Restoration of complex V deficiency caused by a novel deletion in the human TMEM70 gene normalizes mitochondrial morphology

Abstract

We report a fragmented mitochondrial network and swollen and irregularly shaped mitochondria with partial to complete loss of the cristae in fibroblasts of a patient with a novel TMEM70 gene deletion, which could be completely restored by complementation of the TMEM70 genetic defect. Comparative genomics analysis predicted the topology of TMEM70 in the inner mitochondrial membrane, which could be confirmed by immunogold labeling experiments, and showed that the TMEM70 gene is not restricted to higher multi-cellular eukaryotes. This study demonstrates that the role of complex V in mitochondrial cristae morphology applies to human mitochondrial disease pathology.

Introduction

Complex V (or the mitochondrial (mt) ATP synthase, EC 3.6.3.14), uses the proton gradient to generate ATP during oxidative phosphorylation. Complex V is a multisubunit complex consisting of two functional domains, F₁ and F_o, connected by two stalks. The catalytic F₁ domain is composed of subunits α, β, γ, δ, ε and a loosely attached IF1 inhibitor protein. The membrane-embedded F_o domain consists of an additional ten subunits, a (or subunit 6), b, c, d, e, f, g, OSCP, A6L (or subunit 8), and F6 and functions as a proton channel (Collinson et al., 1996, Houstek et al., 2009). Subunits 6 and 8 of the F₀ domain are encoded by the mtDNA genes MT-ATP6 and MT-ATP8, respectively. All other complex V subunits are encoded by the nuclear DNA. Mitochondria are enclosed by a double membrane. The inner membrane is composed of two subdomains: the inner boundary membrane and the cristae membrane. Cristae are invaginations of the inner membrane that are connected to the inner boundary membrane by narrow tubular structures, so-called crista junctions (Frey et al., 2002). It has been shown that complex V of mammalian mitochondria is arranged in long rows of dimeric supercomplexes (Strauss et al., 2008). Ribbons of complex V dimers are common to all eukaryotes (Strauss et al., 2008). The role of complex V dimers in the formation of tubular cristae has been hypothesized by Allen (1995). The link between the dimerization of mitochondrial complex V, through subunits e, g, and – as described just recently – subunits i, k, and the biogenesis of cristae has been provided by studying yeast cells (Paumard et al., 2002, Wagner et al., 2010). Complex V oligomers are found either on the crest of lamellae, or along the length of tubular cristae and introduce a positive curvature to the inner mitochondrial membrane (Strauss et al., 2008). In a recent yeast study, this effect has been shown to be attributed to the action of subunit e and subunit g (Rabl et al., 2009). Moreover, it has been suggested that the formation of cristae and crista junctions in mitochondria depends on antagonism between Fcj1 (formation of crista junction protein 1) and subunits e and g (Rabl et al., 2009). In HeLa cells, it has been shown that IF1 overexpression increases mitochondrial cristae formation and dimerization of complex V (Campanella et al., 2008). Other components such as prohibitins or OPA1 or others yet to be identified also could contribute to crista junction and cristae tip formation (Rabl et al., 2009, Zick et al., 2009). Complex V mutations have been described in MT-ATP6 (see, among many others, (Morava et al., 2006)), in MT-ATP8 (Jonckheere et al., 2008), in the nuclear encoded F₁-specific assembly gene ATP12 (De Meirleir et al., 2004), and in the nuclear encoded structural subunit ε (ATP5E) (Mayr et al., 2010). A common splice site mutation and an isolated frameshift mutation were described in the TMEM70 gene particularly in a homogeneous ethnic group (Romanies), and it has been shown that TMEM70 is required to maintain normal expression levels of complex V (Cizkova et al., 2008). The TMEM70 gene, located on chromosome 8q21.11, consists of three exons and encodes a 260 amino acid (AA) protein, transmembrane protein 70. GFP-tagged TMEM70 has been shown to be localized in mitochondria (Calvo et al., 2006). The exact molecular function of the TMEM70 protein is not known at present (Houstek et al., 2009), although it has been suggested that TMEM70 is involved in complex V biogenesis (Cizkova et al., 2008). TMEM70 homologues have been found in genomes of multi-cellular eukaryotes and plants, while it has been described to be absent from yeast and other fungi (Houstek et al., 2009).