Determinants of amino acid transporter oligomerization in membranes

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ABSTRACT The Amino Acid-Polyamine-organoCation (APC) superfamily of membrane transport proteins is one of the largest families of solute carriers. They are involved in nutrient and amino acid transport across all kingdoms of life. In humans, they are essential for amino acid transport in kidneys, neurotransmitter re-uptake in synapses, movement of substances across the blood-brain barrier, and many other physiological roles. While their functions are diverse, they adopt a common topological arrangement known as the "LeuT-fold", involving an inverted topology repeat of 5 + 5 transmembrane helices, with an additional 2+ helices at the C-terminus that engage in oligomerization in some isoforms. In recent years, structures of many LeuT-fold homologues have been solved and in nearly every state along the transport cycle. When examining these structures, a major question stands out - why are LeuT-fold transporters observed in diverse oligomeric forms (i.e. monomers, dimers and trimers) despite possessing the same subunit fold? We hypothesize that oligomerization plays a role in APC transporter regulation, and that all LeuT-fold transporters possess an innate potential to dimerize in membranes due to the conserved subunit structure. However, the diversity of observed oligomers arises because of different stabilities due to differences in protein and lipid contact surfaces. To investigate this, we will study a pair of APC transporters that provide a model system for the rigorous investigation of this question. These are the arginine/agmatine antiporter AdiC and glutamate/GABA antiporter GadC. These two transporters are evolutionarily related within the same sub-family, and are both involved in extreme acid resistance in E. coli, yet, AdiC has only ever been observed as a dimer, while GadC is monomeric. In addition, these proteins are easy to purify and can be studied functionally in reconstituted systems, providing rigorous model systems to study dimerization determinants of LeuT-fold amino-acid transporters. In this investigation, we will use a combination of interdisciplinary studies that have allowed us to examine dimerization stability of other membrane protein systems, such as the CLC Cl-/H+ antiporter and the dual-topology Fluc F- ion channel. Computational studies will map out protein partners, interactions with water or lipids, and membrane structure in associated and dissociated forms. Single-molecule photobleaching analysis experiments will be carried out to measure the equilibrium dimerization affinity of subunits in membranes while testing key protein and lipid variables identified in the computational analysis. In Aim 1, we will map out what defines AdiC dimerization, and in Aim 2, identify the defining features that make GadC monomeric, with a final test to engineer GadC as a functional dimer. This rigorous compare/contrast study between two model LeuT-fold transporters will provide a fundamental understanding of oligomerization mechanisms relevant to all APC transporters, including those that are important in human health and disease.