Abstract
All but thirteen mammalian mitochondrial proteins are encoded by the nuclear genome, translated in the cytosol and then imported into the mitochondria. For a significant proportion of the mitochondrial proteins, import is coupled with the cleavage of a presequence called the transit peptide, and the formation of a new N-terminus. Determination of the neo N-termini has been investigated by proteomic approaches in several systems, but generally in a static way to compile as many N-termini as possible. In the present study, we have investigated how the mitochondrial proteome and N-terminome react to chemical stimuli that alter mitochondrial metabolism, namely zinc ions and rapamycin. To this end, we have used a strategy that analyzes both internal and N-terminal peptides in a single run, the dN-TOP approach. We used these two very different stressors to sort out what could be a generic response to stress and what is specific to each of these stressors. Rapamycin and zinc induced different changes in the mitochondrial proteome. However, convergent changes to key mitochondrial enzymatic activities such as pyruvate dehydrogenase, succinate dehydrogenase and citrate synthase were observed for both treatments. Other convergent changes were seen in components of the N-terminal processing system and mitochondrial proteases. Investigations into the generation of neo-N-termini in mitochondria showed that the processing system is robust, as indicated by the lack of change in neo N-termini under the conditions tested. Detailed analysis of the data revealed that zinc caused a slight reduction in the efficiency of the N-terminal trimming system and that both treatments increased the degradation of mitochondrial proteins. In conclusion, the use of this combined strategy allowed a detailed analysis of the dynamics of the mitochondrial N-terminome in response to treatments which impact the mitochondria.
Highlights
A Combined N-terminomics and Shotgun Proteomics Approach to Investigate the Responses of Human Cells to Rapamycin and Zinc at the Mitochondrial Level*□S
We have investigated how the mitochondrial proteome and N-terminome react to chemical stimuli that alter mitochondrial metabolism, namely zinc ions and rapamycin
Modulation of the Mitochondrial Proteome by Rapamycin and Zinc—A classical differential analysis using internal peptides was performed to investigate the effects of rapamycin and zinc on the mitochondrial proteome after a 24 h treatment
Summary
A Combined N-terminomics and Shotgun Proteomics Approach to Investigate the Responses of Human Cells to Rapamycin and Zinc at the Mitochondrial Level*□S. N-terminomics approaches have been used to characterize the new N-termini in various organisms, from yeast to mammals (10 –12) These studies have shown that besides the major mitochondrial processing peptidase that cleaves the transit peptide, the system includes aminopeptidases that trim the N-terminal end to create ragged termini and stabilize the proteins [10, 13]. Determination of the neo-N-termini of mitochondrial proteins is still an active research field (10 –12), nothing is yet known about the robustness of the mitochondrial protein processing system under conditions of mitochondrial stress, nonlethal stress It is not known, for example, whether errors occur in the precursor cleavage during stress, From the ‡Laboratoire de Spectrometrie de Masse BioOrganique (LSMBO), Universitede Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France; §Chemistry and Biology of Metals, Univ. Combined Mitochondrial N-terminomics and Shotgun Proteomics or how the other components of the mitochondrial protein processing system behave in such conditions
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