Nucleotide-induced conformational changes of PMP70, an ATP binding cassette transporter on rat liver peroxisomal membranes.
Nucleotide-induced conformational changes of the 70-kDa peroxisomal membrane protein (PMP70) were investigated by means of limited-trypsin digestion. Rat liver peroxisomes preincubated with various nucleotides were subsequently digested by trypsin. The digestion products were subjected to immunoblot analysis with an anti-PMP70 antibody that recognizes the carboxyl-terminal 15 amino acids of the protein. PMP70 was initially cleaved in the boundary region between the transmembrane and nucleotide-binding domains and a carboxyl-terminal 30-kDa fragment resulted. The fragment in turn was progressively digested at the helical domain between the Walker A and B motifs. The fragment, however, could be stabilized with MgATP or MgADP. In contrast to MgATP, MgATP-gammaS protected whole PMP70 as well as the fragment. The 30-kDa fragment processed by trypsin was recovered in the post-peroxisomal fraction as a complex with a molecular mass of about 60 kDa irrespective of the presence of MgATP. These results suggest that PMP70 exists as a dimer on the peroxisomal membranes and the binding and hydrolysis of ATP induce conformational changes in PMP70 close to the boundary between the transmembrane and nucleotide binding domains and the helical domain between the Walker A and B motifs.
Kashiwayama
Yoshinori
Y
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan.
Morita
Masashi
M
Kamijo
Keiju
K
Imanaka
Tsuneo
T
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D019281:Dimerization; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020675:Peroxisomes / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; C489272:Abcd3 protein, rat; D008565:Membrane Proteins; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D014357:Trypsin