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

Abstract

The transport system b(0,+) mediates reabsorption of dibasic amino acids and cystine in the kidney. It is made up of two disulfide-linked membrane subunits: the carrier, b(0,+)AT and the helper, rBAT (related to b(0,+) amino acid transporter). rBAT mutations that impair biogenesis of the transporter cause type I cystinuria. It has been shown that upon assembly, b(0,+)AT prevents degradation and promotes folding of rBAT; then, rBAT traffics b(0,+)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 b(0,+) is unknown. In the present study, we studied these points. We first identified the five N-glycans of rBAT. Elimination of the N-glycan Asn(575), 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 Asn(575) 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 b(0,+). Further, double-mutant cycle analysis showed partial additivity of the effects of the Asn(679) loop residue and the N-glycan Asn(575) on transporter maturation, indicating that they may interact during system b(0,+) biogenesis. These data highlight the important role of the N-glycan Asn(575) and the C-terminal disulfide loop of rBAT in biogenesis of the rBAT-b(0,+)AT heterodimer.

Heteromeric amino acid transporters. In search of the molecular bases of transport cycle mechanisms

“…Recently, it has been reported that the N-glycan N575 of human rBAT is indispensable for early traffic, and the C-terminal loop of this heavy subunit is relevant for the stability and/or early traffic of rBAT-b 0, + AT [47]. A possible interaction between the N575 N-glycan and the C-terminal loop has been proposed to be important for the biogenesis of this heterodimer [47].…”

“…Assembly with b 0, + AT prevents ER-associated degradation (ERAD) of unassembled rBAT and is required for the oxidative folding of the rBAT-ED, which proceeds via the formation of three consecutive disulfide bonds (see 'The heavy subunits of HATs'), two of them -the domain B disulfide and the disulfide connecting domain C with the C-terminal tail -essential for biogenesis [46]. Recently, it has been reported that the N-glycan N575 of human rBAT is indispensable for early traffic, and the C-terminal loop of this heavy subunit is relevant for the stability and/or early traffic of rBAT-b 0, + AT [47]. A possible interaction between the N575 N-glycan and the C-terminal loop has been proposed to be important for the biogenesis of this heterodimer [47].…”

Ca2+-mediated higher-order assembly of b0,+AT–rBAT is a key step for system b0,+biogenesis and cystinuria

“…Human rBAT has an additional glycan on Asn575', which is critical for rBAT maturation (23). Asn575' in ovine rBAT is not glycosylated, due to the replacement of Ser577' to Asn577' (Fig.…”

“…This structural difference is caused by the bulky amino acids Tyr425' and Tyr579' pushing away Asn575' in human rBAT, which are replaced by smaller amino acids Ser425' and Ser579' in ovine rBAT. Thus, human rBAT has an additional glycan for stabilizing the domain A-C interface, which explains why disrupting this glycan causes premature degradation of system b 0,+ in humans (23).…”

“…7b), and our structure indicates that these substitutions affect the domain A-TMD and A-C interfaces and probably destabilize the assembled system b 0,+ . Human rBAT possesses an additional N-glycan on Asn575', which stabilizes the domain A-C interface and is indispensable in the maturation of other glycans (23). The C-terminal peptide of rBAT forms a β -hairpin that mediates interactions with b 0,+ AT and the lipid bilayer, which explains how the L678P mutant affects protein stability (23) (Fig.…”