RESUMO
Limited proteolysis, called protein processing, is an essential post-translational mechanism that controls protein localization, activity, and in consequence, function. This process is prevalent for mitochondrial proteins, mainly synthesized as precursor proteins with N-terminal sequences (presequences) that act as targeting signals and are removed upon import into the organelle. Mitochondria have a distinct and highly conserved proteolytic system that includes proteases with sole function in presequence processing and proteases, which show diverse mitochondrial functions with limited proteolysis as an additional one. In virtually all mitochondria, the primary processing of N-terminal signals is catalyzed by the well-characterized mitochondrial processing peptidase (MPP). Subsequently, a second proteolytic cleavage occurs, leading to more stabilized residues at the newly formed N-terminus. Lately, mitochondrial proteases, intermediate cleavage peptidase 55 (ICP55) and octapeptidyl protease 1 (OCT1), involved in proteolytic cleavage after MPP and their substrates have been described in the plant, yeast, and mammalian mitochondria. Mitochondrial proteins can also be processed by removing a peptide from their N- or C-terminus as a maturation step during insertion into the membrane or as a regulatory mechanism in maintaining their function. This type of limited proteolysis is characteristic for processing proteases, such as IMP and rhomboid proteases, or the general mitochondrial quality control proteases ATP23, m-AAA, i-AAA, and OMA1. Identification of processing protease substrates and defining their consensus cleavage motifs is now possible with the help of large-scale quantitative mass spectrometry-based N-terminomics, such as combined fractional diagonal chromatography (COFRADIC), charge-based fractional diagonal chromatography (ChaFRADIC), or terminal amine isotopic labeling of substrates (TAILS). This review summarizes the current knowledge on the characterization of mitochondrial processing peptidases and selected N-terminomics techniques used to uncover protease substrates in the plant, yeast, and mammalian mitochondria.
RESUMO
The threat of global warming makes uncovering mechanisms of plant tolerance to long-term moderate heat stress particularly important. We previously reported that Arabidopsis (Arabidopsis thaliana) plants lacking mitochondrial proteases FTSH4 or OMA1 suffer phenotypic changes under long-term stress of 30°C, while their growth at 22°C is not affected. Here we found that these morphological and developmental changes are associated with increased accumulation of insoluble mitochondrial protein aggregates that consist mainly of small heat-shock proteins (sHSPs). Greater accumulation of sHSPs in ftsh4 than oma1 corresponds with more severe phenotypic abnormalities. We showed that the proteolytic activity of FTSH4, and to a lesser extent of OMA1, as well as the chaperone function of FTSH4, is crucial for protecting mitochondrial proteins against aggregation. We demonstrated that HSP23.6 and NADH dehydrogenase subunit 9 present in aggregates are proteolytic substrates of FTSH4, and this form of HSP23.6 is also a substrate of OMA1 protease. In addition, we found that the activity of FTSH4 plays an important role during recovery from elevated to optimal temperatures. Isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analyses, along with identification of aggregation-prone proteins, implicated mitochondrial pathways affected by protein aggregation (e.g. assembly of complex I) and revealed that the mitochondrial proteomes of ftsh4 and oma1 plants are similarly adapted to long-term moderate heat stress. Overall, our data indicate that both FTSH4 and OMA1 increase the tolerance of plants to long-term moderate heat stress by reducing detergent-tolerant mitochondrial protein aggregation.
