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Chaperones of F1-ATPase.

Ludlam A, Brunzelle J, Pribyl T, Xu X, Gatti DL, Ackerman SH - J. Biol. Chem. (2009)

Bottom Line: One important feature of this model was the prediction that as long as Atp11p is bound to beta and Atp12p is bound to alpha, the two F(1) subunits cannot interact at either the catalytic site or the noncatalytic site interface.Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to alpha and beta prevents further interactions between these F(1) subunits.However, Atp11p and Atp12p do not resemble alpha or beta, and it is instead the F(1) gamma subunit that initiates the release of the chaperones from alpha and beta and their further assembly into the mature complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

ABSTRACT
Mitochondrial F(1)-ATPase contains a hexamer of alternating alpha and beta subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to beta and alpha. In the absence of Atp11p and Atp12p, the hexamer is not formed, and alpha and beta precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F(1) assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486-1493) hypothesized that the chaperones themselves look very much like the alpha and beta subunits, and proposed an exchange of Atp11p for alpha and of Atp12p for beta; the driving force for the exchange was expected to be a higher affinity of alpha and beta for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to beta and Atp12p is bound to alpha, the two F(1) subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to alpha and beta prevents further interactions between these F(1) subunits. However, Atp11p and Atp12p do not resemble alpha or beta, and it is instead the F(1) gamma subunit that initiates the release of the chaperones from alpha and beta and their further assembly into the mature complex.

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Growth of S. cerevisiae mutants on nonfermentable and fermentable carbons. S. cerevisiae mutants disrupted at the genetic locus for Atp11p (Δatp11) or Atp12p (Δatp12) were transformed with a plasmid producing C. glabrata Atp11p or P. denitrificans Atp12p, and evaluated for growth on glucose and nonfermentable ethanol-glycerol (EG) plates. Wild type W303, the deletion strains Δatp11 and Δatp12, and the transformants Δatp11/YEp-cgatp11 and Δatp12/pRS316-pdatp12 were grown overnight in liquid YPD (2% glucose, 2% peptone, 1% yeast extract). The next day the cultures were adjusted to A600 = 1.0, and then serially diluted by a factor of 2. Five μl of each dilution were applied to a YPD and an EG plate (2% ethanol, 3% glycerol, 2% peptone, 1% yeast extract), and the plates were incubated at 30 °C. After 48 h, the deletion strain Δatp12 does not grow at all on EG, whereas Δatp11 displays a leaky phenotype. The transformant Δatp11/YEp-cgatp11, which produces from a multicopy plasmid C. glabrata Atp11p (56% identity with S. cerevisiae) with its own mitochondrial targeting sequence, grows on EG almost as well as the wild type. The transformant Δatp12/pRS316-pdatp12, which produces a chimeric protein in which P. denitrificans Atp12p (23% identity with S. cerevisiae) is fused to the mitochondrial targeting sequence of S. cerevisiae Atp11p, shows growth on EG, despite the plasmid being single copy; transformants of Δatp12 with the same chimeric allele expressed from a multicopy plasmid grow almost as well as the wild type (data not shown).
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Figure 2: Growth of S. cerevisiae mutants on nonfermentable and fermentable carbons. S. cerevisiae mutants disrupted at the genetic locus for Atp11p (Δatp11) or Atp12p (Δatp12) were transformed with a plasmid producing C. glabrata Atp11p or P. denitrificans Atp12p, and evaluated for growth on glucose and nonfermentable ethanol-glycerol (EG) plates. Wild type W303, the deletion strains Δatp11 and Δatp12, and the transformants Δatp11/YEp-cgatp11 and Δatp12/pRS316-pdatp12 were grown overnight in liquid YPD (2% glucose, 2% peptone, 1% yeast extract). The next day the cultures were adjusted to A600 = 1.0, and then serially diluted by a factor of 2. Five μl of each dilution were applied to a YPD and an EG plate (2% ethanol, 3% glycerol, 2% peptone, 1% yeast extract), and the plates were incubated at 30 °C. After 48 h, the deletion strain Δatp12 does not grow at all on EG, whereas Δatp11 displays a leaky phenotype. The transformant Δatp11/YEp-cgatp11, which produces from a multicopy plasmid C. glabrata Atp11p (56% identity with S. cerevisiae) with its own mitochondrial targeting sequence, grows on EG almost as well as the wild type. The transformant Δatp12/pRS316-pdatp12, which produces a chimeric protein in which P. denitrificans Atp12p (23% identity with S. cerevisiae) is fused to the mitochondrial targeting sequence of S. cerevisiae Atp11p, shows growth on EG, despite the plasmid being single copy; transformants of Δatp12 with the same chimeric allele expressed from a multicopy plasmid grow almost as well as the wild type (data not shown).

Mentions: Crystals of C. glabrata Atp11p and P. denitrificans Atp12p were obtained after screening several homologous proteins, including those from Homo sapiens, Mus musculus, S. cerevisiae, Candida albicans, Kluveromyces lactis, Rhodobacter capsulatus, and Arabidopsis thaliana. C. glabrata Atp11p and P. denitrificans Atp12p are 79.9 and 44.2% similar and 56.3% and 23.4% identical to the corresponding proteins in S. cerevisiae (Fig. 1). When produced from episomal plasmids C. glabrata Atp11p and P. denitrificans Atp12p rescue the respiratory defect of S. cerevisiae atp11 or atp12 deletion strains (Table 1; Fig. 2), suggesting that the mechanism by which the chaperones facilitate the assembly of F1 is maintained across evolutionary lines.


Chaperones of F1-ATPase.

Ludlam A, Brunzelle J, Pribyl T, Xu X, Gatti DL, Ackerman SH - J. Biol. Chem. (2009)

Growth of S. cerevisiae mutants on nonfermentable and fermentable carbons. S. cerevisiae mutants disrupted at the genetic locus for Atp11p (Δatp11) or Atp12p (Δatp12) were transformed with a plasmid producing C. glabrata Atp11p or P. denitrificans Atp12p, and evaluated for growth on glucose and nonfermentable ethanol-glycerol (EG) plates. Wild type W303, the deletion strains Δatp11 and Δatp12, and the transformants Δatp11/YEp-cgatp11 and Δatp12/pRS316-pdatp12 were grown overnight in liquid YPD (2% glucose, 2% peptone, 1% yeast extract). The next day the cultures were adjusted to A600 = 1.0, and then serially diluted by a factor of 2. Five μl of each dilution were applied to a YPD and an EG plate (2% ethanol, 3% glycerol, 2% peptone, 1% yeast extract), and the plates were incubated at 30 °C. After 48 h, the deletion strain Δatp12 does not grow at all on EG, whereas Δatp11 displays a leaky phenotype. The transformant Δatp11/YEp-cgatp11, which produces from a multicopy plasmid C. glabrata Atp11p (56% identity with S. cerevisiae) with its own mitochondrial targeting sequence, grows on EG almost as well as the wild type. The transformant Δatp12/pRS316-pdatp12, which produces a chimeric protein in which P. denitrificans Atp12p (23% identity with S. cerevisiae) is fused to the mitochondrial targeting sequence of S. cerevisiae Atp11p, shows growth on EG, despite the plasmid being single copy; transformants of Δatp12 with the same chimeric allele expressed from a multicopy plasmid grow almost as well as the wild type (data not shown).
© Copyright Policy - open-access
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Show All Figures
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Figure 2: Growth of S. cerevisiae mutants on nonfermentable and fermentable carbons. S. cerevisiae mutants disrupted at the genetic locus for Atp11p (Δatp11) or Atp12p (Δatp12) were transformed with a plasmid producing C. glabrata Atp11p or P. denitrificans Atp12p, and evaluated for growth on glucose and nonfermentable ethanol-glycerol (EG) plates. Wild type W303, the deletion strains Δatp11 and Δatp12, and the transformants Δatp11/YEp-cgatp11 and Δatp12/pRS316-pdatp12 were grown overnight in liquid YPD (2% glucose, 2% peptone, 1% yeast extract). The next day the cultures were adjusted to A600 = 1.0, and then serially diluted by a factor of 2. Five μl of each dilution were applied to a YPD and an EG plate (2% ethanol, 3% glycerol, 2% peptone, 1% yeast extract), and the plates were incubated at 30 °C. After 48 h, the deletion strain Δatp12 does not grow at all on EG, whereas Δatp11 displays a leaky phenotype. The transformant Δatp11/YEp-cgatp11, which produces from a multicopy plasmid C. glabrata Atp11p (56% identity with S. cerevisiae) with its own mitochondrial targeting sequence, grows on EG almost as well as the wild type. The transformant Δatp12/pRS316-pdatp12, which produces a chimeric protein in which P. denitrificans Atp12p (23% identity with S. cerevisiae) is fused to the mitochondrial targeting sequence of S. cerevisiae Atp11p, shows growth on EG, despite the plasmid being single copy; transformants of Δatp12 with the same chimeric allele expressed from a multicopy plasmid grow almost as well as the wild type (data not shown).
Mentions: Crystals of C. glabrata Atp11p and P. denitrificans Atp12p were obtained after screening several homologous proteins, including those from Homo sapiens, Mus musculus, S. cerevisiae, Candida albicans, Kluveromyces lactis, Rhodobacter capsulatus, and Arabidopsis thaliana. C. glabrata Atp11p and P. denitrificans Atp12p are 79.9 and 44.2% similar and 56.3% and 23.4% identical to the corresponding proteins in S. cerevisiae (Fig. 1). When produced from episomal plasmids C. glabrata Atp11p and P. denitrificans Atp12p rescue the respiratory defect of S. cerevisiae atp11 or atp12 deletion strains (Table 1; Fig. 2), suggesting that the mechanism by which the chaperones facilitate the assembly of F1 is maintained across evolutionary lines.

Bottom Line: One important feature of this model was the prediction that as long as Atp11p is bound to beta and Atp12p is bound to alpha, the two F(1) subunits cannot interact at either the catalytic site or the noncatalytic site interface.Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to alpha and beta prevents further interactions between these F(1) subunits.However, Atp11p and Atp12p do not resemble alpha or beta, and it is instead the F(1) gamma subunit that initiates the release of the chaperones from alpha and beta and their further assembly into the mature complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

ABSTRACT
Mitochondrial F(1)-ATPase contains a hexamer of alternating alpha and beta subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to beta and alpha. In the absence of Atp11p and Atp12p, the hexamer is not formed, and alpha and beta precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F(1) assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486-1493) hypothesized that the chaperones themselves look very much like the alpha and beta subunits, and proposed an exchange of Atp11p for alpha and of Atp12p for beta; the driving force for the exchange was expected to be a higher affinity of alpha and beta for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to beta and Atp12p is bound to alpha, the two F(1) subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to alpha and beta prevents further interactions between these F(1) subunits. However, Atp11p and Atp12p do not resemble alpha or beta, and it is instead the F(1) gamma subunit that initiates the release of the chaperones from alpha and beta and their further assembly into the mature complex.

Show MeSH
Related in: MedlinePlus