Determinism and contingencies shaped the evolution of mitochondrial protein import

Abstract

Significance All mitochondria import most of their proteins from the cytosol. Even though the targeting signals of imported proteins are well conserved within eukaryotes, this is not the case for the mitochondrial outer membrane receptors that recognize these signals. Here we compare the substrate preferences of protein import receptors from the parasitic protozoans Trypanosoma brucei and Trichomonas vaginalis, as well as from yeast. Using biochemical and proteomic analysis, combined with complementation experiments, we show that evolutionarily unrelated receptors can share the same substrate preferences. Moreover, we provide evidence that receptors sharing the same domain structure and topology can have different substrate specificity. In summary, our study illustrates how determinism and contingencies have shaped the evolution of mitochondrial import receptors. 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.