Mitochondrial protein import requires outer membrane receptors that evolved independently in different lineages. Here we used quantitative proteomics and in vitro binding assays to investigate the substrate preferences of ATOM46 and ATOM69, the two mitochondrial import receptors of Trypanosoma brucei The results show that ATOM46 prefers presequence-containing, hydrophilic proteins that lack transmembrane domains (TMDs), whereas ATOM69 prefers presequence-lacking, hydrophobic substrates that have TMDs. Thus, the ATOM46/yeast Tom20 and the ATOM69/yeast Tom70 pairs have similar substrate preferences. However, ATOM46 mainly uses electrostatic, and Tom20 hydrophobic, interactions for substrate binding. In vivo replacement of T. brucei ATOM46 by yeast Tom20 did not restore import. However, replacement of ATOM69 by the recently discovered Tom36 receptor of Trichomonas hydrogenosomes, while not allowing for growth, restored import of a large subset of trypanosomal proteins that lack TMDs. Thus, even though ATOM69 and Tom36 share the same domain structure and topology, they have different substrate preferences. The study establishes complementation experiments, combined with quantitative proteomics, as a highly versatile and sensitive method to compare in vivo preferences of protein import receptors. Moreover, it illustrates the role determinism and contingencies played in the evolution of mitochondrial protein import receptors.

Biochemistry and Functional Proteomics Institute of Biology 2 Faculty of Biology University of Freiburg 79104 Freiburg Germany

Biochemistry and Functional Proteomics Institute of Biology 2 Faculty of Biology University of Freiburg 79104 Freiburg Germany;

Department of Chemistry Biochemistry and Pharmaceutical Sciences University of Bern CH 3012 Bern Switzerland

Department of Chemistry Biochemistry and Pharmaceutical Sciences University of Bern CH 3012 Bern Switzerland;

Department of Parasitology Faculty of Science Charles University BIOCEV 12843 Prague Czech Republic

Signalling Research Centres BIOSS and CIBSS University of Freiburg 79104 Freiburg Germany

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