A purified, artificial precursor protein was used as a transport vehicle to test the tolerance of the mitochondrial protein import system. The precursor was a fusion protein consisting of mouse dihydrofolate reductase linked to a yeast mitochondrial presequence; it contained a unique cysteine as its COOH-terminal residue. This COOH-terminal cysteine was covalently coupled to either a stilbene disulfonate derivative or, with the aid of a bifunctional cross-linker, to one of the free amino groups of horse heart cytochrome c. Coupling to horse heart cytochrome c generated a mixture of branched polypeptide chains since this cytochrome lacks a free alpha-amino group. Both adducts were imported and cleaved by isolated yeast mitochondria. The mitochondrial protein import machinery can thus transport more complex structures and even highly charged "membrane-impermeant" organic molecules. This suggests that transport occurs through a hydrophilic environment.
Mitochondria can import artificial precursor proteins containing a branched polypeptide chain or a carboxy-terminal stilbene disulfonate.
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D Vestweber, G Schatz; Mitochondria can import artificial precursor proteins containing a branched polypeptide chain or a carboxy-terminal stilbene disulfonate.. J Cell Biol 1 December 1988; 107 (6): 2045–2049. doi: https://doi.org/10.1083/jcb.107.6.2045
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