Human mitochondrial protein import proteomics - Proteinase K control

ABSTRACT: Most mitochondrial proteins are encoded in the nucleus and require mitochondrial import after translation in the cytosol. A number of mutations in the mitochondrial protein import machinery have been shown to lead to human pathologies. However, a lack suitable tools to measure mitochondrial protein uptake has prevented determination of import rates across the mitochondrial proteome and identification of specific proteins affected by perturbation. Here, we introduce a pulsed-SILAC based proteomics approach that includes a booster signal to selectively increase the sensitivity for mitochondrial proteins, enabling dynamic analysis of mitochondrial protein uptake at the global scale. We applied this method to determine protein uptake kinetics and examined how inhibitors of different mitochondrial import machineries affect protein uptake in sub-mitochondrial compartments. Monitoring changes in translation and protein uptake upon mitochondrial membrane depolarization revealed that protein uptake was extensively modulated the import- and translation machineries, via activation of the integrated stress response. Strikingly, uptake changes were not uniform with three groups of protein identified that showed no changes in uptake, or changes driven by reduced translation or import capacity. This study provides with a quantitative proteomics method to monitor mitochondrial protein uptake at a global scale to provide with insight into uptake rearrangements upon perturbation.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Hela Cell

SUBMITTER: Jonas Michaelis  

LAB HEAD: Christian Münch

PROVIDER: PXD024066 | Pride | 2021-12-20

REPOSITORIES: Pride