Nucleotide binding and nucleotide hydrolysis properties of the ABC transporter MRP6 (ABCC6).
Mutations in the MRP gene family member MRP6 cause pseudoxanthoma elasticum (PXE) in humans, a disease affecting elasticity of connective tissues. The normal function of MRP6, including its physiological substrate(s), remains unknown. To address these issues, recombinant rat Mrp6 (rMrp6) was expressed in the methylotrophic yeast Pichia pastoris. The protein was expressed in the membrane fraction as a stable 170 kDa protein. Its nucleotide binding and hydrolysis properties were investigated using the photoactive ATP analogue 8-azido-[alpha-(32)P]ATP and compared to those of the drug efflux pump MRP1. rMrp6 can bind 8-azido-[alpha-(32)P]ATP in a Mg(2+)-dependent and EDTA-sensitive fashion. Co(2+), Mn(2+), and Ni(2+) can also support 8-azido-[alpha-(32)P]ATP binding by rMrp6 while Ca(2+), Cd(2+), and Zn(2+) cannot. Under hydrolysis conditions (at 37 degrees C), the phosphate analogue beryllium fluoride (BeF(x)()) can stimulate trapping of the 8-azido-[alpha-(32)P]adenosine nucleotide in rMrp6 (and in MRP1) in a divalent cation-dependent and temperature-sensitive fashion. This suggests active ATPase activity, followed by trapping and photo-cross-linking of the 8-azido-[alpha-(32)P]ADP to the protein. By contrast to MRP1, orthovanadate-stimulated nucleotide trapping in rMrp6 does not occur in the presence of Mg(2+) but can be detected with Ni(2+) ions, suggesting structural and/or functional differences between the two proteins. The rMrp6 protein can be specifically photolabeled by a fluorescent photoactive drug analogue, [(125)I]-IAARh123, with characteristics similar to those previously reported for MRP1 (1), and this photolabeling of rMrp6 can be modulated by several structurally unrelated compounds. The P. pastoris expression system has allowed demonstration of ATP binding and ATP hydrolysis by rMrp6. In addition to providing large amounts of active protein for detailed biochemical studies, this system should also prove useful to identify potential rMrp6 substrates in [(125)I]-IAARh123 photolabeling competition studies, as well as to study the molecular basis of PXE mutations, which are most often found in the NBD2 of MRP6.
Cai
Jie
J
Department of Biochemistry, McGill Cancer Center, McGill University, 3655 Sir William Osler Promenade, Montreal, Quebec, Canada H3G 1Y6.
Daoud
Roni
R
Alqawi
Omar
O
Georges
Elias
E
Pelletier
Jerry
J
Gros
Philippe
P
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism; D001608:Beryllium / Q000494:pharmacology; D002413:Cations, Divalent / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005459:Fluorides / Q000494:pharmacology; D006868:Hydrolysis / Q000187:drug effects; D007457:Iodine Radioisotopes / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D020112:Rhodamine 123 / Q000378:metabolism; D014638:Vanadates / Q000378:metabolism
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001386:Azides; D001608:Beryllium; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D005459:Fluorides; D007457:Iodine Radioisotopes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D020112:Rhodamine 123; D014638:Vanadates; C102029:ABCC6 protein, human; C035982:beryllium fluoride; C017040:8-azidoadenosine 5'-triphosphate