Investigating Disease‐Causing Mutations in SLC4 Transporter Homologs

Abstract

The SLC4 family of transporters includes the archetypal transporter Band 3, or Anion Exchanger 1 (AE1), a bicarbonate and chloride ion exchanger essential for human respiration and the most abundant membrane protein in red blood cells. Mutations in SLC4 transporters such as AE1 can lead to diseases such as hereditary spherocytosis or renal tubular acidosis. The SLC4 family shares sequence and structural homology with borate transporters, which are found in plants and fungi and regulate cellular borate levels to protect against borate toxicity. Here we generated mutations in the S. cerevisiae borate transporter Bor1 homologous with known disease‐causing mutations of human AE1, and then tested for borate transport activity in a genetic complementation assay. Our results identify mutants with impaired function but wild‐type expression levels, as well as mutants with impaired function and depleted expression levels. These results are consistent with varying modes of deleterious impact of protein expression, sorting, and function that have been attributed to mutations in human AE1. These data show that mutations equivalent to disease‐causing mutations in human AE1 have major effects in ScBor1, strengthening the functional connection between SLC4 proteins and borate transporters. Additionally, we demonstrate that the ScBor1 D347 residue, which is homologous to the essential E681 of human AE1, can be complemented by the D347E but not D347N substitution, underscoring the necessity of having an acidic residue at this highly conserved functional location. Lastly, we identify several new amino acids critical for function through mutagenesis and complementation studies. These data collectively highlight similarities in the biochemical features of ScBor1 and HsAE1 and help shed light on the mechanisms of borate transporters and their SLC4 homologs.