Zinc binding stabilizes mitochondrial Tim10 in a reduced and import-competent state kinetically.
Tim10 and all the small Tim proteins of the mitochondrial intermembrane space contain a consensus twin CX3C Zn2+-finger motif. While disulphide bond formation between the Cys residues of this motif is essential for complex formation by the small Tim proteins, the specific role of Zn2+-binding during the import and assembly of these proteins is not clear. In this study, we investigated the effects of the biologically relevant thiol-disulphide redox molecule, glutathione, and Zn2+-binding on the oxidative folding of yeast mitochondrial Tim10 using both biochemical and biophysical methods in vitro. We show that, whilst oxidized Tim10 cannot be reduced by reduced glutathione, reduced Tim10 is effectively oxidized at levels of glutathione comparable to those found in the cytosol. The oxidized Tim10 generated in the presence of glutathione is competent for complex formation with its partner protein Tim9, confirming it has a native fold. The standard redox potential of Tim10 at pH 7.4 was determined to be -0.32 V, confirming that Tim10 is a much stronger reductant than glutathione (-0.26 V, at pH 7.4) and could therefore be oxidized rapidly by oxidized glutathione in the cytosol. However, we found that Zn2+-binding can stabilize the reduced Tim10, decreasing the rate of the oxidative folding more than tenfold. In addition, we show that protein disulphide isomerase can catalyse the oxidative folding of Tim10 provided that Zn2+ was removed. We propose that Zn2+-binding is essential to maintain the protein in a reduced and import-competent state in the cytosol, and that zinc has to be removed after the protein is imported into mitochondria to initiate protein oxidative folding and assembly.
Lu
Hui
H
Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK. hui.lu@manchester.ac.uk
Woodburn
Joanna
J
D000098:Acetophenones / Q000494:pharmacology; D000478:Alkylation; D001665:Binding Sites; D002942:Circular Dichroism; D003600:Cytosol / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D005453:Fluorescence; D005978:Glutathione / Q000494:pharmacology*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D017510:Protein Folding; D021381:Protein Transport; D015032:Zinc / Q000378:metabolism*
D000098:Acetophenones; D004220:Disulfides; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C478387:TIM10 protein, E coli; C478388:TIM9 protein, E coli; D005978:Glutathione; D015032:Zinc; C071468:2,2,2',4'-tetrachloroacetophenone; D004229:Dithiothreitol