rBAT-b0,+AT heterodimer is the main apical reabsorption system for cystine in the kidney

Abstract

Mutations in the rBAT and b0,+AT genes cause type I and non-type I cystinuria, respectively. The disulfide-linked rBAT-b0,+AT heterodimer mediates high-affinity transport of cystine and dibasic amino acids (b0,+-like activity) in heterologous cell systems. However, the significance of this heterodimer for cystine reabsorption is unknown, as direct evidence for such a complex in vivo is lacking and the expression patterns of rBAT and b0,+AT along the proximal tubule are opposite. We addressed this issue by biochemical means. Western blot analysis of mouse and human kidney brush-border membranes showed that rBAT and b0,+AT were solely expressed as heterodimers of identical size and that both proteins coprecipitated. Moreover, quantitative immunopurification of b0,+AT followed by SDS-PAGE and mass spectrometry analysis established that b0,+AT heterodimerizes exclusively with rBAT. Together with cystine reabsorption data, our results demonstrate that a decreasing expression gradient of heterodimeric rBAT-b0,+AT along the proximal tubule is responsible for virtually all apical cystine reabsorption. As a corollary of the above, there should be an excess of rBAT expression over that of b0,+AT protein in the kidney. Indeed, complete immunodepletion of b0,+AT did not coprecipitate >20–30% of rBAT. Therefore, another rBAT-associated subunit may be present in latter parts of the proximal tubule.