Expression and processing of the TMEM70 protein

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 29kDa precursor protein that is processed to a 21kDa 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.317-2A>G mutation thus confirming that TMEM70 biosynthesis is prevented in these patients.

Mitochondrial ATP synthase: architecture, function and pathology

“…The N-terminal part of the cytosolic 29 kDa precursor is cleaved to a 21 kDa mature mitochondrial protein (Hejzlarova et al 2011). Analysis of submitochondrial fractions has shown that TMEM70 is associated with the inner mitochondrial membrane (Hejzlarova et al 2011). It has been demonstrated that TMEM70 is required to maintain normal expression levels and activity of complex V (Cizkova et al 2008).…”

“…It has been demonstrated that TMEM70 is required to maintain normal expression levels and activity of complex V (Cizkova et al 2008). It however does not interact directly with holocomplex V (Hejzlarova et al 2011). A transient binding to complex V assembly intermediates has been proposed (Cameron et al 2011).…”

“…A transient binding to complex V assembly intermediates has been proposed (Cameron et al 2011). Since TMEM70 is a low abundant mitochondrial protein, like the ATP11 and ATP12 assembly factors, a regulatory role of TMEM70 in complex V biogenesis has been suggested (Hejzlarova et al 2011). Further studies are necessary to clarify the exact mechanism by which TMEM70 defects lead to complex V deficiency.…”

Nuclear Genes Causing Mitochondrial Cardiomyopathy

“…The exact role of TMEM70 in ATP synthase assembly is not known. Recent evidence indicates that TMEM localizes to the inner mitochondrial membrane and does not appear to have a direct physical interaction with ATP synthase, but rather exists in a dimeric form [76]. TMEM70 is estimated to constitute up to 30 % of inner membrane-bound proteins and mutations lead to loss of integrity of the inner mitochondrial membrane and ultrastructural abnormalities.…”

Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder

“…The most common nuclear genetic cause of complex V deficiency, however, is associated with TMEM70 (MIM: 612418), 46 which encodes a protein required for the biogenesis and stability of complex V 47 . The presentation of disorders of complex V has often been described as an early-onset encephalocardiomyopathy that is typically observed in individuals with TMEM70 mutations 46, 48, 49.…”