The assembly factor Atp11p binds to the beta-subunit of the mitochondrial F(1)-ATPase

Atp11p is a protein of Saccharomyces cerevisiae required for the assembly of the F(1) component of the mitochondrial F(1)F(0)-ATP synthase. This study presents evidence that Atp11p binds selectively to the beta-subunit of F(1). Under conditions in which avidin-Sepharose beads specifically adsorbed biotinylated Atp11p from yeast mitochondrial extracts, the F(1) beta-subunit coprecipitated with the tagged Atp11p protein. Binding interactions between Atp11p and the entire beta-subunit of F(1) or fragments of the beta-subunit were also revealed by a yeast two-hybrid screen: Atp11p bound to a region of the nucleotide-binding domain of the beta-subunit located between Gly(114) and Leu(318). Certain elements of this sequence that would be accessible to Atp11p in the free beta-subunit make contact with adjacent alpha-subunits in the assembled enzyme. This observation suggests that the alpha-subunits may exchange for bound Atp11p during the process of F(1) assembly.     

Atp11p and Atp12p are assembly factors for the F(1)-ATPase in human mitochondria.

Atp11p and Atp12p were first described as proteins required for assembly of the F(1) component of the mitochondrial ATP synthase in Saccharomyces cerevisiae (Ackerman, S. H., and Tzagoloff, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4986-4990). Here we report the isolation of the cDNAs and the characterization of the human genes for Atp11p and Atp12p and show that the human proteins function like their yeast counterparts. Human ATP11 spans 24 kilobase pairs in 9 exons and maps to 1p32.3-p33, while ATP12 contains > or =8 exons and localizes to 17p11.2. Both genes are broadly conserved in eukaryotes and are expressed in a wide range of tissues, which suggests that Atp11p and Atp12p are essential housekeeping proteins of human cells. The information reported herein will be useful in the evaluation of patients with ascertained deficiencies in the ATP synthase, in which the underlying biochemical defect is unknown and may reside in a protein that influences the assembly of the enzyme.     

A purified subfragment of yeast Atp11p retains full molecular chaperone activity

Atp11p is a molecular chaperone of the mitochondrial matrix that participates in the biogenesis pathway to form F1, the catalytic unit of the ATP synthase. Affinity tag pull-down assays and yeast two-hybrid screens have shown that Atp11p binds to free beta subunits of F1 (Wang, Z. G., and Ackerman, S. H. (2000) J. Biol. Chem. 275, 5767-5772). This binding action prevents the beta subunit from associating with itself in non-productive complexes and fosters the formation of a (alpha beta)3 hexamer. Following the premise that Atp11p action is mediated primarily through a surface (as opposed to specific amino acids, as in an enzyme active site), solving its three-dimensional structure so that we may learn how the shape of the protein influences its function is a high priority. Recombinant yeast Atp11p has proven refractory for such analysis because of the presence of a disordered region in the protein. In this article, we show that removal of 67 residues from the amino terminus of recombinant Atp11p yields a subfragment of the protein (called Atp11pTRNC) that retains molecular chaperone function as determined in vitro with both a surrogate substrate (reduced insulin) and the natural substrate (F1 beta). Moreover, preliminary 15N-1H heteronuclear single quantum coherence spectra obtained with Atp11pTRNC indicate that the truncated protein is well ordered and amenable to structure determination by nuclear magnetic resonance.     

Atp10p assists assembly of Atp6p into the F0 unit of the yeast mitochondrial ATPase

The F(0)F(1)-ATPase complex of yeast mitochondria contains three mitochondrial and at least 17 nuclear gene products. The coordinate assembly of mitochondrial and cytosolic translation products relies on chaperones and specific factors that stabilize the pools of some unassembled subunits. Atp10p was identified as a mitochondrial inner membrane component necessary for the biogenesis of the hydrophobic F(0) sector of the ATPase. Here we show that, following its synthesis on mitochondrial ribosomes, subunit 6 of the ATPase (Atp6p) can be cross-linked to Atp10p. This interaction is required for the integration of Atp6p into a partially assembled subcomplex of the ATPase. Pulse labeling and chase of mitochondrial translation products in vivo indicate that Atp6p is less stable and more rapidly degraded in an atp10 null mutant than in wild type. Based on these observations, we propose Atp10p to be an Atp6p-specific chaperone that facilitates the incorporation of Atp6p into an intermediate subcomplex of ATPase subunits.     

Atp11p and Atp12p are chaperones for F(1)-ATPase biogenesis in mitochondria

The bioenergetic needs of aerobic cells are met principally through the action of the F(1)F(0) ATP synthase, which catalyzes ATP synthesis during oxidative phosphorylation. The catalytic unit of the enzyme (F(1)) is a multimeric protein of the subunit composition alpha(3)beta(3)(gamma)(delta) epsilon. Our work, which employs the yeast Saccharomyces cerevisiae as a model system for studies of mitochondrial function, has provided evidence that assembly of the mitochondrial alpha and beta subunits into the F(1) oligomer requires two molecular chaperone proteins called Atp11p and Atp12p. Comprehensive knowledge of Atp11p and Atp12p activities in mitochondria bears relevance to human physiology and disease as these chaperone actions are now known to exist in mitochondria of human cells.     

Characterization of ATP12, a yeast nuclear gene required for the assembly of the mitochondrial F1-ATPase.

Mitochondrial F1-ATPase is an oligomeric enzyme composed of five distinct subunit polypeptides. The alpha and beta subunits make up the bulk of protein mass of F1. In Saccharomyces cerevisiae both subunits are synthesized as precursors with amino-terminal targeting signals that are removed upon translocation of the proteins to the matrix compartment. Recently, two different complementation groups (G13, G57), consisting of yeast nuclear mutants with defective F1, have been described. Biochemical analyses indicate that the mutational block in both groups of mutants affects a critical step needed for the assembly of the alpha and beta subunits into the F1 oligomer after their transport into mitochondria. In this study the ATP12 gene representative of the nuclear respiratory-deficient mutant of S. cerevisiae (pet) complementation group G57 has been cloned and the encoded product partially characterized. The ATP12 reading frame is 975 base pairs long and codes for a protein of Mr = 36,587. The ATP12 protein is not homologous to the subunits of F1 whose sequences are known, nor does it exhibit significant primary structure similarity to any known protein. In vitro import assays indicate that ATP12 protein is synthesized as a precursor approximately 3 kDa larger than the mature protein. The mitochondrial localization of the protein has been confirmed by Western blot analysis of mitochondrial proteins with an antibody against a hybrid protein expressed from a trpE-ATP12 fusion. Fractionation of mitochondria indicates further that the ATP12 protein is either a minor component of the matrix compartment or is weakly bound to the matrix side of the inner membrane. The molecular weight of the native protein, estimated from its sedimentation properties in sucrose gradients, is at least two times larger than the monomer. This suggests that the ATP12 protein is probably part of a larger complex.     

The alpha-subunit of the mitochondrial F(1) ATPase interacts directly with the assembly factor Atp12p

The Atp12p protein of Saccharomyces cerevisiae is required for the assembly of the F(1) component of the mitochondrial F(1)F(0) ATP synthase. In this report, we show that the F(1) alpha-subunit co-precipitates and co-purifies with a tagged form of Atp12p adsorbed to affinity resins. Moreover, sedimentation analysis indicates that in the presence of the F(1) alpha-subunit, Atp12p behaves as a particle of higher mass than is observed in the absence of the alpha-subunit. Yeast two-hybrid screens confirm the direct association of Atp12p with the alpha-subunit and indicate that the binding site for the assembly factor lies in the nucleotide-binding domain of the alpha-subunit, between Asp133 and Leu322. These studies provide the basis for a model of F(1) assembly in which Atp12p is released from the alpha-subunit in exchange for a beta-subunit to form the interface that contains the non-catalytic adenine nucleotide-binding site.     

An accessible hydrophobic surface is a key element of the molecular chaperone action of Atp11p

Atp11p is a soluble protein of mitochondria that binds unassembled beta subunits of the F(1)-ATPase and prevents them from aggregating in the matrix. In this report, we show that Atp11p protects the insulin B chain from aggregating in vitro and therefore acts as a molecular chaperone. The chaperone action of Atp11p is mediated by hydrophobic interactions. An accessible hydrophobic surface in Atp11p was identified with the environment-sensitive fluorescent probe 1,1'-bis(4-anilino-5-napththalenesulfonic acid (bis-ANS). The spectral changes of bis-ANS in the presence of Atp11p indicate that the probe binds to a nonpolar region of the protein. Furthermore, the dye quenches the fluorescence of Atp11p tryptophan residues in a concentration-dependent manner. Although up to three molecules of bis-ANS can bind cooperatively to Atp11p, the binding of only one dye molecule is sufficient to virtually eliminate the chaperone activity of the protein.     

A mutation altering the kinetic responses of the yeast mitochondrial F1-ATPase

Nucleotide-binding proteins, including the mitochondrial F1-ATPase, the ras proteins, and the G-proteins, contain a homologous glycine-rich sequence that is thought to constitute part of the active site. This study reports the effects of a single amino acid replacement of Thr197 to Ser197, which is located at the hinge region of this putative loop, in the yeast Saccharomyces cerevisiae F1-ATPase. This replacement resulted in a 3-fold increase in the specific activity of the enzyme, eliminated the stimulatory effects of oxyanions, and modulated the effects of the inhibitor NaN3 while having little effect on the uni-site ATPase. These results indicate a role of the glycine-rich loop in many of the kinetic responses of the F1-ATPase.     

The human homologue of the yeast mitochondrial AAA metalloprotease Yme1p complements a yeast yme1 disruptant

In yeast, three AAA superfamily metalloproteases (Yme1p, Afg3p and Rca1p) are localized to the mitochondrial inner membrane where they perform roles in the assembly and turnover of the respiratory chain complexes. We have investigated the function of the proposed human orthologue of yeast Yme1p, encoded by the YME1L gene on chromosome 10p. Transfection of both HEK-293EBNA and yeast cells with a green fluorescent protein-tagged YME1L cDNA confirmed mitochondrial targeting. When expressed in a yme1 disruptant yeast strain, YME1L restored growth on glycerol at 37 degrees C. We propose that YME1L plays a phylogenetically conserved role in mitochondrial protein metabolism and could be involved in mitochondrial pathologies.     

Isolation of a gene for a regulatory 15-kDa subunit of mitochondrial F1F0-ATPase and construction of mutant yeast lacking the protein

A gene coding for yeast 15-kDa protein, a regulatory factor of mitochondrial F1F0-ATPase, was isolated. The cloned gene was disrupted in vitro and mutant strains that did not contain the 15-kDa protein were constructed by transformation of yeast cells with the disrupted gene. The ATP-synthesizing activity of the mutant mitochondria was the same as that of wild-type cells, suggesting that the 15-kDa protein is not required for mitochondrial oxidative phosphorylation. Collapse of the membrane potential induced ATP-hydrolyzing activity of F1F0-ATPase of the mutant mitochondria but not of normal mitochondria. Activation of the enzyme was also observed during incubation of submitochondrial particles from mutant cells, but not of those from wild-type cells. Thus, it is inferred that the 15-kDa protein supports the action of an intrinsic ATPase inhibitor of the ATP-hydrolyzing activity of the enzyme upon de-energization of mitochondrial membranes.     

Nuclear genes encoding the yeast mitochondrial ATPase complex. Analysis of ATP1 coding the F1-ATPase alpha-subunit and its assembly

Mitochondria prepared from the yeast nuclear pet mutant N9-84 lack a detectable F1-ATPase activity. Genetic complementation of this mutant with a pool of yeast genomic DNA in the yeast Escherichia coli shuttle vector YEp13 restored its growth on a nonfermentable carbon source. Mitochondria prepared from the transformed host contained an 8-fold higher than normal level of the F1 alpha-subunit and restored ATPase activity to 50% that of the wild-type strain. Deletion and nucleotide sequence analysis of the complementing DNA on the plasmid revealed a coding sequence designated ATP1 for a protein of 544 amino acids which exhibits 60 and 54% direct protein sequence homology with the proton-translocating ATPase alpha-subunits from tobacco chloroplast and E. coli, respectively. In vitro expression and mitochondrial import experiments using this ATP1 sequence showed that additional amino-terminal sequences not present in the comparable plant and bacterial subunits function as transient sequences for import.     

Ni-chelate-affinity purification and crystallization of the yeast mitochondrial F1-ATPase

The yeast mitochondrial ATPase has been genetically modified to include a His(6) Ni-affinity tag on the amino end of the mature beta-subunit. The modified beta-subunit is imported into the mitochondrion, properly processed to the mature form, and assembled into a mature and fully active ATP synthase. The F(1)-ATPase has been purified from submitochondrial particles after release from the membrane with chloroform, followed by Ni-chelate-affinity and gel filtration chromatography. The final enzyme is a homogeneous preparation with full activity and no apparent degradation products. This enzyme preparation has been used to obtain crystals that diffract to better than 2.8 A resolution.     

Dual role of the mitochondrial chaperone Mdj1p in inheritance of mitochondrial DNA in yeast

Mdj1p, a homolog of the bacterial DnaJ chaperone protein, plays an essential role in the biogenesis of functional mitochondria in the yeast Saccharomyces cerevisiae. We analyzed the role of Mdj1p in the inheritance of mitochondrial DNA (mtDNA). Mitochondrial genomes were rapidly lost in a temperature-sensitive mdj1 mutant under nonpermissive conditions. The activity of mtDNA polymerase was severely reduced in the absence of functional Mdj1p at a nonpermissive temperature, demonstrating the dependence of the enzyme on Mdj1p. At a permissive temperature, the activity of mtDNA polymerase was not affected by the absence of Mdj1p. However, under these conditions, intact [rho(+)] genomes were rapidly converted to nonfunctional [rho(-)] genomes which were stably propagated in an mdj1 deletion strain. We propose that mtDNA polymerase depends on Mdj1p as a chaperone in order to acquire and/or maintain an active conformation at an elevated temperature. In addition, Mdj1p is required for the inheritance of intact mitochondrial genomes at a temperature supporting optimal growth; this second function appears to be unrelated to the function of Mdj1p in maintaining mtDNA polymerase activity.     

The leader peptide of yeast Atp6p is required for efficient interaction with the Atp9p ring of the mitochondrial ATPase

Atp6p (subunit 6) of the Saccharomyces cerevisiae mitochondrial ATPase is synthesized with an N-terminal 10-amino acid presequence that is cleaved during assembly of the complex. This study has examined the role of the Atp6p presequence in the function and assembly of the ATPase complex. Two mutants were constructed in which the codons for amino acids 2-9 or 2-10 of the Atp6p precursor were deleted from the mitochondrial ATP6 gene. The concentration of Atp6p and ATPase complex was approximately 2 times less in the mutants. The lower concentration of ATPase complex in the leaderless mutants correlated with less Atp6p complexed with the Atp9p ring of the F0 sector and with accumulation of an Atp6p-Atp8p complex that aggregated into polymers destined for eventual proteolytic elimination. We propose that the presequence either targets Atp6p to the Atp9p or signals insertion of the Atp6p precursor into a microcompartment of the membrane for more efficient interaction with the Atp9p ring. Despite the ATPase deficiency, growth of the leaderless atp6 mutants on respiratory substrates and the efficiency of oxidative phosphorylation were similar to that of wild type, indicating that the mutations did not affect the proton permeability of mitochondria.     

The delta'-subunit of higher plant six-subunit mitochondrial F1-ATPase is homologous to the delta-subunit of animal mitochondrial F1-ATPase.

Mitochondrial F1-ATPases purified from several dicotyledonous plants contain six different subunits of alpha, beta, gamma, delta, delta' and epsilon. Previous N-terminal amino acid sequence analyses indicated that the gamma-, delta-, and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma-subunit, the oligomycin sensitivity-conferring protein and the epsilon-subunit of animal mitochondrial F1F0 complex (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). However, the N-terminal amino acid sequence of the delta'-subunit did not show any obvious homologies with known protein sequences. A cDNA clone for the delta'-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide-hybridization selection of a cDNA library. The 1.0-kilobase-long cDNA contained a 600-base pair open reading frame coding for a precursor for the delta'-subunit. The precursor for the delta'-subunit contained N-terminal presequence of 21-amino acid residues. The mature delta'-subunit is composed of 179 amino acids and its sequence showed similarities of about 31-36% amino acid positional identity with the delta-subunit of animal and fungal mitochondrial F1 and about 18-25% with the epsilon-subunit of bacterial F1 and chloroplast CF1. The sweet potato delta'-subunit contains N-terminal sequence of about 45-amino acid residues that is absent in other related subunits. It is concluded that the six-subunit plant mitochondrial F1 contains the subunit that is homologous to the oligomycin sensitivity-conferring protein as one of the component in addition to five subunits that are homologous to subunits of animal mitochondrial F1.     

The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information.

The NH2 terminus of the yeast F1-ATPase beta subunit precursor directs the import of this protein into mitochondria. To define the functionally important components of this import signal, oligonucleotide-directed mutagenesis was used to introduce a series of deletion and missense mutations into the gene encoding the F1-beta subunit precursor. Among these mutations were three nonoverlapping deletions, two within the 19-amino-acid presequence (delta 5-12 and delta 16-19) and one within the mature protein (delta 28-34). Characterization of the mitochondrial import properties of various mutant F1-beta subunit proteins containing different combinations of these deletions showed that import was blocked only when all three deletions were combined. Mutant proteins containing all possible single and pairwise combinations of these deletions were found to retain the ability to direct mitochondrial import of the F1-beta subunit. These data suggest that the F1-beta subunit contains redundant import information at its NH2 terminus. In fact, we found that deletion of the entire F1-beta subunit presequence did not prevent import, indicating that a functional mitochondrial import signal is present near the NH2 terminus of the mature protein. Furthermore, by analyzing mitochondrial import of the various mutant proteins in [rho-] yeast, we obtained evidence that different segments of the F1-beta subunit import signal may act in an additive or cooperative manner to optimize the import properties of this protein.     

The human TAZ gene complements mitochondrial dysfunction in the yeast taz1Delta mutant. Implications for Barth syndrome

Barth syndrome is a genetic disorder that is caused by different mutations in the TAZ gene G4.5. The yeast gene TAZ1 is highly homologous to human TAZ, and the taz1Delta mutant has phospholipid defects similar to those observed in Barth syndrome cells, including aberrant cardiolipin species and decreased cardiolipin levels. Subcellular fractionation studies revealed that Taz1p is localized exclusively in mitochondria, which supports the theory that tafazzins are involved in cardiolipin remodeling. Because cardiolipin plays an important role in respiratory function, we measured the energy transformation and osmotic properties of isolated mitochondria from the taz1Delta mutant. Energy coupling in taz1Delta mitochondria was dependent on the rate of oxidative phosphorylation, as coupling was diminished when NADH was used as a respiratory substrate but was unaffected when ethanol was the substrate. Membrane stability was compromised in taz1Delta mitochondria exposed to increased temperature and hypotonic conditions. Mitochondria from taz1Delta also displayed decreased swelling in response to ATP, which induces the yeast mitochondrial unspecific channel, and to alamethicin, a membrane-disrupting agent. Coupling was measured in taz1Delta cells containing different splice variants of the human TAZ gene. Only the variant that restores wild type cardiolipin synthesis (lacking exon 5) restored coupling in hypotonic conditions and at elevated temperature. These findings may shed light on the mitochondrial deficiencies observed in Barth syndrome.