Authors

M Gerber²; C E Dewar²; S Oeljeklaus¹; G Klesse²; B Warscheid¹; A Schneider²;  ¹ Department of Biochemistry and Functional Proteomics, Universität Freiburg, Germany;  ² Department of Chemistry, Biochemistry and Pharmaceutical Sciences, Bern, Switzerland

Discussion

Trypanosoma brucei (T. brucei) has a single mitochondrion which makes them a well-suited model organism to study mitochondrial quality control (MQC). MQC is the network of pathways by which eukaryotic cells monitor and maintain the function of their mitochondria. Many examples of convergent evolution have been discovered in T. brucei, and we expect the same to be the case for mechanisms governing MQC.
In a MQC pathway previously characterised in opisthokonts, misfolded, defective or mislocalised proteins in the outer mitochondrial membrane (OMM) are extracted by the ATPase Msp1 to allow for their degradation by the cytosolic proteasome. Yeast Msp1 extracts proteins from a lipid bilayer without additional cofactors or substrate modifications. No stable interactors of Msp1 have been identified in yeast. Substrate motifs are only accessible to Msp1 when its substrate proteins are not in a complex, or ‘orphaned’.
To characterise mitochondrial Msp1 in T. brucei, we identified putative interaction partners that also localize to the OMM, using immunoprecipitation and subsequent SILAC proteomics. Interestingly, some of these proteins are kinetoplastid-specific. We confirm the interaction between these proteins and TbMsp1, as well as between each other, by reciprocal co-immunoprecipitations. Additionally, we have established an assay to assess TbMsp1 function to determine whether these interaction partners are required for efficient substrate degradation. We induced the destabilisation of alpha helical OMM proteins, and found that the degradation of the destabilized OMM protein ATOM19 is dependent on TbMsp1. In this background, we individually knocked down the four OMM TbMsp1 interaction partners. The absence of three of these proteins prevented the efficient degradation of ATOM19. We conclude that, uniquely, mitochondrial TbMsp1 function in MQC is dependent on these three interaction partners. We are now establishing assays for TbMsp1 function in the removal of mislocalised orphan proteins.