Faculty Sponsor: Dr. Bryan Thurtle-Schmidt
Membrane transport proteins are essential cell components that maintain differentiated internal and external environments. The SLC4 family of membrane transport proteins contains borate transporter Bor1 and its human homolog, the bicarbonate transporter Band 3. Through multiple sequence alignment and structural analysis of Saccharomyces cerevisiae Bor1, conserved hydrophilic residues predicted to have functional importance were identified and mutated to an alanine or phenylalanine using site-directed mutagenesis. A genetic complementation assay was used to determine three mutations, Y212A, D272A, and S466A, that disrupted ScBor1 function and displayed a constitutively dead phenotype. While the functional impacts of Y212A and S266A are likely due to protein destabilization, the D272A protein was successfully purified. Purification and migration behavior in size-exclusion chromatography supports that D272A localizes to the membrane and folds correctly, suggesting an essential role for residue 272 in conformational changes of the ScBor1 transporter. Future experiments will attempt purification with residues that display a hypomorphic phenotype.