Targeted Mutation of Lipid‐Binding Sites in ScBor1 Does Not Prevent Dimerization

Abstract

Many transporters and ion channels form higher‐order structural complexes, but the functional significance of such protein complexes often remains unclear. Additionally, many recent studies have implicated lipid‐binding as a key factor in the formation of multimeric membrane protein complexes. The SLC4 transporters carry bicarbonate across membranes, and are typified by Band 3, the most abundant membrane protein in red blood cells. Structures of multiple SLC4 transporters including Band 3 reveal homodimeric structures, but the role dimerization plays in their function remains unknown. In this study we investigate S. cerevisiaeBor1, a borate transporter homologous to human SLC4 transporters. ScBor1 is predominantly a monomer when purified in detergent, but a recent study reveals a possible site for lipids to bind and support dimerization during lipid reconstitution experiments. To better understand the role of multimerization in SLC4 homolog function, we introduced Arg to Ala mutations in ScBor1 designed to abolish a presumed binding site for phospholipids. After protein purification and lipid reconstitution, we assessed multimerization through both oxidative and glutaraldehyde crosslinking experiments. Unlike previous studies, our data show that dimerization of the wild‐type protein is promoted by both phosphatidylcholine and phosphatidylglycerol, and that the mutated protein retains dimerization to the same extent as the wild‐type protein. Further studies will be required to establish the role that dimerization plays in function in the SLC4 family and its homologs.