Studies on Protein Processing for Membrane‐Bound Spinach Leaf Mitochondrial Processing Peptidase Integrated into the Cytochrome bc₁, Complex and the Soluble Rat Liver Matrix Mitochondrial Processing Peptidase

Abstract

The plant mitochondrial processing peptidase (MPP) that catalyses the cleavage of the presequences from precursor proteins during or after protein import is a membrane-bound enzyme that constitutes an integral part of the be, complex of the respiratory chain. In contrast, MPP from mammals is soluble in the matrix space and does not form part of the respiratory chain. In the present study, we have compared the substrate specificity of the isolated spinach leaf bc,lMPP with rat liver MPP using synthetic signal peptides and different mitochondrial precursor proteins. Inhibition studies of processing with synthetic peptides showed a similar inhibition pattern for plant and rat MPP activity. A peptide derived from the presequence of rat liver mitochondrial aldehyde dehydrogenase (ALDH) was a potent inhibitor of the spinach and rat MPP. Two nonprocessed signal peptides, rhodanese and linker-deleted ALDH (a form of ALDH that lacks the RGP linker connecting two helices in the presequence) had lower inhibitory effects towards each protease. The signal peptide from thiolase, another nonprocessed protein, had little inhibitory effect on MPP. Peptides derived from presequence of the plant Nicotiana plunzbaginifolia FIB also showed a similar inhibitory pattern with rat MPP as with spinach MPP processing. In-vitro synthesised precursors of plant N. plunzbaginifolia F,P and rat liver ALDH were cleaved to mature form by both spinach and rat MPP. However, the efficiency of processing was higher with the homologous precursor. Linker-deleted ALDH, rhodanese, and thiolase were not processed by the mammalian or plant MPP. However, both forms of MPP cleaved a mutated form of rhodanese that possesses a typical MPP cleavage motif, RXY S. Addition of the same cleavage motif to thiolase did not result in processing by either MPP. These results show that similar higher-order structural elements upstream from the cleavage site are important for processing by both the membrane-bound plant and the soluble mammalian MPP.Keywords: mitochondrial processing peptidase : signal peptide : protein processing; protein import; cytochrome bc, complex.Kuclear-encoded mitochondrial proteins are translated on cytosolic polyribosomes as precursor proteins. Most of the precursor proteins carry an N-terminal extension, the presequence or signal sequence, that contains information for mitochondrial targeting and sorting. Import of the precursor proteins into the mitochondria is usually accompanied by proteolytic removal of the presequence [l -31 resulting in the production of mature proteins. This step is catalysed by the mitochondrial processing peptidase (MPP) [4-91.MPP, a metal-dependent endopeptidase consisting of two structurally related subunits, a-MPP and b-MPP, forms a heterodimeric complex [lo, 111, in which both subunits are essentialCorrespondrrzce to E. Glaser, Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, SwedenAbbreviaticms. MPP, mitochondrial processing peptidase; bc, complex...

In Vivo Mitochondrial Import

“…These proteins all displayed a 30-kDa band, suggesting that they were processed. This was not observed in our previous in vitro import studies using rat liver mitochondria or processing studies with purified mitochondrial processing peptidase (11,34). A Western blot of isolated mitochondria from HeLa cells expressing either the native or linker-deleted EGFP constructs displayed a 30-kDa band for both proteins (data not shown).…”

Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase

“…The sequences were aligned with core 2 and core 1 subunits (SwissProt‐P23004 and NCBI‐X59692, respectively) of bovine bc 1 complex [protein data bank (PDB) code, 1BGY] (Iwata et al ., 1998) in Phi‐BLAST. The three‐dimensional models of the subunits of MPP were built in MODELLER 4.0 (Šali et al ., 1993) with default model‐building procedures. The quality of the model was checked by WHATIF 97 (Vriend, 1990) and PROCHECK 3.0 (Laskowski et al ., 1993).…”

Feature-extraction from endopeptidase cleavage sites in mitochondrial targeting peptides

“…In addition, we have performed secondary structure predictions to investigate local helix formation in mitochondrial targeting sequences, since mTPs have been shown to have a propensity to adopt amphiphilic ␣-helical structure by NMR experiments and theoretical considerations. 17,20,[25][26][27][28] Our aims were to get a more detailed idea of what the structural MPP and MIP target site features are and to search for possible differences in cleavage site patterns between different groups of organisms. Identification of characteristic target site features in the sequences of mitochondrial precursor proteins may suggest models of enzyme action and could become useful in genome analysis.…”