biostudies-literaturePeikert CDEuropean Research Council15272https://www.ebi.ac.uk/biostudies/studies/S-EPMC5436138biostudies-literatureUnknown8Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing 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 charting an organelle's importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins witNature communicationsCharting organellar importomes by quantitative mass spectrometry.PMC5436138648235Kaser SKnapp BHarsman AMorgenstern MMani JSchneider AOeljeklaus SWenger CWarscheid BPeikert CDfalseCharting organellar importomes by quantitative mass spectrometry.Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing 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 charting an organelle's importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins wit2017-01-01T00:00:00Z2017 May2026-05-01T03:56:51.977Z2019-03-27T02:45:20ZS-EPMC54361382848538810.1038/ncomms15272