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.

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.

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.

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.

The Drosophila gene 2A5 complements the defect in mitochondrial F1-ATPase assembly in yeast lacking the molecular chaperone Atp11p.

Assembly of mitochondrial F1-ATPase in Saccharomyces cerevisiae requires the molecular chaperone, Atp11p. Database searches have identified protein sequences from Schizosaccharomyces pombe and two species of Drosophila that are homologous to S. cerevisiae Atp11p. A cDNA encoding the putative Atp11p from Drosophila yakuba was shown to complement the respiratory deficient phenotype of yeast harboring an atp11::HIS3 disruption allele. Furthermore, the product of this Drosophila gene was shown to interact with the S. cerevisiae F1 beta subunit in the yeast two-hybrid assay. These results indicate that Atp11p function is conserved in higher eukaryotes.

Cloning and sequence analysis of the structural gene for the bc1-type Rieske iron-sulfur protein from Thermus thermophilus HB8.

The structural gene encoding the Rieske iron-sulfur protein from Thermus thermophilus HB8 has been cloned and sequenced. The gene encodes a protein of 209 amino acids that begins with a hydrophilic N-terminus followed by a stretch of 21 hydrophobic amino acids that could serve as a transmembrane helix. The remainder of the protein has a hydrophobicity pattern typical of a water-soluble protein. A phylogenetic analysis of 26 Rieske proteins that are part of bc1 or b6f complexes shows that they fall into three major groups: eubacterial and mitochondrial, cyanobacterial and plastid, and five highly divergent outliers, including that of Thermus. Although the overall homology with other Rieske proteins is very low, the C-terminal half of the Thermus protein contains the signature sequence CTHLGC-(13X)-CPCH that most likely provides the ligands of the [2Fe-2S] cluster. It is proposed that this region of the protein represents a small domain that folds independently and that the encoding DNA sequence may have been transferred during evolution to several unrelated genes to provide the cluster attachment site to proteins of different origin. The role of individual residues in this domain of the Thermus protein is discussed vis-a-vis the three-dimensional structure of the bovine protein (Iwata et al., 1996 Structure 4, 567-579).

Mutational studies with Atp12p, a protein required for assembly of the mitochondrial F1-ATPase in yeast. Identification of domains important for Atp12p function and oligomerization.

The Atp12p protein of Saccharomyces cerevisiae is required for assembly of the F1 moiety of the mitochondrial ATP synthase. The current work has used mutant forms of Atp12p in an effort to learn about amino acids and/or domains that are important for the action of the protein. In one set of studies, the mutant atp12 genes were cloned and sequenced from 13 independent isolates of chemically mutagenized yeast. Of the 10 different mutant alleles that were identified, 9 (8 nonsense and 1 frameshift) lead to the early termination of the protein. A single missense mutation that substitutes lysine for Glu-289 was identified in two of the atp12 strains. Analysis of several Atp12p variants, each with different substitutions at Glu-289, showed that the functional activity of Atp12p is compromised when non-acidic residues are introduced at position 289 in the sequence. In other work, deletion analysis led to the assignment of two domains in Atp12p; the functional domain of the protein was mapped to the sequence between Gln-181 and Val-306, and a structural domain (Asp-307 through Gln-325) was identified that confers Atp12p the ability to oligomerize with other proteins in mitochondria.

Characterization of mutations in the beta subunit of the mitochondrial F1-ATPase that produce defects in enzyme catalysis and assembly.

The ATP2 gene, coding for the beta subunit of the mitochondrial F1-ATPase, was cloned from nine independent isolates of chemically mutagenized yeast. Seven different mutant alleles were identified. In one case the mutation occurs in the mitochondrial targeting sequence (M1I). The remaining six mutations map to the mature part of the beta subunit protein and alter amino acids that are conserved in the bovine heart mitochondrial and Escherichia coli beta subunit proteins. Biochemical analysis of the yeast atp2 mutants identified two different phenotypes. The G133D, P179L, and G227D mutations correlate with an assembly-defective phenotype that is characterized by the accumulation of the F1 alpha and beta subunits in large protein aggregates. Strains harboring the A192V, E222K, or R293K mutations assemble an F1 of normal size that is none-the-less catalytically inactive. The effect of the atp2 mutations was also analyzed in diploids formed by crossing the mutants to wild type yeast. Hybrid enzymes formed with beta subunits containing either the G133D, E222K, or R293K mutations are compromised for steady-state ATPase activity. The display of partial dominance confirms the importance of Gly133 for structural stability and of Glu222 and Arg293 for catalytic cooperativity.

Identification of functional domains in Atp11p. Protein required for assembly of the mitochondrial F1-ATPase in yeast.

The Atp11p protein of Saccharomyces cerevisiae is required for proper assembly of the F1 component of the mitochondrial ATP synthase. The mutant atp11 genes were cloned and sequenced from 12 yeast strains, which are respiratory-deficient due to a defect in Atp11p function. Four of the mutations mapped to the mitochondrial targeting domain (amino-terminal 39 amino acids) of Atp11p. All the genetic lesions found in the mature protein sequence were shown to be nonsense mutations. This result is consistent with the idea that Atp11p activity is provided, principally, by the overall structure of a functional domain, and not by specific amino acid residues in a localized active site. Amino-terminal (Edman) sequence analysis of fragments derived from limited proteolysis of purified Atp11p, and in vivo functional characterization of deletion mutants, were employed to locate the position of the active region in the protein. Three domains, separated by proline-rich sequences, were identified in the mature protein. The active domain of Atp11p was mapped to the sequence between Phe-120 and Asn-174. The domains proximal (Glu-40 through Ser-109) and distal (Arg-183 through Asn-318) to the active region were found to be important for the protein stability inside mitochondria.

Bacterial production and characterization of ATP11, a yeast protein required for mitochondrial F1-ATPase assembly.

ATP11 is a nuclear gene product that is required for assembly of mitochondrial F1-ATPase in the yeast Saccharomyces cerevisiae. ATP11 is synthesized in the yeast cytoplasm with an N-terminal targeting sequence. Following import into mitochondria, the leader sequence is cleaved, generating the functional form of the protein. ATP11 is present in small amounts in yeast mitochondria, which has made it difficult to study its role in F1 assembly. We have developed a bacterial expression system for the overproduction of the mature form of ATP11 and its biotinated derivative, BTATP11. Yeast complementation assays showed that the DNA fragments used to produce ATP11 and BTATP11 in bacteria encode biologically active proteins. The recombinant proteins produced in bacteria were purified to homogeneity and their physical characteristics were shown to be similar to those of the mitochondrial ATP11 protein synthesized in yeast.

Postprandial sleep and thermogenesis in normal men.

Twelve normal male subjects were given low- (16.77 kj/kg) and high- (54.49 kj/kg) calorie liquid carbohydrate lunch meals on 4 days, during which measures of sleep EEG, thermogenesis (heat production), core body temperature, and skin surface temperature were obtained. On 2 days subjects were required to remain awake, and on 2 days sleep was allowed. Both meals were administered in each condition. On the days that subjects were instructed to remain awake, thermogenesis was significantly greater following high-calorie meals than low-calorie meals, and both meal conditions produced levels of thermogenesis that were greater than those observed when sleep was allowed. When given the opportunity, 11 of 12 subjects slept following both low- and high-calorie meals. There was no difference between meal conditions in the total minutes or percent of stages 1, 2, 3/4, or rapid eye movement (REM) sleep following meals. However, the onset of postprandial sleep episodes was associated with the peak of the postprandial rise in thermogenesis, and the occurrence of sleep was followed by precipitous and statistically significant declines in thermogenesis and core body temperature, as well as increases in skin surface temperature. These data suggest that postprandial sleep is associated with rises in thermogenesis, and that its occurrence decreases postprandial heat production and body temperature.

Intake of different concentrations of sucrose and corn oil in preweanling rats.

To investigate the orosensory control of ingestion in preweanling rats, we infused one of the following liquids continuously through anterior sublingual intraoral catheters for 20 min: sucrose solutions (5, 10, 20, and 40%) and corn oil emulsions (6.25, 12.5, 25, 50, and 100%). Pups were tested at 7, 14, 21, and 28 days of age. Each pup was tested only once. Intake of sucrose and of corn oil became increasingly correlated with concentration from 7 to 21 days. Sucrose elicited more intake than water by day 7, whereas corn oil did not until day 14. Low concentrations of sucrose had differential effects on intake by day 14, but low concentrations of corn oil emulsions did not have differential effects on intake until day 21. At all ages, the peak intake of sucrose was significantly larger than the peak intake of corn oil. These differences are probably not based on prior experience because each test of ingestion represented a first exposure to the stimulus. Thus we suggest that the differences are due to the maturation of the orosensory control of ingestion by sucrose and corn oil.

Characterization of ATP11 and detection of the encoded protein in mitochondria of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, expression of functional F1-ATPase requires two proteins encoded by the ATP11 and ATP12 genes. Mutations in either gene block some crucial late step in assembly of F1, causing the alpha and beta subunits to accumulate in mitochondria as inactive aggregates (Ackerman, S. H., and Tzagoloff, A. (1991) Proc. Natl. Acad. Sci. U.S.A. 87, 4986-4990). In the present study we have cloned and determined the sequence of ATP11. The encoded product is protein of 37 kDa with no obvious homology to any known protein. In vitro import assays of ATP11 precursor and immunochemical evidence indicate that the protein is located in mitochondria. A fusion was made between ATP11 and a short sequence coding for 78 amino acids with the biotination signal of bacterial transcarboxylase. The protein expressed from this construct complements atp11 mutants, indicating that the addition of the extra 78 amino acids at the carboxyl terminus of the ATP11 protein does not compromise its function. The hybrid protein is detected in mitochondria with antibodies and with peroxidase-conjugated avidin. Biotinated ATP11 protein can be partially purified by affinity chromatography on monomeric or tetrameric avidin coupled to Sepharose. A fraction eluted from the avidin column and enriched for the biotinated ATP11 protein also contains the alpha and beta subunits of F1-ATPase.

Mitochondrial translational-initiation and elongation factors in Saccharomyces cerevisiae.

C155 and E252 are respiratory-defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes a, a3 and b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild-type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild-type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non-fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2. Disruption of the chromosomal copy of each gene in a wild-type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFM1 gene, coding for the mitochondrial initiation factor, indicates that the amino-terminal 423 residues of the protein are sufficient to promote peptide-chain initiation in vivo.

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.

ATP13, a nuclear gene of Saccharomyces cerevisiae essential for the expression of subunit 9 of the mitochondrial ATPase.

The respiratory deficient nuclear mutant of Saccharomyces cerevisiae, N9-168, assigned to complementation group G95 was previously shown to lack subunit 9, one of the three mitochondrially encoded subunits of the Fo component of the mitochondrial ATPase. As a consequence of the structural defect in Fo, the ATPase activity of G95 mutants is not inhibited by rutamycin. The absence of subunit 9 in N9-168 has been correlated with a lower steady-state level of its mRNA and an increase in higher molecular weight precursor transcripts. These results suggest that the mutation is most likely to affect either translation of the oli1 mRNA or processing of the primary transcript. We have isolated a nuclear gene, designated ATP13, which complements the respiratory defect and restores rutamycin-sensitive ATPase in G95 mutants. Disruption of ATP13 induces a respiratory deficiency which is not complemented by G95 mutants. The nucleotide sequence of ATP13 indicates a primary translation product with an Mapp of 42,897. The protein has a basic amino terminal signal sequence that is cleaved upon import into mitochondria. No significant primary structure homology is detected with any protein in the most recent libraries.

Identification of two nuclear genes (ATP11, ATP12) required for assembly of the yeast F1-ATPase.

Nuclear respiratory-deficient mutants of Saccharomyces cerevisiae (pet mutants) have been screened for defects in the mitochondrial ATPase. Mutants in two complementation groups were found to have 10% or less of wild-type ATPase activity. The two wild-type nuclear genes defined by the mutants have been designated ATP11 and ATP12. The proteins encoded by the two genes are not subunits of the ATPase but rather appear to exercise an important function at a late stage in the synthesis of F1 after transport of the subunits into the internal compartment of mitochondria. Mitochondria of atp11 and atp12 mutants have only marginally reduced levels of the alpha and beta subunits of F1. Both proteins are processed to their mature size but are not part of a native F1 structure or associated with the mitochondrial membrane. The most reasonable explanation for the mutant phenotype is a block in the assembly of the F1 oligomer.

ATP10, a yeast nuclear gene required for the assembly of the mitochondrial F1-F0 complex.

A yeast nuclear gene (ATP10) is reported whose product is essential for the assembly of a functional mitochondrial ATPase complex. Mutations in ATP10 induce a loss of rutamycin sensitivity in the mitochondrial ATPase but do not affect respiratory enzymes. This phenotype has been correlated with a defect in the F0 sector of the ATPase. The wild type ATP10 gene has been cloned by transformation of an atp 10 mutant with a yeast genomic library. The gene codes for a protein of Mr = 30,293. The primary structure of the ATP10 product is not related to any known subunit of the yeast or mammalian mitochondrial ATPase complexes. To further clarify the role of this new protein in the assembly of the ATPase, an antibody was prepared against a hybrid protein expressed from a trpE/ATP 10 fusion gene. The antibody recognizes a 30-kDa protein present in wild type mitochondria. The protein is associated with the mitochondrial membrane but does not co-fractionate either with F1 or with the rutamycin-sensitive F1-F0 complex. These data suggest that the ATP10 product is not a subunit of the ATPase complex but rather is required for the assembly of the F0 sector of the complex.

Plasma tryptophan to large neutral amino acid ratios in depressed and normal subjects.

The ratios of total and free plasma tryptophan to the sum of five large neutral amino acids (LNAAs) were found to be significantly lower in a group of 16 depressed inpatients compared to nine normal subjects after oral loading with L-tryptophan. The group differences in these ratios were significant before, and 2 weeks after starting treatment with a tricyclic antidepressant. Plasma tryptophan ratios and severity of depression were not significantly correlated.