Investigating Voltage Dependence of STP Homologs through Sequence-Guided Mutagenesis
Sugar transport proteins (STPs) are members of the major facilitator superfamily; STPs are transmembrane proteins embedded in the plasma membrane that symport hexose sugars and are proton (H+) coupled. This experiment sought to investigate the structural basis for increased substrate affinity at depolarized potentials in Arabidopsis thaliana’s STP13 and other homologs, as opposed to increased substrate affinity at hyperpolarized potentials in AtSTP1. Phylogenetic analysis followed by multiple sequence alignment of the proteins’ primary sequence revealed areas of conservation within STP13 and other orthologs to mutate. Based on this analysis, reciprocal mutations were introduced to test whether these residues affect voltage dependence. Mutants were cloned into expression vectors, transformed into E. coli and selected with Kanamycin, transcribed into RNA, and expressed in Xenopus laevis oocytes. Electrophysiological properties were assessed using two-electrode voltage clamping across various sugars and membrane potentials. Current analysis is ongoing and inconclusive.