Abstract: Dysfunction of TMEM70 disrupts the biogenesis of ATP synthase and represents the frequent cause of autosomal recessive encephalocardiomyopathy. We used tagged forms of TMEM70 and demonstrated that it has a hairpin structure with the N- and C-termini oriented towards the mitochondrial matrix. On BN-PAGE TMEM70 was detected in multiple forms including dimers and displayed partial overlap with assembled ATP synthase. Immunoprecipitation studies confirmed mutual interactions between TMEM70 molecules but, together with immunogold electron microscopy, not direct interaction with ATP synthase subunits. This indicates that the biological function of TMEM70 in the ATP synthase biogenesis may be mediated through interaction with other protein(s).

Abstract:
TMEM70 protein represents a novel ancillary factor of mammalian ATP synthase. We have investigated
import and processing of this factor in human cells using GFP- and FLAG-tagged forms of TMEM70 and specific
antibodies. TMEM70 is synthesized as a 29 kDa precursor protein that is processed to a 21 kDa mature form.
Immunocytochemical detection of TMEM70 showed mitochondrial colocalization with MitoTracker Red and
ATP synthase. Western blot of subcellular fractions revealed the highest signal of TMEM70 in isolated
mitochondria and mitochondrial location was confirmed by mass spectrometry analysis. Based on analysis of
submitochondrial fractions, TMEM70 appears to be located in the inner mitochondrial membrane, in
accordance with predicated transmembrane regions in the central part of the TMEM70 sequence. Two dimensional
electrophoretic analysis did not show direct interaction of TMEM70 with assembled ATP
synthase but indicated the presence of dimeric form of TMEM70. No TMEM70 protein could be found in cells
and isolated mitochondria from patients with ATP synthase deficiency due to TMEM70 c.317-2ANG mutation
thus confirming that TMEM70 biosynthesis is prevented in these patients.