Characterisation of mitochondrial outer membrane proteins in Arabidopsis thalia

[Truncated abstract] The acquisition of mitochondria is one of the great leaps of evolutionary change. Acquisition of this organelle provided substantial benefits evidenced by the diversity of eukaryotic life present today. However, along with benefits came increased complexity, and the subsequent acquisition of the plastid and centralisation of genetic material in the nucleus compounded this situation, resulting in the need for a complex and highly specific protein import apparatus. While the identity and functions of the mitochondrial protein import apparatus is well characterised in Saccharomyces cerevisiae, the components present in Arabidopsis thaliana are, with the exception of the central import pore, either poorly conserved or entirely absent. The primary import receptor of yeast, TOM20 is not present in the Arabidopsis genome, yet the identification of a similar, though independently evolved protein invited evaluation of its role in protein import. Genetic and biochemical evaluations have previously shown that this protein is encoded by four genes in Arabidopsis, three of which are transcribed. The isolation of Arabidopsis plants deficient in one, two and all three of these genes allowed the evaluation of the role each of these proteins play individually and as a whole in the import of a diverse range of nuclear encoded mitochondrial proteins. In vitro protein import of radio labelled proteins showed that the TOM20 family of Arabidopsis function to accelerate the rate of protein import into mitochondria. Analysis of the individual isoforms in double knockout plants revealed that none of the isoforms of TOM20 individually are essential to this function, though there is some precursor specificity. The maintenance of normal import rates into mitochondria for one of the precursor proteins tested in the absence of TOM20 suggested that a TOM20 independent pathway exists on the outer membrane...