Latent membrane perturbation activity of a mitochondrial precursor protein is exposed by unfolding.
We have purified milligram amounts of an importable mitochondrial precursor protein [the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (DHFR)]. This has made it possible, for the first time, to perform detailed studies on the conformation of a precursor protein and its interaction with lipid membranes. The precursor protein closely resembled authentic mouse DHFR with respect to secondary structure (measured by CD spectra) and stability towards urea (measured by tryptophan fluorescence and enzyme activity). With this precursor protein, the presequence thus does not significantly alter the folding of the attached 'passenger protein'. In contrast to the corresponding presequence peptide, the native precursor exhibited only weak ability to disrupt vesicles with a low mol% of negatively charged lipids, suggesting that the passenger protein masks the amphiphilic properties of the presequence. The membrane-perturbing properties of the precursor were greatly enhanced by increasing the vesicles' content of negatively charged lipid or by denaturing the precursor in 5 M urea. Interaction with vesicles rich in acidic phospholipid was accompanied by partial unfolding of the precursor, suggesting that such a conformational change may also be involved in the interaction of the precursor with the mitochondrial membranes.
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University of Basel, Department of Biochemistry, Switzerland.
Schatz
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D000818:Animals; D002942:Circular Dichroism; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
D004792:Enzyme Precursors; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D003576:Electron Transport Complex IV