Studies on the topology of the protein import channel in relation to the plant mitochondrial processing peptidase integrated into the cytochrome bc1 complex

The mitochondrial processing peptidase (MPP) specifically cleaves N-terminal targeting signals from hundreds of nuclear-encoded, matrix-targeted precursor proteins. In contrast to yeast and mammals, the plant MPP is an integral component of the respiratory cytochrome bq complex. The topology of the protein import channel in relation to MPP/bc(1) in plants was studied using chimeric precursors containing truncated cytochrome b(2) (cyt b(2)) proteins of 55-167 residues in length, fused to dihydrofolate reductase (DHFR). The DHFR domain could be tightly folded by methotrexate (MTX), generating translocation intermediates trapped in the import channel with only the cyt b2 pre-sequence/mature domain protruding into the matrix. Spinach and soybean mitochondria imported and processed unfolded precursors. MTX-folded intermediates were not processed in spinach but the longest (1-167) MTX-folded cyt b(2)-DHFR construct was processed in soybean, while yeast mitochondria successfully processed even shorter MTX-folded constructs. The MTX-folded precursors were cleaved with high efficiency by purified spinach MPP/bc(1) complex. We interpret these results as indicating that the protein import channel is located distantly from the MPP/bc(1) complex in plants, and that there is no link between protein translocation and protein processing.