Abstract:
Mitochondria are essential organelles responsible not only for ATP production but also for diverse cellular processes such as signalling, apoptosis, and metabolism. Mitochondrial biogenesis is highly reliant on the coordinated import and maturation of proteins encoded mostly in the nucleus. Although several mitochondrial protein import pathways have been well characterised, important gaps remain in our understanding of how certain classes of proteins are integrated into mitochondrial membranes and how these pathways differ across eukaryotic cells.
This thesis addresses three aspects of mitochondrial protein biogenesis:
First, I investigated the role of the mammalian proteins MTCH1 and MTCH2 in outer mitochondrial membrane (OMM) protein insertion. Using Saccharomyces cerevisiae strains lacking the MIM complex (composed of Mim1/Mim2), I showed that MTCH1, but not MTCH2, could functionally complement the absence of the yeast insertase. MTCH1 restored growth, steady-state levels of α-helical OMM proteins, TOM complex assembly, and mitochondrial morphology. Experiments utilizing truncation and chimera variants further revealed that the N-terminal region of MTCH1 can enhance the efficiency of mitochondrial targeting of the protein. These findings identify MTCH1 as a bona fide insertase with functional equivalence to the yeast MIM complex, highlighting convergent evolution of OMM α-helical protein insertases.
In a second project, I analysed the proteolytic function of the Schizosaccharomyces pombe supercomplex composed of complexes III and IV of the respiratory chain (CIII₂CIV). Cryo-EM experiments revealed conserved features, including bound cytochrome c and a Zn²⁺ site in Cor1, homologous to the catalytic β-subunit of the mitochondrial processing peptidase (MPP). By performing functional assays, I demonstrated that the purified CIII₂CIV complex possesses proteolytic activity toward mitochondrial precursor proteins, providing the first evidence that CIII₂ in S. pombe has a dual role in both respiration and protein processing.
Finally, I investigated the poorly characterised mammalian TOM40 isoform, TOM40B, which is linked to rare paediatric neurodegenerative disease. Using TOMM40B knockout (KO) cell lines (HEK293T and SH-SY5Y), I found that loss of TOM40B causes slow growth under metabolic stress but does not impair TOM complex assembly or import of selected mitochondrial substrates. High-resolution microscopy comparing control to the KO cells revealed no changes in mitochondrial network, cristae structure, or mtDNA distribution. Preliminary observation show enlarged lysosomes in KO cells, suggesting the possibility of an unexpected link between TOM40B and lysosomal homeostasis. These findings indicate that TOM40B may have a stress dependent function in organelle crosstalk.
Together, this work advances our understanding of mitochondrial protein biogenesis by identifying novel roles for mammalian MTCH1 as an insertase, uncovering proteolytic activity of the S. pombe CIII₂CIV supercomplex, and characterising some cellular consequences of TOM40B deficiency. Collectively, my results highlight both evolutionary conservation and divergence in mitochondrial protein import pathways.
Mitochondria