Rius Mònica, Sala Laura, Chillarón Josep
Department of Physiology and Immunology, Faculty of Biology, University of Barcelona, Av. Diagonal, 643, E-08028, Barcelona, Spain.
Department of Physiology and Immunology, Faculty of Biology, University of Barcelona, Av. Diagonal, 643, E-08028, Barcelona, Spain
Biochem J. 2016 Feb 1;473(3):233-44. doi: 10.1042/BJ20150846. Epub 2015 Nov 4.
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.
转运系统b(0,+)介导肾脏中二元氨基酸和胱氨酸的重吸收。它由两个通过二硫键连接的膜亚基组成：载体b(0,+)AT和辅助蛋白rBAT（与b(0,+)氨基酸转运体相关）。损害该转运体生物合成的rBAT突变会导致I型胱氨酸尿症。研究表明，组装后，b(0,+)AT可防止rBAT降解并促进其折叠；然后，rBAT将b(0,+)AT从内质网（ER）转运至质膜。rBAT的N-聚糖及其与任何其他序列均无同源性的C末端环在b(0,+)系统生物合成中的作用尚不清楚。在本研究中，我们对这些问题进行了研究。我们首先鉴定了rBAT的五个N-聚糖。通过脉冲追踪实验和内切糖苷酶H分析测定，消除N-聚糖Asn(575)而非其他N-聚糖会延迟转运体成熟。此外，仅具有N-聚糖Asn(575)的转运体与野生型相比显示出相似的成熟情况，这表明该N-聚糖对于实现转运体成熟的最大速率是必要且充分的。删除rBAT C末端二硫键环（残基673 - 685）会阻止转运体成熟并促使其降解。丙氨酸扫描诱变揭示了对b(0,+)系统稳定性和/或成熟重要的环残基。此外，双突变循环分析表明Asn(679)环残基和N-聚糖Asn(575)对转运体成熟的影响具有部分加和性，表明它们可能在b(0,+)系统生物合成过程中相互作用。这些数据突出了rBAT的N-聚糖Asn(575)和C末端二硫键环在rBAT - b(0,+)AT异二聚体生物合成中的重要作用。
