Cellular and Functional Analysis of Four Mutations Located in the Mitochondrial ATPase6 Gene Martha Elisa Vazquez-Memije, 1 Teresa Rizza, 2 Maria Chiara Meschini, 2 Claudia Nesti, 2 Filippo Maria Santorelli, 2 and Rosalba Carrozzo 2 * 1 Unidad de Investigacion Medica en Genetica, Centro Medico Nacional, Instituto Mexicano del Seguro Social, Mexico City, Mexico 2 Unit of Molecular Medicine, Bambino Gesu ` Children’s Hospital, Rome, Italy ABSTRACT The smallest rotary motor of living cells, F0F1-ATP synthase, couples proton flow—generated by the OXPHOS system—from the intermembrane space back to the matrix with the conversion of ADP to ATP. While all mutations affecting the multisubunit complexes of the OXPHOS system probably impact on the cell’s output of ATP, only mutations in complex V can be considered to affect this output directly. So far, most of the F0F1-ATP synthase variations have been detected in the mitochondrial ATPase6 gene. In this study, the four most frequent mutations in the ATPase6 gene, namely L156R, L217R, L156P, and L217P, are studied for the first time together, both in primary cells and in cybrid clones. Arginine (‘‘R’’) mutations were associated with a much more severe phenotype than Proline (‘‘P’’) mutations, in terms of both biochemical activity and growth capacity. Also, a threshold effect in both ‘‘R’’ mutations appeared at 50% mutation load. Different mechanisms seemed to emerge for the two ‘‘R’’ mutations: the F1 seemed loosely bound to the membrane in the L156R mutant, whereas the L217R mutant induced low activity of complex V, possibly the result of a reduced rate of proton flow through the A6 channel. J. Cell. Biochem. 106: 878–886, 2009. ß 2009 Wiley-Liss, Inc. KEY WORDS: MITOCHONDRIA; F1F0-ATPase; ATPase6 MUTATIONS; OS-ATPase; LEIGH SYNDROME H uman F0F1-ATP synthase (complex V of the respiratory chain) couples the synthesis of ATP from ADP and inorganic phosphate with the passage of protons from the intermembrane space to the matrix [Elston et al., 1998; Noji and Yoshida, 2001]. This rotary motor is comprised of at least 14 nucleus-encoded subunits (a, b, g, d, e, b, c, d, e, f, g, h, IF1, and OSCP) and two mtDNA- encoded subunits (ATPase 6 and ATPase 8). The F0 portion of the complex is embedded in the mitochondrial inner membrane and contains a ring of c subunits surrounding a central g subunit ‘‘stalk’’ that rotates within the F1 portion of the complex. The latter, in turn, consists of three a/b-dimers protruding into the matrix. ATPase 6, which is also part of F0, forms a channel through which proton flow is coupled with rotation of the c ring [Rastogi and Girvin, 1999; Hutcheon et al., 2001]. Several mutations have been described in the mitochondrial ATPase6 gene. The first description of a mutation, a T > G conversion at nt-8993 (L156R), dates back to 1990 [Holt et al., 1990] and concerned a patient with the neuropathy, ataxia, retinitis pigmentosa (NARP) syndrome. Since then, variable clinical expression between families has been reported [Tatuch et al., 1992], and two main phenotypes identified: NARP and maternally inherited Leigh syndrome (MILS), distinguished by different degrees of heteroplasmy of the T8993G mutation. A different mutation, a T > C transition, at nt-8993 (L156P), has also been found in association with the different clinical presentations of NARP/MILS [de Vries et al., 1993; Santorelli et al., 1994; Fujii et al., 1998]. A similar phenotypic heterogeneity was observed for mutations at nt- 9176 [i.e., T > G (L217R) and T > C (L217P)] [Thyagarajan et al., 1995; Campos et al., 1997; Dionisi-Vici et al., 1998; Carrozzo et al., 2001]. In both cases, the T > G mutation was clinically more severe than the T > C mutation. Moreover, both T > G mutations clearly impaired ATP synthesis in vitro, whereas the T > C mutations displayed a less marked effect on complex V activity [Thyagarajan Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 106:878–886 (2009) 878 Abbreviations used: NARP, neuropathy, ataxia, and retinitis pigmentosa; MILS, maternally inherited Leigh syndrome; OXPHOS, oxidative phosphorylation; ‘‘R,’’ either L156R or L217R mutations; ‘‘P,’’ either L156P or L217P mutations. Grant sponsor: Italian Ministry of Health; Grant sponsor: Ministero degli Esteri-ITALY and Secretaria Relaciones Exteriores-Conacyt-MEXICO. *Correspondence to: Dr. Rosalba Carrozzo, Unit of Molecular Medicine, Bambino Gesu ` Children’s Hospital, Piazza S. Onofrio 4, 00165 Rome, Italy. E-mail: carrozzo@opbg.net Received 21 August 2008; Accepted 15 December 2008  DOI 10.1002/jcb.22055  2009 Wiley-Liss, Inc. Published online 21 January 2009 in Wiley InterScience (www.interscience.wiley.com).