Neutral amino acids are essential for protein synthesis, energy homeostasis, and many vital biochemical pathways. As several neutral amino acids are essential and cannot be synthesised by humans in vivo, their uptake and reuptake by the body's absorbing epithelial layers is of major importance for systemic amino acid homeostasis and human health. Two of the major pathways for the absorption of neutral amino acids in the small intestine and kidney are the Broad Neutral Amino acid Transporters 1

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... nd 3 (B0AT1 and B0AT3). B0AT1 is of medical interest as an indirect regulator of blood glycemia via cellular amino acid starvation response pathways. Absence of B0AT1 results in improved glycemic control and other metabolic effects, revealing it as a potential pharmacological target to treat type II diabetes. Essential to understanding the role of B0AT1 as a global metabolic regulator is its requirement for plasma membrane expression with heteromeric protein partners: collectrin in the kidney and Angiotensin Converting Enzyme 2 (ACE2) in the small intestine. Both proteins are membrane-anchored by a single trans-membrane domain and are homologous. B0AT3 also requires collectrin or ACE2 for membrane expression. Beyond this requirement for plasma membrane expression, however, little is known about the underlying mechanisms of the interaction. This thesis demonstrates additional protein partners of B0AT1, namely Aminopeptidase N (APN), syntaxin 1A, and syntaxin 3. These proteins facilitate changes in the kinetic parameters of neutral amino acid transport, in addition to regulating membrane expression. Moreover, I demonstrate that collectrin is required for catalytic activation of both B0AT1 and B0AT3, while both APN and ACE2 increase B0AT1 and B0AT3 substrate affinity, respectively. ACE2 and APN also form large complexes with B0AT1 at the small intestine brush border membrane. A binding site in both transporters for collectrin is identified as lying in the hydrophobic pocket between TM 5 and 7 of the transporters. Using a mixt [...]