Charting Organellar Importomes by Quantitative Mass Spectrometry – PEX14c

Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analyzing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here, we introduce a method that enables to chart an organelle’s importome. Our method relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import, metabolic labeling and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. An exciting feature of this method is that it overcomes limitations in identifying proteins with dual or multiple locations. We demonstrate the specificity and versatility of this novel ImportOmics method by targeting import machineries in mitochondria and glycosomes demonstrating its high potential for globally studying protein import and inventories of organelles.