Knockout of Tmem70 alters biogenesis of ATP synthase and leads to embryonal lethality in mice

Abstract

TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at ∼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.

A dual function of TMEM70 in OXPHOS: assembly of complexes I and V

“…Moreover, both in the complexomics data and in the two dimensional BN-PAGE we show how, upon lack of TMEM70, the intermediate where F 1 is bound to the membrane-embedded c-subunits octamer 35 is completely absent. While these results are consistent with the ones reported by Vrbacký et al in Tmem70 knockout mouse embryos 28 , by detecting the F 1 -c intermediate in the presence of TMEM70 and its disappearance in the absence of this protein, we are able to specifically link TMEM70 to the formation of the F 1 -c assembly intermediate. This absence can be regarded as an immediate consequence of TMEM70’s impairment and, together with the aforementioned accumulation of c subunits when components of the F 1 module are defective, indicate that TMEM70 stabilizes the c-ring possibly in conjunction with tethering it to the F1 module (see below).…”

“…Mutations in TMEM70 have been reported to severely diminish the content of CV in a large cohort of patients 14,18-26 , and of all nuclear encoded proteins affecting CV, TMEM70 is the most commonly mutated in disease 27 . Moreover, Tmem70 knockout mouse embryos show stalled F 1 module 28 . These observations led to the hypothesis that TMEM70 is a CV assembly factor.…”

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

“…These topographic differences in the biogenesis of OXPHOS complexes may contribute to the preferential accumulation of Complex V at the edge of cristae (Davies et al , 2011; Dudkina et al , 2010), which is presumably important for shaping the mitochondrial inner membrane (Paumard et al , 2002; Strauss et al , 2008). Consistently, mitochondrial ultrastructure was found perturbed in our TMEM70 KO cells and in cells from a patient carrying a premature stop codon after TM2 that destabilizes TMEM70 (Fig.3 and 4), similarly to tissues from a mouse lacking this protein (Vrbacký et al , 2016, 70) and other TMEM70 patients (Braczynski et al , 2015; Diodato et al , 2015; Cameron et al , 2011).…”

“…Although a role for TMEM70 in mammalian ATP synthase biogenesis has been established in recent years (Braczynski et al , 2015; Cameron et al , 2011; Cízková et al , 2008; Jonckheere et al , 2011; Torraco et al , 2012; Vrbacký et al , 2016), its molecular function remained thus far poorly understood. We herein provide evidence that TMEM70 specifically interacts with Su.c before its assembly into the ATP synthase, protects Su.c from degradation, and facilitates its incorporation into the ATP synthase within cristae of the mitochondrial inner membrane.…”

Assembly of the membrane domain of ATP synthase in human mitochondria

“…TMEM70 is a membrane protein that has been suggested to participate in the assembly of the membrane domain of human ATP synthase (32,33), but neither this protein nor any other plausible assembly protein was increased in their association with vestigial complexes A-G ( Fig. 6, SI Appendix, Fig.…”