Role of TMEM65 in the maintenance of nucleoids and mtDNA

TMEM65 is a protein located in the inner mitochondrial membrane. TMEM65 function is still partially unknown, but mutations and depletion of Tmem65 are associated with severe mitochondrial encephalomyopathy. Ablation of TMEM65 results in an increased calcium concentration in the mitochondrial matrix. Indeed, recent studies support a direct role of TMEM65 in calcium homeostasis. Moreover, patients with mutations in TMEM65 show a reduction in the mtDNA copy number. Whether and how these two events are correlated is not known. The aim of this thesis is to study the role of TMEM65 in the maintenance of mtDNA and nucleoids and to investigate if these processes are regulated by mCa2+. An initial intriguing hypothesis proposes that TMEM65 indirectly contributes to the maintenance of mtDNA and nucleoids by regulating the level of mitochondrial calcium. Thus, we hypothesise that the depletion of mCa2+ in Tmem65-/- cells should increase mtDNA copy number and likely nucleoid number. To verify this, we used TMEM65 WT, KO and OE cell lines and treated them with the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) to then study mtDNA copy number by qPCR and nucleoid number by confocal microscopy. A second hypothesis is that TMEM65 directly contributes to the maintenance of mtDNA and nucleoids by regulating mitochondrial transcription or translation through its association with mitoribosomes. To address whether TMEM65 and the mitoribosomes interact, we did a sucrose gradient to verify if the mitoribosomes and TMEM65 are in the same fraction. Our results show that calcium and not directly TMEM65 is responsible for the mtDNA copy number and nucleoid regulation.