Abstract
The iron-sulfur protein of the cytochrome bc1 complex is one of a small number of proteins that are processed in two sequential steps by matrix processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP) during import into Saccharomyces cerevisiae mitochondria. To test whether two-step processing is necessary for import and assembly of the iron-sulfur protein into the cytochrome bc1 complex, we mutagenized the presequence of the iron-sulfur protein to eliminate the original MPP site and replace the MIP site with a new MPP site. The mutated presequence is cleaved and forms mature-sized protein in a single step, and the mature-sized iron-sulfur protein is correctly targeted to the outer side of the inner mitochondrial membrane in vitro. Mutant iron-sulfur protein which is processed to mature size in one step complements the respiratory deficient phenotype of a yeast strain in which the endogenous gene for the iron-sulfur protein is deleted. These results establish that mature-sized iron-sulfur protein can be formed by single-step processing and assembled into a functionally active form in the cytochrome bc1 complex in S. cerevisiae.
Highlights
The majority of mitochondrial proteins are encoded in the nucleus, translated on cytosolic ribosomes, and targeted to the mitochondria by amino-terminal leader presequences [1,2,3]
The iron-sulfur protein (ISP) of the cytochrome bc1 complex is processed in two steps by matrix processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP) during import into Saccharomyces cerevisiae mitochondria [9], whereas the iron-sulfur protein of bovine heart mitochondria is processed in only one step by MPP [10]
This means that the amino terminus of the mature protein is structurally compatible with single-step processing by MPP, and that two-step processing is not necessary for correct targeting and assembly of iron-sulfur protein into the cytochrome bc1 complex in the inner mitochondrial membrane of S. cerevisiae
Summary
The majority of mitochondrial proteins are encoded in the nucleus, translated on cytosolic ribosomes, and targeted to the mitochondria by amino-terminal leader presequences [1,2,3]. This means that the amino terminus of the mature protein is structurally compatible with single-step processing by MPP, and that two-step processing is not necessary for correct targeting and assembly of iron-sulfur protein into the cytochrome bc1 complex in the inner mitochondrial membrane of S. cerevisiae.
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