Assessing Protomer Independence of the Dimeric C4-Dicarboxylate Transporter, Vcindy

Abstract

The divalent anion:sodium symporter (DASS) family includes characterized representatives from bacteria and humans (SLC13 family). DASS family members have been implicated in key physiological roles, including fatty acid synthesis and the transport of Krebs cycle intermediates and sulfate across the cytoplasmic membrane. Disruption of genes encoding DASS family members in mice and flies reveal roles in energy homeostasis, affecting lifespan determination, insulin resistance and obesity in these organisms. VcINDY, from the bacterium Vibrio cholerae is the only DASS family member for which there is high resolution structural information available and functional characterization reveals VcINDY to share key functional characteristics with mammalian DASS family members. VcINDY is a functional dimer exhibiting a large buried surface area at the dimer interface; a feature likely shared by all DASS family members. Characterization of other bacterial members of the family suggests cooperativity between the protomers. This apparent inter-protomer communication is likely transmitted via substrate-induced conformational changes at the dimer interface. If true, this has important mechanistic implications for the entire family. To investigate whether transport by VcINDY is a cooperative process we have introduced cysteine residues at the dimer interface that, under oxidizing conditions or in the presence of crosslinking agents, will staple regions of the interface together, limiting protein movement. Using this procedure we have demonstrated that pinning the interface together in several regions does not abrogate transport activity, indicating that no obligatory conformational changes occur across the dimer interface during transport. Although further confirmatory work is required, these data suggest that the VcINDY protomers function independently of one another during the transport cycle.