rBAT-b(0,+)AT heterodimer is the main apical reabsorption system for cystine in the kidney.
Mutations in the rBAT and b(0,+)AT genes cause type I and non-type I cystinuria, respectively. The disulfide-linked rBAT-b(0,+)AT heterodimer mediates high-affinity transport of cystine and dibasic amino acids (b(0,+)-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 b(0,+)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 b(0,+)AT were solely expressed as heterodimers of identical size and that both proteins coprecipitated. Moreover, quantitative immunopurification of b(0,+)AT followed by SDS-PAGE and mass spectrometry analysis established that b(0,+)AT heterodimerizes exclusively with rBAT. Together with cystine reabsorption data, our results demonstrate that a decreasing expression gradient of heterodimeric rBAT-b(0,+)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 b(0,+)AT protein in the kidney. Indeed, complete immunodepletion of b(0,+)AT did not coprecipitate >20-30% of rBAT. Therefore, another rBAT-associated subunit may be present in latter parts of the proximal tubule.
Fernández
Esperanza
E
Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona 08028, Spain.
Carrascal
Montserrat
M
Rousaud
Ferran
F
Abián
Joaquín
J
Zorzano
Antonio
A
Palacín
Manuel
M
Chillarón
Josep
J
D000042:Absorption; D000595:Amino Acid Sequence; D026923:Amino Acid Transport Systems, Basic; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000737:chemistry; D003553:Cystine / Q000378:metabolism*; D003555:Cystinuria / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007163:Immunosorbent Techniques; D007687:Kidney Tubules, Proximal / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008871:Microvilli / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation
D026923:Amino Acid Transport Systems, Basic; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; C499489:SLC7A9 protein, human; C499488:Slc7a9 protein, mouse; D003553:Cystine