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

Abstract

Mitochondrial ATP-synthesis is catalyzed by a F1Fo-ATP synthase, an enzyme of dual genetic origin enriched at the edge of cristae where it plays a key role in their structure/stability. The enzyme’s biogenesis remains poorly understood, both from a mechanistic and a compartmentalization point of view. The present study provides novel molecular insights into this process through investigations on a human protein called TMEM70 with an unclear role in the assembly of ATP synthase. A recent study has revealed the existence of physical interactions between TMEM70 and the subunit c (Su.c), a protein present in 8 identical copies forming a transmembrane oligomeric ring (c-ring) within the ATP synthase proton translocating domain (FO). Herein we analyzed the ATP-synthase assembly in cells lacking TMEM70, mitochondrial DNA or F1 subunits and observe a reciprocal dependence of TMEM70 and Su.c levels, regardless of the status of other ATP synthase subunits or of mitochondrial bioenergetics. Immunoprecipitation and two-dimensional blue-native/SDS-PAGE reveal that TMEM70 forms large oligomers composed of 8 TMEM70 dimers and that TMEM70 oligomers interact with Su.c not yet incorporated into ATP synthase complexes. Moreover, discrete TMEM70-Su.c complexes with increasing Su.c contents can be detected, suggesting a role for TMEM70 oligomers in the gradual assembly of the c-ring. Furthermore, we demonstrate using expansion super-resolution microscopy the specific localization of TMEM70 at the inner cristae membrane, distinct from the MICOS component MIC60. Taken together, our results show that TMEM70 oligomers provide a scaffold for c-ring assembly and that mammalian ATP synthase is assembled within inner cristae membranes.

TMEM70 and TMEM242 help to assemble the rotor ring of human ATP synthase and interact with assembly factors for complex I

“…In addition, TMEM70 and TMEM242 interact selectively with the c-subunit from among the 18 types of subunits present in the ATP synthase, both of them forming high molecular mass complexes with the c-subunit in the range of 60 to 150 kDa. TMEM70 was also observed in much larger complexes that may either relate to the oligomeric scaffold proposed to aid c 8 -ring assembly (27,28) or, given the tendency of the c-subunit to aggregate in vitro, they could similarly be nonphysiological experimental artifacts. In contrast to TMEM70, TMEM242 has a wider influence as the levels of subunits ATP6, ATP8, j, and k in the vestigial complexes are diminished further in its absence, similar to the effect of removing subunit c (4).…”

The ATP Synthase Deficiency in Human Diseases

“…The biosynthesis of the eukaryotic ATP synthase is a highly organized process that requires the action of specific assembly factors [56,[75][76][77][78][79]. It was shown that mutations in some of these "chaperone" proteins, named ATPA12 and transmembrane protein 70 (TMEM70), can be responsible for secondary ATP synthase deficiencies [80], leading to altered assembly and compromised activity of the enzyme.…”

“…The other important regulatory protein in ATP synthase assembly is TMEM70, localized in the inner mitochondrial membrane [77]. Different mutations have been found over the years for TMEM70, with a broad spectrum of phenotypes and severity.…”

“…The most common features of the syndrome caused by TMEM70 mutations are a severe neonatal lactic acidosis, 3-methlyglutaconinc aciduria, cardiomyopathy, facial dysmorphism and mental retardation [7,81]. Early evidence for the role of TMEM70 in the enzyme assembly came in 2008 [56] and was later confirmed when it was shown that TMEM70 promotes the ATP synthase assembly by interacting with subunit c. This interaction facilitates the incorporation of the c subunit into the rotor structure of the enzyme within the inner mitochondrial membrane [76,77]. The c.317-2A>G mutation, which was firstly reported at the end of the second intron of the TMEM70 gene, resulted in aberrant splicing and the loss of the transcript.…”

TMEM135 is a Novel Regulator of Mitochondrial Dynamics and Physiology with Implications for Human Health Conditions

“…There are several TMEMs in the mitochondria, including two well-studied TMEMs; TMEM70 and TMEM242. TMEM 70 is localized in the inner membrane of the mitochondria and functions as a facilitator of mammalian F1Fo ATP synthase [ 14 , 15 , 16 ]. Given the known role of TMEM70, mutations in TMEM70 lead to oxidative phosphorylation (OXPHOS) deficiencies linked to many mitochondrial diseases that present as neonatal mitochondrial encephalo-cardiomyopathy in humans [ 14 , 15 , 17 , 18 , 19 ].…”