TMEM70 forms oligomeric scaffolds within mitochondrial cristae promoting in situ assembly 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 cring. 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.

A cellular assay to determine the fusion capacity of MFN2 variants linked to Charcot-Marie-Tooth type 2A

“…Variants of human MFN2 were generated by mutagenesis using a QuickChange-derived protocol (Xia et al, 2015). The cDNA encoding wild-type human MFN2 (transcript variant 1, accession NM_014874.4 (Rojo et al, 2002)) was either mutagenized in a 3 kb cloning plasmid (pKSPS (Bahri et al, 2021)) before subcloning into pQCXIB (the retroviral expression vector, Addgene plasmid #22800) or was directly mutagenized in pQCXIB. For convenience, the p.L76P variant was mimicked by the change of two nucleotides (ctg>ccc) instead of one (ctg>ccg).…”

A stable microtubule bundle formed through an orchestrated multistep process controls quiescence exit

“…Cells were then washed three times with PBS. Cells were processed for Expansion Microscopy as previously described in (Bahri et al, 2021).…”

Interplay between Mitochondrial Protein Import and Respiratory Complexes Assembly in Neuronal Health and Degeneration

“…The second transmembrane domain, instead, might be imported in a follow up step through a stop-transfer mechanism via the TIM23 complex as previously described for Cox2 in plants [ 256 ]. Interestingly, two assembly factors previously known for being involved in CI assembly, TMEM70 and TMEM242, were found acting as a scaffold for c-ring assembly [ 257 , 258 ].…”