Cystinuria-specific rBAT(R365W) mutation reveals two translocation pathways in the amino acid transporter rBAT-b0,+AT.
Apical reabsorption of dibasic amino acids and cystine in kidney is mediated by the heteromeric amino acid antiporter rBAT/b(0,+)AT (system b(0,+)). Mutations in rBAT cause cystinuria type A, whereas mutations in b(0,+)AT cause cystinuria type B. b(0,+)AT is the catalytic subunit, whereas it is believed that rBAT helps the routing of the rBAT/b(0,+)AT heterodimeric complex to the plasma membrane. In the present study, we have functionally characterized the cystinuria-specific R365W (Arg(365)-->Trp) mutation of human rBAT, which in addition to a trafficking defect, alters functional properties of the b(0,+) transporter. In oocytes, where human rBAT interacts with the endogenous b(0,+)AT subunit to form an active transporter, the rBAT(R365W) mutation caused a defect of arginine efflux without altering arginine influx or apparent affinities for intracellular or extracellular arginine. Transport of lysine or leucine remained unaffected. In HeLa cells, functional expression of rBAT(R365W)/b(0,+)AT was observed only at the permissive temperature of 33 degrees C. Under these conditions, the mutated transporter showed 50% reduction of arginine influx and a similar decreased accumulation of dibasic amino acids. Efflux of arginine through the rBAT(R365W)/b(0,+)AT holotransporter was completely abolished. This supports a two-translocation-pathway model for antiporter b(0,+), in which the efflux pathway in the rBAT(R365W)/b(0,+)AT holotransporter is defective for arginine translocation or dissociation. This is the first direct evidence that mutations in rBAT may modify transport properties of system b(0,+).
Pineda
Marta
M
Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Barcelona Science Park, Barcelona 08028, Spain.
Wagner
Carsten A
CA
Bröer
Angelika
A
Stehberger
Paul A
PA
Kaltenbach
Simone
S
Gelpí
Josep Ll
JL
Martín Del Río
Rafael
R
Zorzano
Antonio
A
Palacín
Manuel
M
Lang
Florian
F
Bröer
Stefan
S
D019943:Amino Acid Substitution / Q000235:genetics* / Q000502:physiology; D026962:Amino Acid Transport System ASC / Q000502:physiology; D026905:Amino Acid Transport Systems / Q000235:genetics / Q000502:physiology; D026923:Amino Acid Transport Systems, Basic; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology; D045744:Cell Line, Tumor; D003555:Cystinuria / Q000235:genetics* / Q000503:physiopathology; D005260:Female; D006367:HeLa Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000502:physiology; D015778:Minor Histocompatibility Antigens; D009154:Mutation / Q000235:genetics* / Q000502:physiology; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014364:Tryptophan / Q000235:genetics; D014982:Xenopus laevis
D026962:Amino Acid Transport System ASC; D026905:Amino Acid Transport Systems; D026923:Amino Acid Transport Systems, Basic; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D015778:Minor Histocompatibility Antigens; C420506:SLC1A5 protein, human; C499489:SLC7A9 protein, human; D014364:Tryptophan; D001120:Arginine