Distinct classes of trafficking rBAT mutants cause the type I cystinuria phenotype.
Most mutations in the rBAT subunit of the heterodimeric cystine transporter rBAT-b(0,+)AT cause type I cystinuria. Trafficking of the transporter requires the intracellular assembly of the two subunits. Without its partner, rBAT, but not b(0,+)AT, is rapidly degraded. We analyzed the initial biogenesis of wild-type rBAT and type I cystinuria rBAT mutants. rBAT was degraded, at least in part, via the ERAD pathway. Assembly with b(0,+)AT within the endoplasmic reticulum (ER) blocked rBAT degradation and could be independent of the calnexin chaperone system. This system was, however, necessary for post-assembly maturation of the heterodimer. Without b(0,+)AT, wild-type and rBAT mutants were degraded with similar kinetics. In its presence, rBAT mutants showed strongly reduced (L89P) or no transport activity, failed to acquire complex N-glycosylation and to oligomerize, suggesting assembly and/or folding defects. Most of the transmembrane domain mutant L89P did not heterodimerize with b(0,+)AT and was degraded. However, the few [L89P]rBAT-b(0,+)AT heterodimers were stable, consistent with assembly, but not folding, defects. Mutants of the rBAT extracellular domain (T216M, R365W, M467K and M467T) efficiently assembled with b(0,+)AT but were subsequently degraded. Together with earlier results, the data suggest a two-step biogenesis model, with the early assembly of the subunits followed by folding of the rBAT extracellular domain. Defects on either of these steps lead to the type I cystinuria phenotype.
Bartoccioni
Paola
P
Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain.
Rius
Mònica
M
Zorzano
Antonio
A
Palacín
Manuel
M
Chillarón
Josep
J
D026923:Amino Acid Transport Systems, Basic / Q000235:genetics* / Q000378:metabolism*; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics* / Q000378:metabolism*; D003555:Cystinuria / Q000235:genetics*; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D017434:Protein Structure, Tertiary; D021381:Protein Transport
D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; C489857:SLC3A1 protein, human