title: Carrier subunit of plasma membrane transporter is required for oxidative folding of its helper subunit.
abstract: We study the amino acid transport system b(0,+) as a model for folding, assembly, and early traffic of membrane protein complexes. System b(0,+) is made of two disulfide-linked membrane subunits: the carrier, b(0,+) amino acid transporter (b(0,+)AT), a polytopic protein, and the helper, related to b(0,+) amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b(0,+)AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b(0,+)AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally.
authors.affiliation: Department of Physiology and Immunology, Faculty of Biology, University of Barcelona, E-08028 Barcelona, Spain.
authors.forename: Mònica
authors.initials: M
authors.lastname: Rius
authors.forename: Josep
authors.initials: J
authors.lastname: Chillarón
mesh_terms: D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002460:Cell Line; D017931:DNA Primers; D004285:Dogs; D016521:Electrophoresis, Gel, Pulsed-Field; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017510:Protein Folding
chemical_list: D017931:DNA Primers; D026901:Membrane Transport Proteins