The role of N-glycans and the C-terminal loop of the subunit rBAT in the biogenesis of the cystinuria-associated transporter

The transport system b0,+ mediates reabsorption of dibasic amino acids and cystine in the kidney. It is made up of two disulfide-linked membrane subunits: the carrier, b0,+AT and the helper, rBAT (related to b0,+ amino acid transporter). rBAT mutations that impair biogenesis of the transporter cause type I cystinuria. It has been shown that upon assembly, b0,+AT prevents degradation and promotes folding of rBAT; then, rBAT traffics b0,+AT from the endoplasmic reticulum (ER) to the plasma membrane. The role of the N-glycans of rBAT and of its C-terminal loop, which has no homology to any other sequence, in biogenesis of system b0,+ is unknown. In the present study, we studied these points. We first identified the five N-glycans of rBAT. Elimination of the N-glycan Asn575, but not of the others, delayed transporter maturation, as measured by pulse chase experiments and endoglycosidase H assays. Moreover, a transporter with only the N-glycan Asn575 displayed similar maturation compared with wild-type, suggesting that this N-glycan was necessary and sufficient to achieve the maximum rate of transporter maturation. Deletion of the rBAT C-terminal disulfide loop (residues 673–685) prevented maturation and prompted degradation of the transporter. Alanine-scanning mutagenesis uncovered loop residues important for stability and/or maturation of system b0,+. Further, double-mutant cycle analysis showed partial additivity of the effects of the Asn679 loop residue and the N-glycan Asn575 on transporter maturation, indicating that they may interact during system b0,+ biogenesis. These data highlight the important role of the N-glycan Asn575 and the C-terminal disulfide loop of rBAT in biogenesis of the rBAT-b0,+AT heterodimer.