Global mitochondrial protein import proteomics reveal distinct regulation by translation and translocation machinery

Abstract

Summary Most mitochondrial proteins are translated in the cytosol and imported into mitochondria. Mutations in the mitochondrial protein import machinery cause human pathologies. However, a lack of suitable tools to measure protein uptake across the mitochondrial proteome has prevented the identification of specific proteins affected by import perturbation. Here, we introduce mePROD mt , a pulsed-SILAC based proteomics approach that includes a booster signal to increase the sensitivity for mitochondrial proteins selectively, enabling global dynamic analysis of endogenous mitochondrial protein uptake in cells. We applied mePROD mt to determine protein uptake kinetics and examined how inhibitors of mitochondrial import machineries affect protein uptake. Monitoring changes in translation and uptake upon mitochondrial membrane depolarization revealed that protein uptake was extensively modulated by the import and translation machineries via activation of the integrated stress response. Strikingly, uptake changes were not uniform, with subsets of proteins being unaffected or decreased due to changes in translation or import capacity.

The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial protein import by enhancing assembly of the TOM complex

“…The switch of media in a dynamic stable isotope labeling by amino acids in cell culture (SILAC) approach allows for a precise measurement of the turnover rates of individual proteins (Ong et al, 2002, de Godoy et al, 2008). This method proved to be very powerful to determine the import, assembly and degradation of mitochondrial proteins (Bogenhagen and Haley, 2020, Saladi et al, 2020, Schäfer et al, 2021). To this end, we grew wild-type and Δ ubc8 cells in 2% lactate medium (which promotes respiration and gluconeogenesis) to mid-log phase with ‘light’ amino acids (i.e.…”

“…We designed a quantitative proteomics experiment based on dynamic SILAC labeling (Pratt et al, 2002) to follow the changes on the yeast proteome induced by switches from respiratory glucose synthesis to fermentative glucose consumption. Dynamic SILAC labeling has been successfully used in the past to measure the rates of protein turnover on the basis of the relative loss of ‘old’ (in our case light) peptides (Mathieson et al, 2018, Doherty et al, 2009, Dorrbaum et al, 2018, Saladi et al, 2020) or the rates of protein synthesis based on the accumulation of ‘new’ (in our case heavy) peptides (Schäfer et al, 2021). Our results document the potential of metabolic labeling for both aspects:…”

Enhanced immunoprecipitation techniques for the identification of RNA-binding protein partners: IGF2BP1 interactions in mammary epithelial cells

“…Mitochondrial function is in turn an important determinant of clonogenic survival and establishment, also associated with IMP-1 activity ( 68 , 69 ). For example, clustered mitochondria homolog, together with other RBPs, has been shown to regulate the import and expression of a mitochondrial protein network that becomes important under conditions of nutrient deprivation ( 70 , 71 ). Our data indicate that IMP-1 is required for the mitochondrial activity in both mouse mammary EP cells and human breast cancer cells, thus corroborating the identification of many of the mitochondrial ribosomal and complex I component mRNAs as IMP-1 partners.…”

Proteolytic regulation of mitochondrial oxidative phosphorylation components in plants

“…The degradation is mainly following protein import failure, resulting from a decrease in mitochondrial membrane potential, low ATP levels, or the misfolding of mitochondrial precursor proteins [ 35 , 36 ]. Protein import failure can also result from the blockage and disruption of the translocase of outer membrane (TOM) complex, the main protein import pore of the outer membrane [ 35 , 37 , 38 ]. Failure of protein import into mitochondria can lead to protein accumulation that triggers aggregation in the cytosol [ 39 ], therefore degradation of misfolded and aggregated proteins is essential to maintain cellular homeostasis [ 20 , 40 , 41 ].…”