Proteins function at distinct sites within the cell, yet most of them are encoded by the nucleus and synthesised in the cytosol. Thus, newly synthesised mitochondrial proteins have to be transported within the cell. Signal located as amino-terminal extensions of the precursor protein, the so-called presequences, are required to guide the proteins to mitochondria. These signals are removed by the mitochondrial processing peptidase (MPP) after import into the mitochondria.
The aim of the studies presented in this thesis has been to isolate and characterise the MPP from spinach, S.oleracea. The results show that the spinach MPP is a metallo-endopeptidase and in contrast to other organisms, it is membrane-bound and integrated into the bc1 complex of the respiratory chain. Consequently, the bc1 complex of spinach is bifunctional, involved in both respiration and protein processing. Processing is, however, not dependent on respiration or redox state of the bc1 complex. The subunits of the spinach MPP are identical to the matrix exposed core proteins of the bc1 complex.
The MPP is unique as it is a general peptidase removing several hundred presequences, that show no sequence similarity, yet it is specific as it recognises and cleaves at a specific site. In order to study determinants for processing, we have used synthetic peptides corresponding to parts of the native and mutated presequences, site-directed mutagenesis and statistical analysis of presequences.
Results suggest that the MPP recognises a higher-order structure of the presequence in addition to the typical cleavage motifs, containing arginine, serine, alanine and threonine residues. Mutations affecting the secondary structure of the carboxy-terminal portion of the presequence affect the efficiency of processing. Both the membrane bound spinach MPP/bc1 complex and the soluble rat MPP recognise the same structural element prior to the cleavage site. Furthermore, several plant presequences are predicted to form a helix, sometimes followed by an extended structure, prior to the cleavage site. Analysis of presequences by site-directed mutagenesis shows that arginine residues, both close and surprisingly distant to the cleavage site, influence the efficiency of processing of the soybean alternative oxidase but not of the N. plumbaginifolia F1b precursor.
In conclusion, the recognition of the correct cleavage site and the efficiency of processing by MPP is governed by a combination of factors such as secondary structure, flexibility, hydrophobicity and basic amino acid residues of the presequence. Modeling of the interaction of the precursor protein with the MPP, using information from 3D-structural analysis of the bovine bc1 complex, will be important to provide information of the actual cleavage mechanism of the MPP.
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 1998. , 66 p.