Studies on the topology of the protein import channel in relation to the plant mitochondrial processing peptidase integrated into the cytochrome bc(1) complex

The mitochondrial processing peptidase (MPP) specifically cleaves N-termina l targeting signals from hundreds of nuclear-encoded, matrix-targeted precu rsor proteins. In contrast to yeast and mammals, the plant MPP is an integr al 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)) protei ns of 55-167 residues in length, fused to dihydrofolate reductase (DHFR). T he DHFR domain could be tightly folded by methotrexate (MTX), generating tr anslocation intermediates trapped in the import channel with only the cyt b 2 pre-sequence/mature domain protruding into the matrix. Spinach and soybea n mitochondria imported and processed unfolded precursors. MTX-folded inter mediates were not processed in spinach but the longest (1-167) MTX-folded c yt b(2)-DHFR construct was processed in soybean, while yeast mitochondria s uccessfully processed even shorter MTX-folded constructs. The MTX-folded pr ecursors were cleaved with high efficiency by purified spinach MPP/bc(1) co mplex. We interpret these results as indicating that the protein import cha nnel is located distantly from the MPP/bc(1) complex in plants, and that th ere is no link between protein translocation and protein processing.