title: The molecular chaperone, Atp12p, from Homo sapiens. In vitro studies with purified wild type and mutant (E240K) proteins.
abstract: Work in Saccharomyces cerevisiae has shown that Atp12p binds to unassembled alpha subunits of F(1) and in so doing prevents the alpha subunit from associating with itself in non-productive complexes during assembly of the F(1) moiety of the mitochondrial ATP synthase. We have developed a method to prepare recombinant Atp12p after expression of its human cDNA in bacterial cells. The molecular chaperone activity of HuAtp12p was studied using citrate synthase as a model substrate. Wild type HuAtp12p suppresses the aggregation of thermally inactivated citrate synthase. In contrast, the mutant protein HuAtp12p(E240K), which harbors a lysine at the position of the highly conserved Glu-240, fails to prevent citrate synthase aggregation at 43 degrees C. No significant differences were observed between the wild type and the mutant proteins as judged by sedimentation analysis, cysteine titration, tryptophan emission spectra, or limited proteolysis, which suggests that the E240K mutation alters the activity of HuAtp12p with minimal effects on the physical integrity of the protein. An additional important finding of this work is that the equilibrium chemical denaturation curve of HuAtp12p shows two components, the first of which is associated with protein aggregation. This result is consistent with a model for Atp12p structure in which there is a hydrophobic chaperone domain that is buried within the protein interior.
authors.affiliation: Department of Surgery, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: Ayana
authors.initials: A
authors.lastname: Hinton
authors.forename: Domenico L
authors.initials: DL
authors.lastname: Gatti
authors.forename: Sharon H
authors.initials: SH
authors.lastname: Ackerman
mesh_terms: D018833:Chaperonins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D006801:Humans; D024101:Mitochondrial Proteins; D025261:Mitochondrial Proton-Translocating ATPases; D018832:Molecular Chaperones; D009154:Mutation; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification
chemical_list: C110382:ATP12 protein, S cerevisiae; D018076:DNA, Complementary; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D018833:Chaperonins; C484671:ATPAF2 protein, human; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases