Molecular Basis for the Interaction of the Mammalian Amino Acid Transporters B0AT1 and B0AT3 with Their Ancillary Protein Collectrin*

Background: Collectrin is required for membrane expression of the broad neutral amino acid transporters (B0AT1 and -3). Results: Collectrin activates B0AT1 and B0AT3 in a discrete interaction region of the transporters. Conclusion: A potential conserved ancillary protein binding region in B0AT1/B0AT3 mediates collectrin interactions. Significance: This is the first example of a potential common interaction site for multiple solute carrier 6 family ancillary proteins. Many solute carrier 6 (SLC6) family transporters require ancillary subunits to modify their expression and activity. The main apical membrane neutral amino acid transporters in mouse intestine and kidney, B0AT1 and B0AT3, require the ancillary protein collectrin or ACE2 for plasma membrane expression. Expression and activity of SLC6 neurotransmitter transporters are modulated by interaction with syntaxin 1A. Utilizing monocarboxylate-B0AT1/3 fusion constructs, we discovered that collectrin is also necessary for B0AT1 and B0AT3 catalytic function. Syntaxin 1A and syntaxin 3 inhibit the membrane expression of B0AT1 by competing with collectrin for access. A mutagenesis screening approach identified residues on trans-membrane domains 1α, 5, and 7 on one face of B0AT3 as a key region involved in interaction with collectrin. Mutant analysis established residues that were involved in collectrin-dependent functions as follows: plasma membrane expression of B0AT3, catalytic activation, or both. These results identify a potential binding site for collectrin and other SLC6 ancillary proteins.