Bartoccioni Paola, Rius Mònica, Zorzano Antonio, Palacín Manuel, Chillarón Josep
Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain.
Hum Mol Genet. 2008 Jun 15;17(12):1845-54. doi: 10.1093/hmg/ddn080. Epub 2008 Mar 10.
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.
异二聚体胱氨酸转运蛋白rBAT-b(0,+)AT的rBAT亚基中的大多数突变会导致I型胱氨酸尿症。该转运蛋白的运输需要两个亚基在细胞内组装。没有其伙伴rBAT，b(0,+)AT不会迅速降解，但rBAT会迅速降解。我们分析了野生型rBAT和I型胱氨酸尿症rBAT突变体的初始生物合成过程。rBAT至少部分地通过内质网相关蛋白降解（ERAD）途径被降解。在内质网（ER）中与b(0,+)AT组装可阻止rBAT降解，且这一过程可能独立于钙连蛋白伴侣系统。然而，该系统对于异二聚体组装后的成熟是必需的。没有b(0,+)AT时，野生型和rBAT突变体以相似的动力学被降解。在b(0,+)AT存在的情况下，rBAT突变体的转运活性显著降低（L89P）或无转运活性，无法获得复杂的N-糖基化且不能寡聚化，这表明存在组装和/或折叠缺陷。大多数跨膜结构域突变体L89P不能与b(0,+)AT异二聚化并被降解。然而，少数[L89P]rBAT-b(0,+)AT异二聚体是稳定的，这与组装缺陷而非折叠缺陷一致。rBAT胞外结构域的突变体（T216M、R365W、M467K和M467T）能与b(0,+)AT有效组装，但随后被降解。结合早期结果，这些数据提示了一个两步生物合成模型，即亚基先进行早期组装，随后rBAT胞外结构域进行折叠。这两个步骤中任何一个出现缺陷都会导致I型胱氨酸尿症表型。
