The role of MRPL45 and OXA1L in human mitochondrial protein synthesis

Abstract

Mitochondria produce 90% of the adenosine triphosphate (ATP) used by eukaryotic cells as a source of energy. ATP synthesis is carried out by the oxidative phosphorylation (OXPHOS) system whose components are partially-encoded by mitochondrial DNA and translated by mitoribosomes found in the organelle itself. The interaction between mitoribosomes and the inner mitochondrial membrane (IMM) has been claimed to be important for efficient protein synthesis in these organelles, but how this association occurs is still unclear. The aim of this study was to investigate the association between mitoribosomes and IMM in human mitochondria. My attention focused on MRPL45, a component of the mitoribosome that might be a key player of this interaction due to its proximity to the polypeptide exit tunnel and its structural similarity to the IMM-interacting proteins TIM44 and Mba1. During the course of this study, in order to further investigate the interaction, interest in the IMM protein OXA1L arose. The yeast homologue of this protein has been reported to interact with the mitoribosome, offering an interacting point between the IMM and the translation machinery. I performed depletion studies that confirmed the importance of MRPL45 for the stability of the mitoribosomal large subunit (mt-LSU) and mitochondrial translation. With the development of a protocol to investigate membrane-interaction, I demonstrated that MRPL45 is able to interact directly with the IMM. The addition of a FLAG-tag at the C-terminal of MRPL45 did not affect the ability of the protein to interact with the membrane and did not have effects on the homeostasis of the cells. Therefore, immunoprecipitation of MRPL45FLAG was performed in the absence of assembled mitoribosomal subunits to identify potential IMM binding partners. No obvious candidates, however, were detected from mass spectrometry analysis of the immunoprecipitated sample. Modifications of MRPL45 based on structural similarities with TIM44 and Mba1 were performed to identify the membrane-associating domain of the protein. Mutation of charged amino acids on a protruding α-helix of MRPL45 were performed. The resultant protein was completely integrated into the mt-LSU in the absence of the endogenous counterpart, partially rescuing the phenotype observed upon depletion of MRPL45. Membrane-interaction studies showed that the modifications did not affect the ability of this mutant to interact directly with the membrane. Another attempt to disrupt the interaction between MRPL45 and the membrane was performed by expressing a mutant protein lacking 117 amino acids at the N-terminal, which is predicted to correspond to the putative membrane-interacting domain of TIM44. Only a minor proportion of this protein was integrated in the mt-LSU in absence of the endogenous MRPL45, although the mutant protein retained the ability to interact directly with the membrane. The role of another mitochondrial protein, OXA1L, in mitochondrial translation was also investigated in this work. This was motivated by the discovery of a paediatric patient with mutations in the gene encoding OXA1L. Since the published studies showing OXA1L to interact with the mitoribosome were attempted in vitro, I performed immunoprecipitation studies which ii confirmed the ability of OXA1L to associate with the mitoribosome in intact human cells. In order to characterise the role of OXA1L in mitochondrial translation, depletion studies were performed. These suggested that OXA1L was important for the stability of both the large and, unexpectedly, the small mitoribosomal subunit. Due to this surprising result, extensive studies were performed to confirm its robustness, which confirmed the reduction of the steady-state level of the mitoribosomal subunits and of OXPHOS components upon depletion of OXA1L. To conclude, my studies showed the importance of the mitoribosomal protein MRPL45 for the stability and the assembly of the large subunit of the mitochondrial translation machinery, as well as its ability to interact directly with the IMM. Although it was not possible to identify the domain of MRPL45 involved in membrane interaction, insights on the importance of the N-terminal domain for its integration in the mt-LSU were identified. The ability of OXA1L to interact with the mitoribosome in vivo was determined and a role of this protein in the stability and assembly of the mitoribosome was demonstrated.