the ght5 gene plays an important role in the detection and signaling of iron stress.