Oxidative folding and early traffic of the human cystinuria transporter

Resumo

The aim of this work is to gain insight in the understanding of the biogenesis of membrane proteins, specially their folding, assembly and ER-exit. Our model is the human cystinuria transporter rBAT- b0,+AT. Disulfides and N-glycans are crucial for the correct folding, assembly and traffic of proteins. So we identified the disulfides and the N-glycans of the transporter and analysed their role in biogenesis of the transporter. In order to analyse the disulfide connectivity of rBAT, cysteine residues were mutated to serine, and we used mass-tagging of sulfhydryl (-SH) groups with mPEG5000-maleimide (mPEG) under denaturing conditions. A molecule of mPEG attaches to a –SH group, shifting the apparent molecular weight of the protein of interest by aprox. 5 kDa, which is easily detectable in SDS-PAGE. These experiments show that, in the presence of b0,+AT, the rBAT ectodomain is completely oxidised and contains 3 disulfide bonds. The pegylation pattern of C242S and C273A suggest that they form a disulfide bond. Pulse-chase analysis show that mutants C242S to C666S are retained in the ER and degraded, while C673S and C685S displayed slower stability and maturation rate. Interestingly, the double mutant C673S-C685S had a wild-type behaviour. These results strongly suggested that the other 2 disulfides present in rBAT ectodomain are C571-C666 and C673-C685. Further analysis showed that the only disulfide bond that is stably formed in the absence of the others is C242-C273, suggesting that it is the first to form in rBAT. It was also observed that when rBAT is expressed in the absence of b0,+AT, its oxidative folding is impaired, indicating that b0,+AT is necessary for the folding of rBAT. Its main role may be the stabilization of the oxidation of C571 to form the C571-C666 disulfide. This step seems to occur post-translationally and possibly defining the end of the oxidative folding process. In order to analyse the role of the N-glycans of rBAT the N-glycan consensus sites were mutated to serine or alanine. Pulse assays showed that rBAT contains 5 N-glycans. Pulse-chase and Endo H assays showed that all mutants display a wild-type like stability and maturation, except for the S577A mutant. Further studies showed that the N332 probably sustains degradation of unassembled rBAT and that the N-glycan N575 is necessary and sufficient for a wild-type-like maturation rate. The C-terminal loop of rBAT plays a key role in biogenesis as its deletion causes retention in the ER and subsequent degradation of rBAT. Analysis of this C-terminal loop showed that, when mutated, residues S675A, L678A and N679A have a similar maturation defect than the N-glycan mutant S577A and that mutants Y674A, L681A and Y682A showed little maturation and stability, explaining, at least in part, the results obtained when the whole loop is deleted. When expressed in the C673S-C685S background, some single loop mutants show an important decrease in rBAT stability and maturation. This synergistic effect suggests that the disulfide bond masks part of the maturation and stability effects of these loop residues, and that the absence of the disuflide bond potentiate the misfolding and maturation defects caused by these mutations. Finally, the biogenesis effects of some loop residues in the S577A background suggest that residues S675, L678 and N679 interact functionally and/or structurally with the N-glycan N575 and that they may form part of an ER-exit conformational signal in the luminal domain of the transporter.