(Received for publication, October 13, 1995; and in revised form, December 5, 1995)
From the
The Atp11p protein of Saccharomyces cerevisiae is
required for proper assembly of the F 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.
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,
and a peripherally bound moiety, F
. When bound to the
membrane sector, F
catalyzes ATP synthesis/ATP hydrolysis
coupled to proton translocation through the F
; purified
F
(F
-ATPase) functions solely as an ATP
hydrolase.
F contains five different types of subunits,
arranged in the stoichiometric ratio
(1, 2) .
The crystal structure of the bovine F
has been solved at
2.8 Å and shows alternating
and
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
since they have been shown to
be important for binding F
to
F
(4, 5) . An interesting feature of
F
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
catalytic mechanism makes this enzyme
a particularly interesting system in which to study macromolecular
assembly.
The formation of the F oligomer requires
proteins that are not part of the enzyme structure. All of the yeast
F
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
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 assembly is readily detected in atp11 mutants,
whose mitochondria show the accumulation of the F
and
subunits in large protein aggregates(15) . Atp11p action
in F
-ATPase assembly is proposed to occur downstream from
the mitochondrial foldase Hsp60(12, 13) , since there
is no evidence of unprocessed
or
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
subunit (12) .
Atp11p has been identified in complexes with the and
subunits of the enzyme(15) , which suggests that the larger of
the F
proteins interact directly with the assembly factor.
It is possible that Atp11p acts during the initial stages of F
assembly to secure the formation of a stable subcomplex of
and
subunits that, in turn, assembles with the other F
subunits. Evidence for this comes from the analysis of yeast
strains with null mutations in the structural F
genes.
Yeast, which produce only the
or only the
subunit, harbor
the lone subunit in an aggregated form(14) . In contrast, there
is no evidence of
and/or
aggregates in mitochondria
prepared from
(10) ,
(9) , or
(8) null mutants, despite the fact that the absence of any of
these subunits blocks F
assembly. In fact, in
subunit-deficient cells, the
and
subunits show
sedimentation behavior in linear sucrose gradients that suggests the
proteins exist as dimers(10) . Cumulatively, these studies
suggest that
/
subcomplexes are stable once formed. Since the
and
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 (
)
and (
)
complexes. The aggregation
phenotype observed in the
and
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
(17) . Thus, the limited amount of Atp11p present
in mitochondria is probably not sufficient to bind and maintain in
solution
or
protein that accumulates when the partner
subunit is missing.
Here we describe studies that identified the
minimal region of Atp11p capable of promoting F assembly.
This functional domain may represent the region of Atp11p that
interacts with the
and
subunits of F
.
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 40-75,
40-111, and
40-218 Atp11p proteins, and the last
retained amino acid in the R183, I203, G253, and A300 Atp11p proteins.
Since the construction of the Atp11(
40-75)p and
Atp11(
40-218)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.
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.
The atp11 disruption strain, W303ATP11, was transformed with single
copy plasmids that code for Atp11(
40-75)p and
Atp11(
40-111)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
that is properly
assembled and bound to F
, oligomycin-sensitive ATPase
activity indicates the presence of Atp11p that is competent for F
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 W303ATP11 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(40-111)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(
40-75)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(
40-111)p. This observation is analyzed under
``Discussion.''
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(40-75)p and
Atp11(
40-111)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 W303
ATP11, 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(40-75)p,
Atp11(
40-111)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
W303ATP11 with two plasmids; one plasmid coded for Atp11(I203)p
and the other coded for Atp11(
40-218)p. Atp11(I203)p
represents the first two-thirds of Atp11p and contains the active
domain; Atp11(
40-218)p starts at Ser-219 and encompasses the
final third of the protein (see Fig. 1). Western analysis showed
that Atp11(
40-218)p is produced in
stoichiometric
amounts with Atp11(I203)p in yeast mitochondria (data not shown).
Despite the presence of Atp11(
40-218)p, the respiratory
properties observed for the double transformant were identical with
those indicated in Table 5for W303
ATP11 transformed with
only the Atp11(I203)p-producing plasmid. Additional experiments also
showed that, when expressed alone in yeast, Atp11(
40-218)p
does not confer respiratory competence to W303
ATP11. These studies
indicate that the region between Ser-219 and Asn-318 does not have
autonomous function, either in promoting F
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(40-111)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.
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.
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 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(40-111)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
27% 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 114 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(40-75)p showed only
44% the wild type level of activity, while Atp11(
40-111)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
and
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
assembly may overlap, in
part or fully, with the binding site for the
and
subunits.