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.
Highlights
Work in Saccharomyces cerevisiae has shown that Atp12p binds to unassembled ␣ subunits of F1 and in so doing prevents the ␣ subunit from associating with itself in non-productive complexes during assembly of the F1 moiety of the mitochondrial ATP synthase
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
Because unassembled F1 proteins do not accumulate to a significant degree in the cell [6], the Atp12p concentration in mitochondria may be comparable with the concentration of free F1 ␣ subunit protein
Summary
Plasmid Construction—Recombinant plasmids for the production of human Atp12p (HuAtp12p) in Escherichia coli were constructed in the His-tag vector pPROEX HTa (Invitrogen) and were designed to produce the mature form of the protein without the mitochondrial leader peptide. Bacterial colonies harboring the mutant plasmid (called pPROEX/Atp12hE240K) were identified by restriction analysis in an EcoRI/XbaI digest and tested in small scale experiments for isopropyl -D-thiogalactopyranoside-induced production of recombinant protein. One such expressing clone was selected and used for large scale production/purification of the mutant Atp12p protein (HuAtp12pE240K). To identify the principal proteolytic fragment of highest molecular weight, a 100-g sample of HuAtp12p was first digested with either trypsin or chymotrypsin at 1% in 67 l, and after the reaction was quenched, the acid-precipitated protein was resolved in an SDS gel, and transferred to polyvinylidene difluoride membrane (Schleicher & Schuell, WESTRAN®) as described [11].
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