Poster Presentation The 35th Biennial Conference of the Society of Crystallographers in Australia and New Zealand 2024 (Crystal 35)

Structural and biophysical studies of the mitochondrial tRNA import component from Arabidopsis thaliana (#107)

Luke Smithers 1 , Bence Olasz 1 , Genevieve Evans 1 , Anandhi Anandan 1 , Monika Murcha 1 , Crystal Cooper 1 , Alice Vrielink 1
  1. University of Western Australia, Crawley, WA, Australia

Mitochondria are membrane bound organelles in the eukaryotic cell originating through an endosymbiotic event between an archaebacterium and an eubacterium.  As a result, mitochondria have maintained an active genome.  However, the organelle does not contain the full complement of tRNA needed for protein synthesis thus it requires specific nucleic acid importers.  Tric1 acts as a tRNA importer in Arabidopsis thaliana. Tric1 contains an N-terminal membrane bound PRAT (preprotein and amino acid transporters) domain, seen in many proteins involved in transport of proteins and amino acids into mitochondria. Additionally, Tric1 contains a C-terminal SAM (sterile-alpha-motif) domain. SAM domains are small domains which form both homo and heteromeric oligomeric complexes. We determined the crystal structure of the SAM domain of Tric1 to better understand the features of the protein that facilitates tRNA binding and import.  We observed a helical superstructure of 6 SAM domain monomers per helical turn in the crystal lattice (comprising two asymmetric units).  The roles of specific amino acids (Asp235 and Gly241) in subunit interactions were tested by site directed mutagenesis, crystal structure analyses and by measuring the ability of the mutant proteins to bind tRNA using electrophoretic mobility shift assays.  To further understand the impact of the mutated residues on protein function, complementation assays in planta were also carried out.   Arabidopsis tric1:tric2 knockout cell lines were complemented with wild type Tric1, and with a double mutant (Asp235Ala:Gly241Glu) and the ability to restore developmentally delayed phenotype measured.  Finally, we used AlphaFold2 to further probe the oligomeric structure of full length Tric1. The results of our studies suggest that Tric1 likely forms a cyclic hexameric structure with a positively charged inner pore of appropriate dimensions to accommodate a tRNA molecule facilitating the movement of tRNA across the mitochondrial membrane. These results are now being used to guide structural and biophysical experiments of Tric1 in complex with tRNA and alone. 

  1. Olasz, B., Smithers, L., Evans, G. L., Anandan, A., Murcha, M. W., Vrielink A. Structural analysis of the SAM domain of the Arabidopsis mitochondrial tRNA import receptor. Journal of Biological Chemistry, 300(5). 2024.