They play a role in the respiratory chain, the citric acid cycle, genome maintenance and translation, the urea cycle, protein folding, and stress response. Oct1-dependent substrate proteins were found in the mitochondrial matrix and inner membrane, and their functions are quite diverse. Oct1 cleaves an octapeptide from the N-terminus of the preprotein intermediate, thus generating the mature protein ( Gakh et al., 2002). This leads to the conversion of a destabilizing N-terminus into a stable one, thereby preventing premature degradation of substrate proteins ( Vögtle et al., 2009).Īnother protease, which cleaves precursor intermediates generated by MPP, is the matrix-located mitochondrial intermediate peptidase (MIP), octapeptidyl aminopeptidase 1 (Oct1). Icp55 typically removes one amino acid residue from preprotein intermediates that carry a destabilizing amino acid residue at their N-terminus according to the N-end rule for bacterial proteins ( Mogk et al., 2007 Varshavsky, 2008). A further intermediate cleaving peptidase, Icp55, which cleaves preprotein intermediates after initial processing by MPP has recently been identified ( Naamati et al., 2009 Vögtle et al., 2009). This can be performed, for example, by the inner membrane peptidase (IMP) or the rhomboid protease processing of cytochrome c peroxidase (Pcp1), which act to regulate submitochondrial sorting of preproteins ( Esser et al., 2002 Koppen and Langer, 2007). Some preproteins can undergo a second processing event after MPP cleavage. Most presequences are removed upon import by the mitochondrial processing peptidase (MPP) located in the matrix ( Witte et al., 1988 Gavel and von Heijne, 1990 Glaser et al., 1998 Gakh et al., 2002 Habib et al., 2007 Huang et al., 2009 Yogev and Pines, 2011). Those preproteins possess N-terminal positively charged extensions that form amphipathic α-helices required for targeting and import into the organelle. Approximately 70% of these mitochondrial preproteins use the presequence pathway for import into mitochondria, involving the translocase of the outer membrane (TOM) and the presequence translocase of the inner membrane (TIM23) complexes ( Neupert and Herrmann, 2007 Endo and Yamano, 2009 Vögtle et al., 2009 van der Laan et al., 2010 Schmidt et al., 2010). Virtually all mitochondrial proteins are synthesized on cytosolic ribosomes and are imported into the organelle in a posttranslational manner. Thus Oct1 converts unstable precursor intermediates generated by MPP into stable mature proteins. We compared the stability of intermediate and mature forms of Oct1 substrate proteins in organello and in vivo and found that Oct1 cleavage increases the half-life of its substrate proteins, most likely by removing destabilizing amino acids at the intermediate's N-terminus. Inspection of the Oct1 substrates revealed that the N-termini of the intermediates typically carry a destabilizing amino acid residue according to the N-end rule of protein degradation, whereas mature proteins carry stabilizing N-terminal residues. In this paper, we report the identification of a novel Oct1 substrate protein with an unusual cleavage motif. However, the function of this second cleavage step is elusive. A subset of precursors with a characteristic amino acid motif is additionally processed by the mitochondrial intermediate peptidase (MIP) octapeptidyl aminopeptidase 1 (Oct1), which removes an octapeptide from the N-terminus of the precursor intermediate. The vast majority of presequences are proteolytically removed by the mitochondrial processing peptidase (MPP) localized in the matrix. Most mitochondrial proteins are encoded in the nucleus as precursor proteins and carry N-terminal presequences for import into the organelle.
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