The C‐terminal positively charged region of subunit 8 of yeast mitochondrial ATP synthase is required for efficient assembly of this subunit into the membrane F₀sector

Abstract

This paper deals with a truncated derivative of subunit 8 of yeast mitochondrial ATP synthase in which a conserved positively charged residue (Lys47) has been removed by site-directed mutagenesis together with the Cterminal residue (Leu48). This derivative has been expressed as a chimaeric precursor N9L/Y8-1 (K47-STP) carrying an N-terminal cleavable leader sequence (N9L), fused by a short bridging sequence to the truncated subunit-8 passenger protein. Allotopic expression of N9L/YS-l(K47-STP) in vivo in an aapl mit-host yeast strain lacking endogenous subunit 8 leads to partial restoration of bioenergetic function in the transformant strain denoted T475. Import and assembly studies were carried out in vitro using target mitochondria from strain YGL-1 partially depleted in subunit 8 ; such controlled depletion has been previously shown to be required for the efficient assembly (monitored immunochemically) of full-length subunit 8 imported in vitro as the precursor N9L/Y8-1. It was found that N9L/YS-l(K47-STP) synthesized in vitro was imported successfully into YGL-1 mitochondria, but no significant assembly of the truncated subunit 8 was observed in these or any other mitochondria tested. The bioenergetic defects in T475 mitochondria are ascribed to the impaired assembly of the subunit-8 variant in vivo, resulting from the truncation at Lys47. In consequence, T475 mitochondria behave as though partially depleted of subunit 8. This conclusion was supported by the ability of isolated T475 mitochondria to provide a vehicle for the efficient import and assembly of subunit 8 processed form full-length N9L/Y8-1. Two related aspects of import and assembly have been addressed as part of the analysis of truncated subunit 8. First, mitochondria from strain T2-1, an aapl mit-mutant genetically reconstituted by allotopic expression of N9L/ Y8-1, were also found to be effective in the in vitro assembly of subunit 8 derived from imported N9L/Y8-1. This suggests an intramitochondrial shortage of subunit 8 delivered by allotopic expression of N9L/Y8-1 in vivo, which may underlie the incomplete restoration of energy coupling in T2-1 mitochondria compared to those of wild-type yeast. Second, on allotopic expression of N9L/Y8-2 (containing subunit 8 directly fused to N9L) in the aapl mithost, a rescued transformant strain T10-1 was generated which displays bioenergetic defects superficially similar to those of T475. Processed subunit 8 clearly assembled into the ATP synthase of isolated YGL-1 mitochondria, in spite of the relatively weak import of N9L/Y8-2 in vitro. This demonstrates the ability of the in vitro assembly system to distinguish assembly properties of variants of subunit 8, such as N9L/YS-l(K47-STP) and N9LIY8-2.The Fa sector of the yeast mitochondrial ATP synthase (mtATPase) provides a versatile system for the study of the assembly of integral membrane proteins into energy-transducing enzyme complexes [l]. Previous studies of mutants of Saccharomyces cerevisiae, affected in the three mitochondrial genes encoding Fa s...

The Molecular Neighborhood of Subunit 8 of Yeast Mitochondrial F1F0-ATP Synthase Probed by Cysteine Scanning Mutagenesis and Chemical Modification

“…Sources of mitochondria were as follows. function (23,27). Cysteine substitutions for any of these 4 conserved amino acids had no demonstrable effect on cell growth or ATP hydrolase activity, suggesting that in yeast none of these residues is individually required for mtATPase function.…”

“…Allotopic expression (24), whereby a mitochondrial gene is recoded for nuclear expression with subsequent delivery of the protein back to mitochondria, has enabled our laboratory to undertake a detailed molecular genetic approach to investigate the structure and function of Y8. Detailed studies on genetically modified Y8 variants, allotopically expressed in yeast lacking endogenous Y8, have suggested that the N-terminal motif of Y8 plays a functional role in mtATPase (25)(26)(27) while the positively charged amino acid region at the C terminus of Y8 is involved in both the assembly and function of the F 0 sector (27)(28)(29). A surprising feature of Y8 is the functional accommodation of charged amino acid residues within the CHD (30 -33).…”

Modifying Mitochondrial Genetics

“…Through AE, a term first coined in respect to mtDNA gene expression in 1986 (Gearing and Nagley, 1986;Grasso et al, 1991), this phenomenon has led to speculation, experimentation, and subsequent mouse models . AE as a potential gene therapy was postulated as a strategy for either circumventing or correcting diseases involving mtDNA mutation (DiMauro and Mancuso, 2007;Kyriakouli et al, 2008) and as a means to overcome the absence of animal models reflecting mtDNA mutations in human disease (see .…”

“…AE as a potential gene therapy was postulated as a strategy for either circumventing or correcting diseases involving mtDNA mutation (DiMauro and Mancuso, 2007;Kyriakouli et al, 2008) and as a means to overcome the absence of animal models reflecting mtDNA mutations in human disease (see . Accordingly, AE was initially demonstrated in cultured cells (Gearing and Nagley, 1986;Manfredi et al, 2002;Zullo et al, 2005) and in somatic tissues following delivery via viral vector (Qi et al, 2007;Guy et al, 2009). Our efforts resulted in the first germ line-competent transgenic mouse models of AE .…”

Expression of Protein Tyrosine Phosphatase-like Molecule ICA512/IA-2 Induces Growth Arrest in Yeast Cells and Transfected Mammalian Cell Lines

“…Growth rates of recombinant S. cerevisiae strains were estimated in triplicate using the supplemented Sacc salts medium above, and the red filter of a KlettSummerson spectrophotometer corresponding to an approximate optical density of 600 nm, as described [18]. The strains expressing ICA512/IA-2 and ICA512/IA-2-i.c.…”