title: 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.
authors.affiliation: Department of Physiology and Immunology, Faculty of Biology, University of Barcelona, Av. Diagonal, 643, E-08028, Barcelona, Spain.
authors.forename: Mònica
authors.initials: M
authors.lastname: Rius
authors.affiliation: Department of Physiology and Immunology, Faculty of Biology, University of Barcelona, Av. Diagonal, 643, E-08028, Barcelona, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Sala
authors.affiliation: Department of Physiology and Immunology, Faculty of Biology, University of Barcelona, Av. Diagonal, 643, E-08028, Barcelona, Spain jchillaron@ub.edu.
authors.forename: Josep
authors.initials: J
authors.lastname: Chillarón
mesh_terms: D020816:Amino Acid Motifs; D026923:Amino Acid Transport Systems, Basic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D003553:Cystine / Q000378:metabolism; D003555:Cystinuria / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism*
chemical_list: D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D011134:Polysaccharides; C489857:SLC3A1 protein, human; D003553:Cystine
keywords: N-linked glycosylation; cystinuria; endoplasmic reticulum; folding; trafficking; transporter