©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Functional Domains in Atp11p
PROTEIN REQUIRED FOR ASSEMBLY OF THE MITOCHONDRIAL F(1)-ATPase IN YEAST (*)

(Received for publication, October 13, 1995; and in revised form, December 5, 1995)

Zhen-Guo Wang Sharon H. Ackerman (§)

From the Departments of Surgery and Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Atp11p protein of Saccharomyces cerevisiae is required for proper assembly of the F(1) 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.


INTRODUCTION

The ATP synthase is the key enzyme involved in energy production in aerobic organisms(1, 2) . The enzyme is composed of two oligomeric units, an integral membrane component, F(0), and a peripherally bound moiety, F(1). When bound to the membrane sector, F(1) catalyzes ATP synthesis/ATP hydrolysis coupled to proton translocation through the F(0); purified F(1) (F(1)-ATPase) functions solely as an ATP hydrolase.

F(1) contains five different types of subunits, arranged in the stoichiometric ratio alpha(3)beta(3)(1, 2) . The crystal structure of the bovine F(1) has been solved at 2.8 Å and shows alternating alpha and beta subunits surrounding a central helical structure made up of the amino and carboxyl termini of the subunit(3) . The and subunits, which are not resolved in the current structure(3) , are presumed to be located near the base of F(1) since they have been shown to be important for binding F(1) to F(0)(4, 5) . An interesting feature of F(1) is that maximal catalytic rates are achieved through cooperative subunit interactions in response to ligand binding(1, 2) . The degree to which protein-protein interactions affect F(1) catalytic mechanism makes this enzyme a particularly interesting system in which to study macromolecular assembly.

The formation of the F(1) oligomer requires proteins that are not part of the enzyme structure. All of the yeast F(1) subunits are nuclear gene products(6, 7, 8, 9, 10) . With the exception of (8) , the other four subunits are synthesized as larger precursors and the amino-terminal mitochondrial targeting sequences are removed by the matrix protease (11) when the proteins are imported into mitochondria. The intramitochondrial steps in F(1) biosynthesis include subunit folding, which is assisted by the molecular chaperone Hsp60 (12, 13) , and assembly of the enzyme oligomer. Previous work with yeast mutants has shown that the oligomer assembly is dependent on the products of the ATP11 and the ATP12 genes(14) . The amino acid sequences deduced from ATP11(15) and ATP12(16) do not show homology with known proteins. Thus, the encoded ``assembly factors'' are considered to be distinct from the class of molecular chaperones that have been described previously (13) .

In this communication, we report work that focuses on the ATP11 gene product (called Atp11p). Native Atp11p is a soluble, monomeric protein of the mitochondrial matrix(15, 17) . Initially, the protein is synthesized in the cytoplasm with an amino-terminal mitochondrial targeting sequence(15) . Intramitochondrial proteolytic cleavage of the precursor protein is predicted to occur at Pro-39 to generate a 32-kDa mature protein that begins with residue, Glu-40(17) . Defective F(1) assembly is readily detected in atp11 mutants, whose mitochondria show the accumulation of the F(1) alpha and beta subunits in large protein aggregates(15) . Atp11p action in F(1)-ATPase assembly is proposed to occur downstream from the mitochondrial foldase Hsp60(12, 13) , since there is no evidence of unprocessed alpha or beta in mitochondria of atp11 mutants. In contrast to this, disruption of Hsp60 activity correlates with the intramitochondrial accumulation of both the mature and precursor forms of the beta subunit (12) .

Atp11p has been identified in complexes with the alpha and beta subunits of the enzyme(15) , which suggests that the larger of the F(1) proteins interact directly with the assembly factor. It is possible that Atp11p acts during the initial stages of F(1) assembly to secure the formation of a stable subcomplex of alpha and beta subunits that, in turn, assembles with the other F(1) subunits. Evidence for this comes from the analysis of yeast strains with null mutations in the structural F(1) genes. Yeast, which produce only the alpha or only the beta subunit, harbor the lone subunit in an aggregated form(14) . In contrast, there is no evidence of alpha and/or beta aggregates in mitochondria prepared from (10) , (9) , or (8) null mutants, despite the fact that the absence of any of these subunits blocks F(1) assembly. In fact, in subunit-deficient cells, the alpha and beta subunits show sedimentation behavior in linear sucrose gradients that suggests the proteins exist as dimers(10) . Cumulatively, these studies suggest that alpha/beta subcomplexes are stable once formed. Since the alpha and beta proteins precipitate out of solution in mitochondria of atp11 mutants(14) , it is conceivable that Atp11p is required to shield ``sticky'' sites on these subunits that would otherwise favor the formation of dead-end (alpha) and (beta) complexes. The aggregation phenotype observed in the alpha and beta subunit null strains (see above) may result from the fact that the mitochondrial concentration of Atp11p is three orders of magnitude lower relative to F(1)(17) . Thus, the limited amount of Atp11p present in mitochondria is probably not sufficient to bind and maintain in solution alpha or beta protein that accumulates when the partner subunit is missing.

Here we describe studies that identified the minimal region of Atp11p capable of promoting F(1) assembly. This functional domain may represent the region of Atp11p that interacts with the alpha and beta subunits of F(1).


EXPERIMENTAL PROCEDURES

Yeast Strains and Growth Media

The genotypes and sources of the mutant and wild type yeast strains used in the present study are listed in Table 1. Chemically induced mutants were obtained as described(19) . Escherichia coli RR1 (proA leuB lacY galK xyl-5 mtl-1 ara-14 rpsL supE hsdS ) was the host bacterial strain for the recombinant plasmid constructions. Yeast was grown in the following media: YPD (2% glucose, 2% peptone, 1% yeast extract), YPGal (2% galactose, 2% peptone, 1% yeast extract), YEPG (3% glycerol, 2% ethanol, 2% peptone, 1% yeast extract), WO (2% glucose, 0.67% yeast nitrogen base without amino acids (Difco)). Amino acids and other growth requirements were added at a final concentration of 20 µg/ml. The solid media contained 2% agar in addition to the components described above.



Preparation of Yeast Mitochondria

Yeast were grown aerobically in liquid YEPG or YPGal at 30 °C to early stationary phase. The method of Faye et al.(20) was used to prepare mitochondria with the exception that Zymolyase, instead of Glusulase, was used to digest the cell wall. Phenylmethylsulfonyl fluoride was added to 10 µg/ml final concentration during the cell-breaking step to minimize proteolysis.

Solubilization of Atp11p from Mitochondria

Mitochondria were suspended at 10 mg/ml in 10 mM Tris-HCl, pH 8.0. Sodium deoxycholate was added to 1 mg/ml, and phenylmethylsulfonyl fluoride (final concentration 10 µg/ml) was added to minimize proteolysis of the solubilized proteins. Following a 15-min incubation at 0 °C, the suspension was centrifuged for 30 min at 4 °C at 50,000 rpm in a Beckman 70Ti rotor.

DNA Sequencing

The oligonucleotide primers used for sequencing and for constructing atp11 deletion/truncation mutants are listed in Table 2. Genomic DNAs were purified from yeast and amplified by PCR (^1)using the primers 2 and 12. The 1010-bp product was gel-purified and sequenced directly by the dideoxy method (22) with Sequenase (United States Biochemical Corp.). Both strands of two separate PCR products were sequenced for each atp11 mutant.



Plasmid Constructions

The plasmids used in this study are described in Table 3. Atp11p is numbered from 1 to 318, where residue 1 is the initiator methionine in the primary translation product(15) . In cases where sequences were removed from the 3` end of the ATP11 gene, the plasmids and the encoded products are named according to where the mutant protein is truncated. This is indicated by the single-letter code and number (15) of the last retained amino acid. The plasmids coding for atp11 sequences in which internal deletions were made, and the encoded proteins, are designated by the Delta symbol, followed by the span of ATP11 codons removed. The plasmid nomenclature also includes the letter ``m'' or ``s'' to designate a multi or single copy vector. Table 3also shows the number of amino acids that are deleted from the carboxyl or amino terminus of Atp11p in each of the plasmid-borne mutant proteins. The details of the plasmid constructions are provided in a footnote. (^2)All of the plasmids whose construction utilized PCR (plasmids pG13/BK1.5, pG13/I203, pG13/R183, pG13Delta40-75, and pG13Delta40-218) were sequenced to verify that there were no atp11 mutations introduced by the method.



Determination of Doubling Times for Yeast Grown in Liquid YEPG

Yeast that showed the ability to grow on plates containing nonfermentable carbon sources (YEPG, ethanol-glycerol media) were pregrown on YEPG plates and used to inoculate liquid YEPG cultures. The growth progression of the cells was followed by turbidity measurements with a Klett colorimeter. Doubling times were calculated by fitting an exponential function to the growth curves obtained from plots of Klett units versus time.

Analysis of Atp11p Tryptic Peptides

One hundred microgram samples of purified recombinant Atp11p protein were incubated in 400 µl with increasing concentrations of trypsin for 30 min, in 10 mM Tris-HCl, pH 7.5, at 37 °C. The peptide fragments were separated on a 10% SDS-polyacrylamide gel and then transferred to Immobilon P membrane (Millipore) as described(24) . The amino-terminal 10 amino acids of the membrane-bound peptides were sequenced by Edman degradation at the Macromolecular Core facility of Wayne State University School of Medicine.

Assays

The number of free sulfhydryl groups in Atp11p was determined with the Ellman reagent, 5,5`-dithiobis(2-nitrobenzoic acid) (25) , using an extinction coefficient at 412 nm of 13,400 M for the 5-nitro-2-thiobenzoate liberated in the reaction. Protein concentrations were estimated by the method of Lowry et al.(26) . ATPase activity was measured by the colorimetric determination of inorganic phosphate as described previously(14) . The amount of mutant Atp11p activity was determined by dividing the percent wild type level of oligomycin-sensitive ATPase activity by the percent wild type level of Atp11p protein detected in mitochondria prepared from yeast transformants that produce Atp11p from plasmids.

Miscellaneous Procedures

Standard techniques were used for restriction endonuclease analysis of DNA, purification and ligation of DNA fragments, and transformations of and recovery of plasmid DNA from E. coli(27) . Yeast transformations employed the LiAc procedure(28) . The method of Laemmli (29) was used for SDS-polyacrylamide gel electrophoresis. Western blotting followed the procedure of Schmidt et al.(30) . To obtain antibody against Atp11p, an aqueous solution of recombinant Atp11p, purified from a bacterial expression system(17) , was administered to rabbits along with complete Freund's adjuvant. The immune serum was used at a 1:1000 dilution.


RESULTS

Characterization of the atp11 Mutants Obtained by Chemical Mutagenesis

Twelve independent yeast isolates, with mutations in the ATP11 gene, were obtained by chemical mutagenesis(19) . These strains are respiratory-deficient due to a defect in the F(1)-ATPase assembly pathway(14) . To determine the type of genetic lesions that affect Atp11p function, the mutant genes were cloned and sequenced from the atp11 strains (see ``Experimental Procedures''). All of the mutants were found to be defective for producing a full-length protein. Table 4shows the position of the mutations in the nucleotide and protein sequences. All of the atp11 strains, except C15, were found to have nonsense mutations that converted the relevant amino acid to a translational stop; C15 has a missense mutation at the first nucleotide that converts the methionine initiator to a leucine residue. Four of the atp11 strains (C15, E716, E741, C203) have mutations in the mitochondrial leader sequence of the primary translation product. The genetic lesions in the other eight strains occur in the coding region for the mature protein, which should lead to the synthesis of Atp11p deleted for either 52, 82, 108, 114, 115, 135, or 175 amino acids from the carboxyl terminus (Table 4). The positions where these mutant proteins are predicted to terminate are indicated with arrows in the sequence of Atp11p shown in Fig. 1. Mitochondrial samples were prepared from the atp11 mutants, whose lesions occur in the mature portion of the protein, and probed for Atp11p in Western blots. The truncated Atp11p proteins present in yeast strains E381, E824, and N224 were visualized by Western analysis; the mutant proteins that are predicted to have geq114 amino acids deleted from the carboxyl terminus were not detected in the blot (data not shown).




Figure 1: Description of Atp11p deletion mutants. The primary translation product of the ATP11 gene (15) is shown in single-letter code. The amino-terminal mitochondrial targeting domain is shown in the dotted box; Pro-39 is estimated to be the site where cleavage occurs to generate the mature form of Atp11p(17) . The names of the atp11 strains, which were obtained by chemical mutagenesis, are highlighted in the black boxes; the arrows indicate the amino acid residue that is mutated in each of the strains. The white boxes bear the names of the plasmid-borne Atp11p deletion mutants. Arrows are used to indicate the first Atp11p amino acid in the Delta40-75, Delta40-111, and Delta40-218 Atp11p proteins, and the last retained amino acid in the R183, I203, G253, and A300 Atp11p proteins. Since the construction of the Atp11(Delta40-75)p and Atp11(Delta40-218)p encoding plasmids resulted in the addition of sequence between the mitochondrial leader and the mature part of the protein, either or both of these proteins might have amino acids coded for in the linker sequence preceding the Atp11p amino acid that is indicated in the figure (see Footnote 2).



Although only a small number of chemically induced atp11 mutants was available for analysis, we found noteworthy that there were no amino acid substitutions identified in the mature protein sequence that correlated with the loss of Atp11p function. This result was interpreted as an indication that the activity of Atp11p may depend primarily on the overall structural features of its functional domain, rather than on specific amino acid residues.

Proteolytic Mapping of Atp11p

The premise that hypersensitive protease digestion sites occur at the protein surface in between compact domains provided the rationale for analyzing Atp11p proteolytic fragments in order to obtain information on the location of stable structures in the protein. Fig. 2, lanes 2-7, shows the results obtained when recombinant Atp11p purified from a bacterial expression system (17) was digested with limiting amounts of trypsin. The recombinant protein sequence is identical with the mature form of native Atp11p, with the exception that the amino-terminal amino acid is a glycine rather than a glutamate residue(17) . Although sequence analysis reveals 38 trypsin cleavage sites in recombinant Atp11p, the small amount of trypsin used in the experiment allowed identification of the sites in the protein that are hypersensitive to proteolysis.


Figure 2: Western blot showing the pattern of Atp11p proteolytic products obtained in vitro. Five micrograms of purified Atp11p protein were incubated in 20 µl (final concentration 8 µM) with increasing concentrations of trypsin for 30 min, in 10 mM Tris-HCl, pH 7.5, at 37 °C. The reactions were stopped by the addition of SDS gel loading buffer, and the samples were run on a 12% SDS-polyacrylamide gel. The amount of trypsin added, and the resultant molar concentration of the protease, were as follows for the samples shown in lanes 1-7: 0, 0.5 ng (1 nM), 1.5 ng (3 nM), 5 ng (10 nM), 15 ng (30 nM), 50 ng (100 nM), and 150 ng (300 nM). Gel electrophoresis and Western blotting were performed as described under ``Experimental Procedures.'' After probing with anti-Atp11p, the blot was reacted with horseradish peroxidase-conjugated anti-rabbit antibody, and the bands were visualized using a colorimetric assay with 4-chloro-1-naphthol. The migration of molecular mass markers (given in kilodaltons) is shown in the right-hand margin of the figure.



The amino-terminal 10 amino acids were sequenced from four of the tryptic peptides to locate the positions of cleavage. At the lowest trypsin concentrations used, sequential cleavages occur first at Arg-75 and Lys-106, giving rise to the two largest digestion products seen in Fig. 2, lanes 2-4. As the amount of trypsin is increased, the amino terminus of Atp11p is further shortened by 10 amino acids by digestion at Lys-117 yielding a polypeptide that starts at Val-118 (Fig. 2, lane 6, lower band of the doublet). Commensurate with this, there is digestion at the carboxyl end of the protein to produce shorter tryptic peptides (Fig. 2, lanes 6 and 7) that retain Val-118 at the amino terminus. The smaller fragments are likely produced by sequential digestion at Lys-308 and Lys-299, which are the basic residues closest to the carboxyl terminus of Atp11p (see Fig. 1).

There is a significant amount of protein detected for the tryptic peptide that encompasses the sequence between Val-118 and Lys-299 when 8 µM recombinant Atp11p is exposed to 0.3 µM trypsin (lane 7, Fig. 2). This is noteworthy since there are 17 trypsin cleavage sites in this protein fragment. Since it may be expected that the active region of Atp11p forms a stable structure in the protein, the observed pattern of limited proteolysis suggests that the functional domain of Atp11p maps distal to Lys-117 and does not include the first 78 amino acids of the mature protein.

Characterization of Yeast Strains That Produce Atp11p with Amino-terminal Deletions

The consequence of removing amino-terminal sequences from Atp11p was examined directly by evaluating the respiratory properties of yeast that produce mutant forms of the protein whose sequences mimic the tryptic peptide fragments described above. Nested deletions were made in the ATP11 sequence to produce the Atp11(Delta40-75)p and Atp11(Delta40-111)p mutant proteins (Table 3). Following cleavage of the leader sequence in yeast, the mitochondrial form of these proteins would be deleted for 36 or 72 amino acids from the mature amino terminus (Table 3) and would begin at Glu-76 or Asp-112, respectively (see Fig. 1).

The atp11 disruption strain, W303DeltaATP11, was transformed with single copy plasmids that code for Atp11(Delta40-75)p and Atp11(Delta40-111)p, and the respiratory properties of the transformants were analyzed. Yeast cells that produce either protein were shown to grow on nonfermentable substrates (ethanol-glycerol media) with doubling times approximately 1.4 times that of yeast that produces full-length Atp11p from a single copy plasmid (Table 5). Mitochondria were prepared from yeast cells grown in ethanol-glycerol media and assayed for ATPase activity in the absence and presence of the inhibitor oligomycin (Table 5). Since oligomycin only inhibits the ATPase activity of F(1) that is properly assembled and bound to F(0), oligomycin-sensitive ATPase activity indicates the presence of Atp11p that is competent for F(1) assembly. The mitochondrial samples were also probed for Atp11p by Western analysis (Fig. 3) to determine the level of mutant protein relative to wild type control cells, and this information was used to quantitate the relative amounts of Atp11p activities in each of the strains (Table 5). The use of Western analysis to quantitate the intramitochondrial levels of Atp11p is based on the assumption that the distribution of epitopes recognized by the polyclonal Atp11p antibody is uniform along the length of the protein. This appears to be a valid assumption since Atp11p tryptic peptides, of decreasing size, gave comparable values for band intensities when the proteins were stained with either the antibody or Coomassie Blue.




Figure 3: Western blot of mitochondria prepared from yeast transformants that produce Atp11p proteins from plasmids. Mitochondria were prepared from W303DeltaATP11 transformants grown in liquid YEPG. These strains produce the Atp11p proteins indicated in the figure. Equivalent amounts of total mitochondrial protein (40 µg) were loaded in each lane of a 12% SDS-polyacrylamide gel. Gel electrophoresis, Western blotting, and immunodecoration with Atp11p were done as described in the legend to Fig. 2. The migration of molecular mass markers (given in kilodaltons) is shown in the right-hand margin of the figure.



The analysis of the mutant proteins deleted for residues from the mature amino terminus showed that Atp11(Delta40-111)p is fully active. This maps the location of the functional domain more than 70 amino acids from the amino terminus of the protein. Curiously, Atp11(Delta40-75)p only showed 44% the wild type level of activity despite the fact that fewer amino acids were removed from the amino terminus to produce this mutant protein relative to the deletion made in constructing Atp11(Delta40-111)p. This observation is analyzed under ``Discussion.''

Characterization of Yeast Strains That Produce Atp11p with Carboxyl-terminal Deletions

To map the carboxyl-terminal boundary of the functional domain, single and multicopy plasmids were constructed that carry atp11 inserts deleted for sequences from the 3` end of the gene (Table 3) and used to transform W303DeltaATP11. These plasmids encode the mutant proteins, Atp11(A300)p, Atp11(G253)p, Atp11(I203)p, and Atp11(R183)p, which are deleted, respectively, for 18, 65, 115, and 135 amino acids from the carboxyl terminus (Table 3). The last retained amino acid of these mutant proteins is indicated with an arrow in the sequence shown in Fig. 1.

The properties of yeast cells that produce Atp11p truncated at the carboxyl terminus were evaluated in a manner similar to that described above for the amino-terminal deletion strains. In contrast to Atp11(Delta40-75)p and Atp11(Delta40-111)p, none of the Atp11p proteins deleted for carboxyl sequences conferred respiratory competence to the mutant host strain when produced from a single copy plasmid. This was not unexpected in view of the respiratory-deficient phenotype of yeast that harbors the atp11 nonsense alleles in single copy. The analysis was repeated with strains that carry the 3`-deleted ATP11 genes on multicopy plasmids. Overproducing Atp11(G253)p did not rescue the respiratory-deficient phenotype of W303DeltaATP11, although Western analysis confirmed that the mutant protein was present in mitochondria (data not shown). However, the transformants that overproduce Atp11(A300)p, Atp11(I203)p, and Atp11(R183)p were shown to grow in ethanol-glycerol media, with rates that were 60-77% of the wild type rate (Table 5). The mitochondrial level of the mutant proteins produced from multicopy plasmids (Table 5) was determined from the Western blot shown in Fig. 3. Mitochondria from the strain that overproduces Atp11(A300)p routinely show a cross-reacting band that runs with an apparent higher molecular weight than what is expected for the mutant protein. This could represent precursor protein that has not been processed to the mature form. The activities for Atp11(A300)p, Atp11(I203)p, and Atp11(R183)p were extrapolated to be, respectively, 44%, 40%, and 27% of the level observed for the wild type protein (Table 5).

Additional studies that examined the physical properties of the mutant proteins provided an indication that the values reported for Atp11(I203)p and Atp11(R183)p activity may be underestimated. Native Atp11p is a component of the mitochondrial matrix that is almost quantitatively released to the soluble fraction when mitochondria are extracted with 0.1% sodium deoxycholate. The solubility properties of the mutant Atp11p proteins were evaluated in two separate mitochondrial samples prepared from each transformant. Atp11(Delta40-75)p, Atp11(Delta40-111)p, Atp11(A300)p, and Atp11(G253)p were readily visualized in Western blots of the soluble mitochondrial extracts (data not shown). However, it was necessary to overexpose the x-ray film to detect Atp11(I203)p and Atp11(R183)p in the blot. Furthermore, the protein detected in these two samples was largely degraded. These results suggest that Atp11(I203)p and Atp11(R183)p are, inherently, more labile relative to wild type Atp11p and to the other mutant proteins that were analyzed. Such lability could be indicative of improper protein folding. Thus, it is possible that the signals for Atp11(I203)p and Atp11(R183)p in Western blots of whole mitochondria (Fig. 3) could represent nonhomogenous populations in which only a portion of the protein is in the native, active conformation.

The ability of the carboxyl region of Atp11p to stabilize the functional domain in trans, and thus enhance the activity of Atp11p carboxyl-terminal deletion mutants, was explored by co-transforming W303DeltaATP11 with two plasmids; one plasmid coded for Atp11(I203)p and the other coded for Atp11(Delta40-218)p. Atp11(I203)p represents the first two-thirds of Atp11p and contains the active domain; Atp11(Delta40-218)p starts at Ser-219 and encompasses the final third of the protein (see Fig. 1). Western analysis showed that Atp11(Delta40-218)p is produced in stoichiometric amounts with Atp11(I203)p in yeast mitochondria (data not shown). Despite the presence of Atp11(Delta40-218)p, the respiratory properties observed for the double transformant were identical with those indicated in Table 5for W303DeltaATP11 transformed with only the Atp11(I203)p-producing plasmid. Additional experiments also showed that, when expressed alone in yeast, Atp11(Delta40-218)p does not confer respiratory competence to W303DeltaATP11. These studies indicate that the region between Ser-219 and Asn-318 does not have autonomous function, either in promoting F(1) assembly or in enhancing the activity of Atp11(I203)p when co-produced from a separate plasmid. The fact that the stability-enhancing function of the carboxyl region of Atp11p is only apparent in the full-length protein is consistent with our idea that the primary role of this sequence is to secure proper folding of the mature protein.

The data described thus far provide evidence that the active region of Atp11p is within the region of the sequence that is bordered by Asp-112 and Arg-183, which are, respectively, the mature amino- and carboxyl-terminal amino acids of Atp11(Delta40-111)p and Atp11(R183)p. Attempts were made to express the functional domain of Atp11p as an individual entity. However, plasmids constructed to produce solely this portion of the protein did not show evidence of the encoded product in mitochondria (data not shown). Apparently, the loss in protein stability that is observed when carboxyl-terminal sequences are removed (see above) is even more pronounced when the amino-terminal region of the mature protein is also deleted. These results suggest that both segments of Atp11p that flank the functional domain are important to maintain normal levels of Atp11p inside mitochondria.

Characteristics of the Primary Sequence of Atp11p

Fig. 4shows the hydropathy profile for Atp11p. A schematic drawing of the protein is shown above the plot that indicates 4 discrete domains, which include the amino-terminal mitochondrial targeting domain (Met-1 through Pro-39), and three domains in the mature portion of the protein that are each separated by proline-rich sequences. The most proximal domain of mature Atp11p (Glu-40 through Ser-109) consists of 70 amino acids. The hydrophilic character of this region is consistent with its sensitivity to proteolytic enzymes. The next section of the protein (Phe-120 through Asn-174) corresponds to the active region of Atp11p. This span of sequence is characterized by the presence of two hydrophobic segments in the distal portion of the domain. The second proline-rich segment adjoins the active region with the carboxyl domain of the protein (Arg-183 through Asn-318). The hydropathy profile suggests that this region is, overall, polar in character.


Figure 4: Hydropathy plot for Atp11p. The algorithm of Kyte and Doolittle (31) was used to obtain the hydropathy plot for Atp11p that is shown in the lower portion of the figure. The position of the two cysteine residues in the Atp11p sequence is shown by arrows. The open boxes in the diagram at the top of the figure represent the Atp11p amino-terminal targeting, proximal, active, and carboxyl domains. The residue number for the amino acids that comprise the amino- and carboxyl-terminal boundaries of the domains are indicated (residue numbering as per (15) ). The Pro-rich sequences that are proposed to form the linker regions between the proximal, active, and carboxyl domains are shown with single-letter code.



There are two cysteine residues in Atp11p that could potentially form a disulfide bridge linking the active and carboxyl domains of the protein. However, both sulfhydryl groups of the protein were titrated with the Ellman reagent(25) . Interestingly, when the chemical modification is performed under native conditions (10 mM Tris-HCl, pH 8.0), only 1 mol of cysteine per mol of Atp11p is detected. The second cysteine residue is titrated when the protein is denatured in 6 M guanidine HCl. The cysteine residue that is inaccessible to solvent in native Atp11p is most likely Cys-156, which is located in the hydrophobic stretch of the active domain and presumably buried in the native protein structure.


DISCUSSION

Sequence analysis of 12 independent isolates of yeast, harboring chemically induced mutations in the ATP11 gene, disclosed nine nonsense alleles and one mutant gene with a missense mutation that changes the initiating codon (Table 4). The nucleotide changes in these strains were of the type expected (G and C transitions) (Table 4) to result from mutagenesis with ethylmethanesulfonate and nitrosoguanidine(19) . However, the prevalence of nonsense mutations (9 out of 10 mutant alleles) was unusual since this is not a common feature of the pet mutant collection from which these strains were obtained(19) . A possible explanation for this is that amino acid replacements that may have been sustained during the mutagenesis did not impair the ability of Atp11p to assemble F(1) in an amount sufficient to support growth on the selective media used in the genetic screen. This observation has led us to postulate that Atp11p activity may not be dependent on specific, individual amino acids, but rather on the overall conformation of its functional domain. As a first step toward examining the mechanistic features of this domain, we have determined its boundaries in the protein.

The work described places the Atp11p functional domain between Phe-120 and Asn-174. Several lines of evidence support this assignment. The proteolytic mapping follows the rationale that hypersensitive protease digestion sites occur peripheral to stable domains in proteins. This concept has proven true in work with other proteins(32, 33) . Exposure of recombinant Atp11p to trypsin showed that the region between Val-118 and Lys-299 exists as a protease-resistant domain. Deletion studies have provided a more fine-tuned mapping of the active region. In accord with the proteolysis analysis, deleting the first 72 amino acids from the amino terminus of mature Atp11p produces a fully functional mutant protein (Atp11(Delta40-111)p), which begins at Asp-112. The active region is expected to end proximal to Arg-183, on the basis that the mutant protein Atp11(R183)p retains geq27% of the wild type level of Atp11p activity (Table 5). Finally, the assignment of Phe-120 and Asn-174 as the amino- and carboxyl-terminal amino acids of the functional domain is based on features of the Atp11p sequence. There are Pro-rich sequences immediately proximal to Phe-120 and distal to Asn-174. These sequences may serve as interdomain connecting regions.

Both the amino and carboxyl domains of Atp11p were found to be nonessential for Atp11p activity. However, several results indicate that these regions are important for extending the life of Atp11p inside mitochondria: 1) none of the chromosomally encoded mutant proteins, predicted to be deleted for geq114 amino acids from the carboxyl terminus, were detected by Western analysis; 2) it was necessary to produce the carboxyl-terminal Atp11p deletion proteins from multicopy plasmids in order to detect their activity; 3) the plasmid-borne Atp11(I203)p and Atp11p(R183)p, which are deleted for >115 amino acids from the carboxyl terminus, were found to be unusually labile following their solubilization from mitochondria; 4) Atp11p, deleted for both amino and carboxyl sequences, was not detected in strains that produce the double mutant from multicopy plasmids.

A curious finding was that shorter deletions from either the mature amino or the carboxyl termini of Atp11p were found to coincide with lower levels of Atp11p activity relative to mutant proteins in which longer deletions were made. For example, Atp11(Delta40-75)p showed only 44% the wild type level of activity, while Atp11(Delta40-111)p was shown to be fully functional. Another case was apparent with Atp11(G253)p, which is completely inactive despite the fact that it retains more carboxyl sequence relative to the active, mutant proteins, Atp11(I203)p and Atp11(R183)p. Without the availability of detailed structural information for Atp11p, it is difficult to explain these results. We offer the argument that ``less is better''; there may be structural interference imposed when part of the amino-terminal and carboxyl domains are removed and this is more detrimental to Atp11p function than removing the domains entirely.

As reviewed in the Introduction, previous studies support the idea that Atp11p binds transiently to F(1) alpha and beta subunits so that improper subunit interactions are avoided during assembly of the enzyme. This putative activity is similar to the ``capping'' mechanism that has been described for the periplasmic chaperone, PapD, which promotes proper assembly of pili in Gram-negative bacteria(34) . The region of Atp11p that was shown in the present work to be essential for F(1) assembly may overlap, in part or fully, with the binding site for the alpha and beta subunits.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant GM48157. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and requests for reprints should be addressed. Tel.: 313-577-8645; Fax: 313-577-7642; :sackerm{at}cms.cc.wayne.edu.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); wt, wild type.

(^2)
Plasmids coding for Atp11p deleted for sequences from the carboxyl terminus are described as follows. Atp11(A300)p is encoded by plasmids pG13/A300m and pG13/A300s. pG13/A300m was made by first digesting the 2µ vector YEp352 (21) with EcoRI, treating the linear fragment with mung bean nuclease, and digesting it with BamHI. The cut vector was then ligated to a 1.1-kb BamHI-StuI fragment prepared from pG13/ST11 (this plasmid carries the wild type ATP11 gene with 5`- and 3`-flanking sequences). To make pG13/A300s, pG13/A300m was first cut with AseI, blunt-ended with Klenow, then cut with BamHI to produce a 1.2-kb fragment that was ligated to the SmaI and BamHI sites of the CEN vector pRS316(23) . Atp11(G253)p is coded for by pG13/G253m and pG13/G253s. For these constructions, YEp352 and pRS316, respectively, were cut with XbaI, blunt-ended with Klenow, digested with BamHI, and ligated with a 0.9-kb BamHI-MscI fragment prepared from pG13/ST11. The plasmid/insert junctions were sequenced in pG13/A300m, pG13/A300s, pG13/G253m, and pG13/G253s to confirm that they encoded truncated forms of Atp11p. The polymerase chain reaction was used to create additional atp11 plasmids. Unless otherwise indicated, Taq polymerase was used for PCR. Atp11(I203)p is encoded by pG13/I203m. For this synthesis, the primers M13 1212 and 13 were used with the template, pG13/ST19, which carries the full-length atp11-6 allele (see Table 1). The resultant 885-bp PCR fragment was digested with BamHI and PstI and ligated to BamHI, PstI-cut YEp352. Atp11(R183)p is coded for in plasmid pG13/R183m. For its construction, plasmid pG13/ST11 was used as the template with the primers 12 and 14 to produce a 608-bp product that was digested with BamHI and ligated to the BamHI site of YEp352. Plasmids coding for Atp11p deleted for sequences from the mature amino terminus are described as follows. Atp11(Delta40-75)p is produced by pG13Delta40-75s. For this construction, PCR was first used to make the plasmid, pG13/EK1.2. The primers 5 and YEp351-1 were used with pG13/ST11 as template to produce a 1.2-kb DNA fragment that was then cut with EcoRI and KpnI and ligated to similarly cut pRS316. Next, a 309-bp fragment was synthesized by PCR using the primers 3 and 4 with pG13/ST11 (template), and the DNA was digested with SmaI and BamHI and finally ligated to pG13/EK1.2 cut with the same enzymes. This construction deletes ATP11 codons 40-75. There are 3 amino acids introduced in between the mitochondrial leader and mature portion of the Atp11p protein coded for by pG13Delta40-75s. The inclusion of these residues results from the addition of the sequence, GGGCTGCAG, which codes for Gly-Leu-Gln. Atp11(Delta40-111)p is coded for in plasmid pG13Delta40-111s. To make this plasmid, a 1.1-kb Sau3A-KpnI fragment was purified from pG13/BK1.5s (described below) and ligated with a 4.9-kb BamHI-KpnI fragment also prepared from pG13/BK1.5s. The resultant recombinant plasmid was opened at the BamHI site, blunt-ended with Klenow, cut with SstI, and ligated with a 340-bp SstI-SmaI fragment purified from plasmid pG13Delta40-75s to give pG13Delta40-111s. No amino acids are introduced at the junction between the mitochondrial leader and mature sequence of the Atp11p protein that is coded for by the pG13Delta40-111s plasmid. Atp11(Delta40-218)p is encoded by pG13Delta40-218s and pG13Delta40-218m. The former plasmid was constructed using Pfu DNA polymerase (Stratagene) for PCR with the primers 9 and YEp351-1 and plasmid pG13/ST11 as the template. The 700-bp product was digested with KpnI to give a fragment containing one blunt and one sticky end, which was ligated with a 5.2-kb fragment of pG13Delta40-75s that had been prepared with a blunt and a sticky end following EcoRI digestion, a Klenow reaction, and digestion with KpnI. The insert of pG13Delta40-218s was prepared as a blunt/sticky end fragment by digestion with KpnI, filling in with T(4) polymerase, and digestion with BamHI, and ligated to BamHI-SmaI-cut YEp351 (21) to give pG13Delta40-218m. There are 5 amino acids introduced in between the mitochondrial leader and mature portion of the Atp11p protein coded for by the pG13Delta40-218 plasmids. Their inclusion results from the addition of the sequence, GGGCTGCAGGAATTT, which codes for Gly-Leu-Gln-Glu-Phe. The wild type Atp11p control used is produced by pG13/BK1.5s. The first step to make this plasmid used pG13/ST11 as the PCR template with the primers 1 and 6. The 129-bp product was then digested with SmaI and EcoRI and ligated to the SmaI, EcoRI sites of plasmid pG13Delta40-75s. The insert in pG13/BK1.5s has the same sequence as wild type ATP11 with the exception of 3 nucleotide changes that arise from the insertion of cloning sites. The 5` primer used (Primer 1, Table 2) introduces a SmaI site that introduces a silent change in codon 39 (CCA CCC) and changes codon 40 from GAG to GGG. The replacement of Glu-40 with a glycine residue does not affect Atp11p activity(17) . The 3` primer used (No. 6, Table 2) introduces an EcoRI site, resulting in a silent codon change (TTT TTC) at Phe-77.


ACKNOWLEDGEMENTS

We gratefully acknowledge Michal Ram for sequencing the amino terminus of the Atp11p proteolytic fragments and Michael White for providing us with the purified recombinant Atp11p protein. We also wish to thank Domenico Gatti, Richard Needleman, Amy Roth, and Alexander Tzagoloff for their critical evaluation of the manuscript.


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