The mitochondrial targeting presequence of the Rieske iron-sulfur protein is processed in a single step after insertion into the cytochrome bc1 complex in mammals and retained as a subunit in the complex.

U Brandt,L Yu,C.A Yu,B.L Trumpower

doi:10.1016/s0021-9258(18)52883-0

Abstract

The amino acid sequence of subunit 9 of the bovine heart cytochrome bc1 complex is identical to the 78-amino acid presequence that is removed post-translationally from the Rieske iron-sulfur protein as it is imported and targeted to the mitochondrial cytochrome bc1 complex. Iron-sulfur protein precursor, generated by in vitro transcription and translation, is processed to mature size in a single step when incubated with rat liver mitochondria, and generates a peptide that comigrates on SDS-polyacrylamide gel electrophoresis with subunit 9. These results suggest that the Rieske protein is processed in a single proteolytic step after it is inserted into the cytochrome bc1 complex in mammals, and that the processed presequence remains as a subunit of the complex. This is apparently the first instance in which a cleaved targeting presequence has been shown to be retained in the cell, possibly exhibiting a second function in addition to its function in protein trafficking.

Highlights

21- or %-amino acid presequence' from the precursor ironsulfur protein, after which a mitochondrial intermediate protease (MIP)removes an octapeptide to generate mature length iron-sulfur apoprotein (5)
Amino acid presequence thatis removed post-translationally from the Rieske iron-sulfur protein as it is importedandtargeted to themitochondriacl ytochrome b c 1 complex.Iron-sulfurproteinprecursor, generatedby in vitro transcription and translationi,s processed to mature size in a single step when incubated with rat liver mitochondria,andgeneratesa peptidethatcomigratesonSDS-polyacrylamide gel electrophoresis with subunit 9
Materials-Cytochrome bc, complex was purified from yeast strain W303-1A (7),and bovine heart &CI complex (8) was a gift from H

Summary

Subunit in the Complex*

Proteolytic cleavage sites in mitochondrial precursor proteins, Hendrick and co-workers ( 6 )identified a 3-amino acid motif,. Amino acid presequence thatis removed post-translationally from the Rieske iron-sulfur protein as it is importedandtargeted to themitochondriacl ytochrome b c 1 complex.Iron-sulfurproteinprecursor, generatedby in vitro transcription and translationi,s processed to mature size in a single step when incubated with rat liver mitochondria,andgeneratesa peptidethatcomigratesonSDS-polyacrylamide gel electrophoresis with subunit 9. Sequencing reactions were analyzed on 0.4-0.8-mm field piration and photosynthesisI.n eukaryotes the Rieske protein gradient gels containing 6% acrylamide, 7 M urea in 90 mM Tris, 90 is encoded on the nuclear genome, synthesized on cytoplasmic ribosomes, and imported post-translationally into the mitochondria or chloroplasts, where itis insertedinto the bcl complex in the energy transducing membrane (1,2).

RESULTS

CAG GCC GCG GTCGCC GCC ACC TCG GAG

GAG AGC AGT GAG GCT CGti