Identification of a Nuclear Gene (FMC1) Required for the Assembly/Stability of Yeast Mitochondrial F1-ATPase in Heat Stress Conditions

Abstract

We have identified a yeast nuclear gene (FMC1) that is required at elevated temperatures (37°C) for the formation/stability of the F 1 sector of the mitochondrial ATP synthase. Western blot analysis showed that Fmc1p is a soluble protein located in the mitochondrial matrix. At elevated temperatures in yeast cells lacking Fmc1p, the ␣-F 1 and ␤-F 1 proteins are synthesized, transported, and processed to their mature size. However, instead of being incorporated into a functional F 1 oligomer, they form large aggregates in the mitochondrial matrix. Identical perturbations were reported previously for yeast cells lacking either Atp12p or Atp11p, two specific assembly factors of the F 1 sector (Ackerman, S. H., and Tzagoloff, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4986 -4990), and we show that the absence of Fmc1p can be efficiently compensated for by increasing the expression of Atp12p. However, unlike Atp12p and Atp11p, Fmc1p is not required in normal growth conditions (28 -30°C). We propose that Fmc1p is required for the proper folding/stability or functioning of Atp12p in heat stress conditions. F 1 F o -ATP synthases play a major role in cellular energy production. They are found in the plasma membranes of bacteria, thylakoid membranes of chloroplasts, and in the inner membrane of mitochondria. They use a proton gradient across their host membrane to produce ATP from ADP and inorganic phosphate (1, 2). This enzyme contains two distinct parts, called F o and F 1 . The F o mediates the transmembrane transport of protons, and the synthesis of ATP takes place on the F 1 .The F 1 contains five different types of subunits in the stoichiometric ratio ␣ 3 ␤ 3 ␥␦⑀ (3, 4). The three-dimensional structures of F 1 from bovine heart (5), rat liver (6) and yeast (7) show that the ␣-and ␤-subunits alternate in a hexagonal array with a central cavity occupied by the amino and carboxyl termini of the ␥-subunit. The interfaces between the ␣-and ␤-subunits form three catalytic and three noncatalytic nucleotide binding sites.In the yeast Saccharomyces cerevisiae, the F 1 subunits are encoded in the nucleus (8 -12), synthesized in the cytoplasm, imported into mitochondria as unfolded polypeptide chains (13), and then folded in the mitochondrial matrix with the help of Hsp60p and Hsp10p (14). The oligomerization of the F 1 monomers is assisted by two proteins called Atp12p and Atp11p. These interact directly with the ␣-F 1 and ␤-F 1 proteins, respectively (15, 16). In yeast strains lacking either Atp11p or Atp12p, the ␣-F 1 and ␤-F 1 proteins aggregate in the mitochondrial matrix (17). Thus it is believed that Atp12p and Atp11p facilitate the formation of ␣␤ heterodimers by protecting these two F 1 subunits from non-productive interactions (16).We report in this study the identification of Fmc1p, a novel protein required for the formation or stability of the F 1 oligomer. Like Atp11p and Atp12p, its absence also results in the aggregation of the ␣-F 1 and ␤-F 1 proteins. However, this is seen only at elevated temperatures (37°...

Impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast

“…Autophagic response was already reported to be induced by misfolded proteins. 29,30 However, if the Dfmc1 model used here effectively accumulates a-F1 and b-F1 as large matricial aggregates under autophagy-promoting conditions 20,21 (heat stress and anaerobiosis), such aggregates also accumulate in cells grown under aerobiosis (Figure 2d), without induction of autophagy. Hence, protein misfolding is not the 'stressinducing' event of autophagy in our study.…”

“…Therefore, Fmc1p is also required, although only under heat stress conditions (371C), for the proper assembly of a-F1 and b-F1 subunits of mitochondrial ATP synthase. 20 Cells lacking Atp12p or Atp11p, or cells lacking Fmc1p and grown at restrictive temperature, accumulate large electron-dense particles in the mitochondrial matrix, which were shown by ultrastructural and immunocytochemical analysis to be composed almost exclusively of mature F1 a and/or b subunits (i.e. processed to remove the amino-terminal targeting peptide).…”

“…processed to remove the amino-terminal targeting peptide). [20][21][22] These mutants hence lack functional ATP synthase. Such a defect becomes a major issue in respiration-deficient cells, where the maintenance of an electrical potential across the mitochondrial inner membrane (DC) is no longer provided by the respiratory chain, but should be generated by the reverse functioning of the mitochondrial ATP synthase and the mitochondrial ATP/ADP carrier (ANC): normally in nonrespiring cells, the ANC imports ATP and exports ADP, and the F1-catalysed ATP hydrolysis is coupled to proton extrusion toward the mitochondrial inter-membrane space, thus maintaining a weaker DC.…”

“…However, in Dfmc1 cells grown under such conditions, F1 sector was not correctly assembled into a functional Fo-F1 ATPase, since DC collapse prevents Atp1p import into mitochondria; non imported Atp1p might still be trapped into aggregates. [20][21][22] Such an enrichment of proteins in clusters might provide a clue for a more efficient degradation of Atp1p compared to Por1p. It is nonetheless noteworthy that in both cases, this degradation was largely prevented in the macroautophagy-deficient double mutant Dfmc1 Datg5.…”

The Saccharomyces cerevisiae ATP22 Gene Codes for the Mitochondrial ATPase Subunit 6-Specific Translation Factor

“…Three genes ATP11 (Ackerman and Tzagoloff 1990), ATP12 (Ackerman and Tzagoloff 1990), and FMC1 (Lefebvre- Legendre et al 2001) code for mitochondrial proteins that aid assembly of the a-and b-subunits of F 1 ATPase into a catalytically active oligomer. The other nine genes affect expression of the F 0 unit of the ATPase.…”