TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme

Abstract

Biogenesis of F1Fo ATP synthase, the key enzyme of mitochondrial energy provision, depends on transmembrane protein 70 (TMEM70), localized in the inner mitochondrial membrane of higher eukaryotes. TMEM70 absence causes severe ATP‐synthase deficiency and leads to a neonatal mitochondrial encephalo‐cardiomyopathy in humans. However, the exact biochemical function of TMEM70 remains unknown. Using TMEM70 conditional knockout in mice, we show that absence of TMEM70 impairs the early stage of enzyme biogenesis by preventing incorporation of hydrophobic subunit c into rotor structure of the enzyme. This results in the formation of an incomplete, pathologic enzyme complex consisting of F1 domain and peripheral stalk but lacking Fo proton channel composed of subunits c and a. We demonstrated direct interaction between TMEM70 and subunit c and showed that overexpression of subunit c in TMEM70‐/‐ cells partially rescued TMEM70 defect. Accordingly, TMEM70 knockdown prevented subunit c accumulation otherwise observed in F1‐deficient cells. Altogether, we identified TMEM70 as specific ancillary factor for subunit c. The biologic role of TMEM70 is to increase the low efficacy of spontaneous assembly of subunit c oligomer, the key and rate‐limiting step of ATP‐synthase biogenesis, and thus to reach an adequately high physiologic level of ATP synthase in mammalian tissues.—Kovalčíkova, J., Vrbacký, M., Pecina, P., Tauchmannová, K., Nůsková, H., Kaplanová, V., Brázdová, A., Alán, L., Eliáš, J., Čunátová, K., Kořínek, V., Sedlacek, R., Mráček, T., Houštěk, J. TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme. FASEB J. 33, 14103‐14117 (2019). http://www.fasebj.org

TMEM70 forms oligomeric scaffolds within mitochondrial cristae promotingin situassembly of mammalian ATP synthase proton channel

“…In addition, we have shown that this is accompanied by a drastic drop in Su.c levels (see Fig. 2-4), which is a unique feature of TMEM70 deficiency and corroborates a very recent report (Kovalčíková et al , 2019). Whether the lack in Su.c was caused by a bioenergetic failure due to the compromised ability of the ATP synthase to assemble in the absence of TMEM70 and/or to the lack of another mitochondrial component(s) whose assembly/stability would be TMEM70-dependent was not known.…”

“…Recently, the presence of TMEM70 in pull down assays using a FLAG-version of su.c was reported (Kovalčíková et al , 2019), indicating the existence of possible interactions between Su.c and TMEM70. In these assays most of the Su.c pool was immunoprecipitated, which made it difficult to estimate the fraction of Su.c that interacts with TMEM70.…”

“…Studies by the group of J. Houstek have provided support to this hypothesis (Andersson et al , 1997; Houstek et al , 1995). On the other hand, important variations in the content of TMEM70 in mouse tissues have been reported recently (Kovalčíková et al , 2019). Therefore, the TMEM70-dependent assembly/stability of Su.c revealed in this study indicates that TMEM70 may play a key role in ATP synthase biogenesis modulation.…”

Optic Atrophy-associated TMEM126A is an assembly factor for the ND4-module of Mitochondrial Complex I

“…While an MRI conducted was considered normal, the patient was reported to have moderate hypertrophic cardiomyopathy, which is a more common presentation for patients with complex I deficiency (Frazier et al, 2020;Frazier et al, 2019;Gorman et al, 2016;Hanein et al, 2009). Analysis of TMEM126A-interacting proteins revealed an association with TMEM70, a protein long known to play a role in assembly of ATP synthase through incorporation of subunit c into the complex V rotor (Kovalčíková et al, 2019;Spiegel et al, 2011), but more recently also implicated in assembly of the complex I ND4-module (PD-a intermediate) (Guerrero-Castillo et al, 2017;Sánchez-Caballero et al, 2020).…”

MLQ is responsible for stabilisation of subunit a in the holoenzyme of mammalian ATP synthase

“…The above CNE profiles of ATP synthase indicated possible alterations in supercomplex stability. It is well known, that less stable ATP synthase complexes can be disintegrated during separation at harsher/more stringent conditions of electrophoresis such as upon exposure to Coomassie Blue Dye (Wittig, Meyer et al 2010, Hejzlarova, Kaplanova et al 2015, Kovalcikova, Vrbacky et al 2019). Thus, BNE was further used to assess stability of ATP synthase complexes (Figure 4).…”