From the
The highly conserved motif of Saccharomyces cerevisiae H
The yeast plasma membrane H
The
functionally important Lys-474 is located within the sequence motif
KGAP, which is fully conserved among all eukaryotic cation-transporting
ATPases. Lys-474 corresponds to the amino acid modified in animal
ATPases by the active site inhibitor fluorescein isothiocyanate
(FITC)
To gain insight
into the role of Lys-474 in ATPase function and identify interacting
domains during the catalytic cycle, we have performed an intragenic
suppression analysis of the K474R mutation. Location of an
allele-specific suppressor mutation suggests direct interaction of the
KGAP motif with the phosphorylation domain. In addition, this reversion
analysis led to the identification of three allele-nonspecific
suppressors.
To express the mutant ATPases,
transformants were grown in galactose medium to a final absorbance of 3
at 660 nm, diluted 100-fold and grown for 24 h at 30 °C in glucose
medium.
The second-site mutations
were located within specific restriction fragments of the ATPase gene
by constructing chimeric genes containing portions of both the K474R
and revertant genes and by testing whether these constructs supported
growth on acid glucose medium. The chimeric genes were constructed as
described (Eraso and Portillo, 1994).
The DNA fragments conferring
on the K474R mutant the ability to grow on acid glucose medium were
sequenced completely by the dideoxy method (Sanger et al.,
1977) using 17-mer oligonucleotide primers starting at different
positions on the fragment.
All the mutant
constructions were verified by sequencing.
To ascertain whether ability to
grow on acid glucose medium was due to full reversion or to second-site
mutations, we took advantage of the destruction by the K474R mutation
of an MseI restriction endonuclease site present in the
wild-type allele. Plasmid DNA isolated from revertants and analyzed
showed that ten of 99 reversions were due to a second-site mutation in
the K474R mutant allele.
DNA sequence analysis of 20 full revertants
showed that, in 50% of these cases lysine restoration at position 474
occurred by a transition AGA to AAA specific of hydroxylamine (Freese,
1971), and in the remainder, Lys-474 restoration resulted from two
simultaneous nucleotide changes which restored the wild-type codon AAG.
A possible explanation for this alteration is the replacement of the
AGA codon by ectopic gene conversion between PMA1 and PMA2 (Harris
et al., 1993).
The position of the second-site mutations
was determined by constructing chimeric genes (see
``Materials and Methods''). After localizing each
mutation to a single restriction fragment, the changed base pair was
determined by DNA sequence analysis of the entire fragment. The
positions of the second-site mutations are shown in Fig. 1. Two
second-site mutations (A165V and V169I/D170N) were localized at the end
of the predicted membrane-spanning helix 2 in the stalk region
(MacLennann et al., 1985; Serrano, 1989; Wach et al.,
1992), one substitution (V396I) was found near the Asp-378 residue,
which is phosphorylated during catalysis (Serrano, 1991), three
mutations (V484I, V484I/E485K and E485K/E486K) were located 10 residues
away from the Lys-474 residue and another (P536L) was situated within
the proposed ATP binding domain (Portillo and Serrano, 1988; Taylor and
Green, 1989; Clarke et al., 1990; Capieaux et al.,
1993).
Before analyzing the effect of the new
mutations on the kinetic properties of the ATPase, ATPase activity
levels and FITC sensitivity of the K474R revertants, were determined.
Table II summarizes these characteristics of the enzyme from mutant
cells. The K474R mutant ATPase showed a low level of ATP hydrolysis and
high resistance to FITC inhibition as described (Portillo and Serrano,
1989). Although second-site mutations did not restore wild-type levels
of activity, they increased the ATPase activity relative to the K474R
mutant enzyme. Since K474R substitution affects neither the
enzyme's stability nor its targeting to the plasma membrane
(Portillo and Serrano, 1989), it seems reasonable to think that
second-site mutations improve the efficiency of the mutant enzymes. In
addition, all revertant enzymes exhibited an increased sensitivity to
FITC inhibition, suggesting alteration of the FITC binding site.
I shows that
the amount of mutant enzyme quantified by immunoassay in purified
plasma membranes was similar to that of the wild type, except for
A165V, V169I/D170N and P536L mutants, which exhibited increased plasma
membrane enzyme.
Mutant ATPase containing the K474R mutation showed
a very low V
All mutant enzymes containing only the second-site
mutations were found to increase the V
The FITC sensitivity of four of the mutant enzymes
was modified; the V484I, V484I/E485K, E485K/E486K, and P536L mutation
produced an enzyme more resistant than the wild type. This suggests
that amino acids Val-484, Glu-485, Glu-486, and Pro-536 could form part
of the FITC binding pocket or are able to alter this domain.
Vanadate is a P-ATPase inhibitor of which seems to mimic the
transition state for aspartyl-phosphate hydrolysis (Josephson and
Cantley, 1977). Mutations V484I, V484I/E485K, and E485K/E486K (together
with the previously described A165V and V169I/D170N) rendered an ATPase
with increased resistance to vanadate suggesting that amino acids
Ala-165, Val-169, Asp-170, Val-484, Glu-485, and Glu-486 contribute to
or alter the vanadate (P
Erythrosine B
inhibits the ATP hydrolysis catalyzed by the yeast
H
Mutations
at the Lys-474 residue greatly decrease the steady-state level of the
phosphorylated intermediate formed during the catalytic cycle of the
enzyme (Portillo and Serrano, 1988, 1989). We measured the level of
phosphorylated intermediate under steady-state conditions to determine
whether or not it was affected by the second-site mutations. All mutant
enzymes containing the second-site mutation showed phosphoenzyme levels
similar to those of the wild-type enzyme (data not shown).
The pH
dependence of the mutant enzymes is shown in Fig. 2. Two types of
pH profile were observed. Mutations A165V and P536L rendered an ATPase
with a pH optimum displaced to the acid range (Eraso and Portillo,
1993), while mutant enzymes bearing V484I, V484I/E485K, and E485K/E486K
mutations exhibited a pH profile with a pH optimum displaced to the
neutral range. The pH dependence of V169I/D170N and V396I was as in the
wild type.
In this study, revertant analysis of K474R mutation of the
yeast plasma membrane H
The role
of the second-site mutations on enzyme function was evaluated by
creating new pma1 alleles containing only the second-site
mutations and analyzing the kinetic properties of the resultant mutant
enzymes.
The kinetic properties of the V396I enzyme were not
significantly different from those of the wild-type enzyme; thus, the
Val-396 residue does not appear to be essential.
Mutant enzymes
bearing A165V, V169I/D170N, and P536L mutations exhibited kinetic
parameters similar to those described (Eraso and Portillo, 1994). All
of these mutant enzymes showed a V
The V484I,
V484I/E485K, and E485K/E486K mutations resulted in an ATPase with a
complex kinetic behavior. They exhibited a very low V
Suppression is defined as the ability to
support growth on acid glucose media of the RS72 yeast strain. +,
suppression; -, no suppression; ND, combination of mutations was
not done.
We thank Drs. H. Celis, P. Eraso, M. J. Mazón,
and R. Serrano for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-ATPase
KGAP has been proposed to
participate in the formation of the phosphorylated intermediate during
the catalytic cycle (Portillo, F., and Serrano, R. (1988) EMBO J. 7, 1793-1798). In addition, Lys-474 is the FITC binding site
of the yeast enzyme (Portillo, F. and Serrano, R. (1989) Eur. J.
Biochem. 186, 501-507). We have performed an intragenic
suppressor analysis of the K474R mutation to identify the interacting
regions involved in these functions. Random in vitro mutagenesis of the K474R allele resulted in seven suppressor
(second-site) mutations. One mutation (V396I), located 18 residues away
from the Asp-378 residue, which is phosphorylated during catalysis, is
allele-specific. This provides genetic evidence of a direct interaction
between the KGAP motif and the phosphorylation domain during the
catalytic cycle. Three mutations (V484I, V484I/E485K, and E485K/E486K)
are located near Lys-474 and may compense the structural alteration
introduced by the K474R mutation. Two substitutions at the end of the
predicted transmembrane stretch 2 (A165V and V169I/D170N) and another
in the predicted ATP binding domain (P536L) may act as
allele-nonspecific suppressors, as they are also able to suppress a
mutation at the enzyme's carboxyl terminus.
-ATPase belongs to
the family of ion-translocating ATPases characterized by the formation
of a phosphorylated intermediate during the catalytic cycle (Pedersen
and Carafoli, 1987). The proton gradient generated by this ATPase plays
an essential role in active nutrient uptake and intracellular pH
regulation (reviewed by Goffeau and Slayman, 1981 and Serrano, 1985).
Comparison of the amino acid sequence of yeast ATPase with other
cation-transporting ATPases showed that they share several regions of
homology and a common topology (Serrano, 1989; Goffeau and Green, 1990;
Wach et al., 1992). DNA sequence analysis and either random or
directed mutagenesis have been combined to probe structure-function
relationships of this enzyme. These studies have identified several
residues essential for catalysis and regulation of the enzyme (reviewed
by Gaber (1992)) and have led to the proposal of a hypothetical model
relating structure and function in yeast ATPase (Serrano, 1991).
(
)
(Farley et al., 1984; Farley
and Faller, 1985; Filoteo et al., 1987; Mitchinson and Green,
1982; Pardo and Slayman, 1988). Substitution of Arg by Lys at position
474 of the yeast ATPase greatly reduces catalytic activity and FITC
binding (Portillo and Serrano, 1989). Furthermore, mutagenesis of
Lys-474 in this ATPase greatly decreases the steady-state level of the
phosphorylated intermediate of the enzyme, but no change in nucleotide
specificity is observed (Portillo and Serrano, 1988; 1989), indicating
that this residue is not directly involved in ATP binding.
Nevertheless, Lys-474 must lie close to the ATP binding site, since
FITC labeling of this residue inhibits ATP binding.
Yeast Strain and Cell Cultures
Mutant ATPases
were expressed from centromeric plasmids in Saccharomyces
cerevisiae strain RS72 (Cid et al., 1987), a derivative
of BWG1-7A (Guarente et al., 1982) in which the
chromosomal ATPase gene promoter (PMA1) has been replaced by the
galactokinase gene promoter (GAL1). Transformants were grown in medium
with 2% glucose or 2% galactose, 0.7% Yeast Nitrogen Base without amino
acids (Difco), 40 µg/ml adenine and 30 µg/ml histidine. When
indicated, the glucose medium was buffered with 50 m
M succinic
acid, adjusted to pH 3.0 with Tris (acid glucose medium). Solid media
contained 2% agar (Difco).
Mutagenesis and Selection of Revertants
Plasmid
pSB32 (Rose and Broach, 1991) carrying a 5-kb HindIII fragment
containing the yeast ATPase K474R mutant gene (Portillo and Serrano,
1989) was mutagenized with hydroxylamine as described (Serrano et
al., 1986b). Yeast cells were transformed with the
hydroxylamine-treated plasmid using the lithium acetate procedure (Ito
et al., 1983). From 50 µg of mutagenized plasmid, about
16,000 transformants were obtained on galactose plates. Revertants were
selected by replica plating onto acid glucose medium. For each
revertant, plasmid DNA was rescued, amplified into Escherichia coli (Rose et al., 1990) and used to test the revertant
phenotype as above.
Mapping and Sequencing of Revertant Mutants
The
K474R mutation destroys an MseI restriction endonuclease
recognition site. To determine if the reversion occurred at the site of
the original mutation, a 0.49-kb DNA fragment was amplified by the
polymerase chain reaction using plasmid DNA rescued from revertants as
template and subjected to restriction analysis with MseI. The
DNA fragments digested by MseI would indicate that, in the
plasmid DNA rescued from the revertants, the original Lys-474 has been
restored, while a DNA fragment not digested by MseI would
result from a rescued plasmid carrying a second mutation in the ATPase
gene, able to suppress the K474R mutation.
Construction of Mutant ATPase Genes Containing Only the
Second-site Mutation in the Absence of the K474R Mutation
These
genes were constructed in two ways. (i) The fragments containing the
second-site mutation were exchanged with the corresponding fragment in
the wild-type gene. This was the case for A165V, V169I/D170N, V396I
(located on a 0.8-kb EcoRI fragment), and P536L (located on a
2.2-kb XbaI fragment) mutations. (ii) The second-site mutant
genes carrying V484I, V484I/E485K and E485K/E486K mutations were
constructed by site-directed mutagenesis.
Construction of pma1 Alleles Used for Suppression
Analysis
The pma1 alleles in were
constructed as follows. ( a) Mutant ATPase genes containing the
K474R second-site reversions and K474H or K474Q mutations were
generated in two ways: (i) by exchange of the fragments containing the
second-site reversions with the corresponding fragment in the K474H and
K474Q genes, which was the case for A165V, V169I/D170N, V396I, and
P536L; (ii) in the case of V484I, V484I/E485K, and E485K/E486K,
construction of the pma1 alleles by site-directed mutagenesis.
( b) Mutant ATPase genes containing the K474R mutation and
S911A/T912A revertant mutations were created by exchange of either a
2.2-kb XbaI fragment (A565T, G587N, G648S, P669L, and G670S)
or a 0.8-kb EcoRI fragment (A350T and A351T) with the
corresponding fragment in the K474R mutant gene. ( c) Mutant
ATPase genes containing the S911A/T912A mutation and second-site
reversions of the K474R mutation were obtained by exchange of a 2.1-kb
KpnI fragment containing only the second-site mutation with
the corresponding fragment in the S911A/T912A gene.
Site-directed Mutagenesis
A 5.0-kb
HindIII fragment containing either the wild type or K474H and
K474Q ATPase gene were inserted into pSB32 and after XbaI
digestion were religated, resulting in the deletion of the 2.2-kb
XbaI fragment. The excised fragments were subcloned into
M13mp19 and subjected to mutagenesis by the method of Eckstein (Taylor
et al., 1985) with the Mutagenesis System of Amersham
(Amersham Corp.). After mutagenesis, the entire XbaI fragments
were sequenced to confirm that only the sequence changes introduced by
the mutagenic oligonucleotides were obtained. The 2.2-kb XbaI
fragments containing the mutation were liberated from the replicative
form of M13mp19 and inserted into the deleted ATPase genes described
above.
Biochemical Methods
Yeast plasma membranes were
purified from glucose-metabolizing cells by differential and sucrose
gradient centrifugation (Serrano, 1983). ATPase activity was assayed as
described (Serrano, 1988). Immunoquantification of the ATPase in plasma
membrane was performed as described (Eraso and Serrano, 1987). Rabbit
polyclonal antibodies against yeast ATPase (Serrano et al.,
1986a) were affinity purified (Monk et al., 1991). Formation
of the phosphorylated intermediate was performed as described (Vara and
Serrano, 1983) and electrophoresis of phosphoenzyme was as in Niggli
et al. (1979). Protein concentration was measured by the
Bradford method (1976) using the Bio-Rad protein assay reagent and
bovine IgG as standard.
Isolation and Sequence Analysis of K474R Mutant
Revertants
Revertants of the K474R mutant were obtained using
essentially the methodology described (Eraso and Portillo, 1994).
Briefly, the yeast strain RS72 has the genomic copy of the ATPase gene
(PMA1) under the control of a galactose-dependent promoter (Cid et
al., 1987). This strain can grow in glucose only if it is
transformed with a plasmid carrying PMA1. The ATPase with the K474R
mutation supports growth in glucose only if the medium is buffered at
neutral pH, while growth in acid glucose medium is abolished because of
the low activity level of the mutant enzyme (Portillo and Serrano,
1989). The plasmid carrying the K474R mutant allele was mutagenized
with hydroxylamine and introduced into the RS72 strain. Transformants
were grown on galactose medium and tested for growth on acid glucose
medium. Those transformants able to grow in this medium will be full
revertants or have a second mutation in the ATPase gene able to
suppress the K474R mutation. Of 16,000 transformants obtained, 99 were
able to grow on acid glucose medium.
Genetic and Phenotypic Analysis of Revertants
The
A165V, V169I/D170N, and P536L mutations were previously isolated as
suppressors of a mutation (S911A/T912A) which affects activation of the
enzyme by glucose (Eraso and Portillo, 1994). This prompted us to
determine the suppression spectrum of both S911A/T912A and K474R
revertants. We also tested whether the K474R revertants isolated were
specific for the K474R mutation or could suppress other amino acids
substitutions at this position. For this, we created novel combinations
of primary and second-site mutations in cis (see
``Materials and Methods''). The pattern of
intragenic suppression obtained is shown in Table I. Only A165V,
V169I/D170N and P536L reversions could suppress both the S911A/T912A
and K474R mutations. The second-site mutation V396I specifically
suppresses the K474R mutation. Three second-site changes (V484I,
V484I/E485K and E485K/E486K) suppress the substitution of
positive-charged amino acids for the Lys-474 residue. Only second-site
mutation P536L was able to suppress any of the amino acid substitutions
introduced at position 474.
Generation and Biochemical Characterization of Mutant
ATPases Containing Only the Second-site Mutations
To further
characterize the role of the second-site mutations in enzyme function,
we created new pma1 alleles containing only the second-site
mutation in the absence of the K474R substitution (see
``Material and Methods''). The effect of the
second-site mutation on the ATPase function was evaluated by analyzing
the kinetic parameters of the mutant enzymes. Table III and Fig. 2
summarize the behavior of these mutants.
and high resistance to FITC
inhibition. Other kinetic properties assayed appeared to be essentially
wild type.
relative
to the K474R enzyme. In three cases (A165V, V169I/D170N, and P536L),
the V
was even higher than that of the wild type
as described (Eraso and Portillo, 1994). Three other mutant enzymes
(V484I, V484I/E485K, and E485K/E486K) exhibited an altered
K
for ATP. The nucleotide binding
specificity of all mutant enzymes was essentially as in the wild type
(data not shown).
) binding site.
-ATPase by binding to the enzyme's ATP binding
site (Wach and Gräber, 1991). Enzymes bearing V484I, V484I/E485K,
E485K/E486K, and P536L mutations were more resistant to erythrosine B
inhibition than the wild-type, suggesting that these mutations disturb
the ATP binding domain structure. It is interesting to note that these
mutant enzymes were also resistant to inhibition by FITC.
Figure 2:
Effect of pH on the ATPase activity of
wild-type and mutant enzymes. ATPase activity was assayed in purified
plasma membrane from glucose-metabolizing yeast cells with 2 m
M ATP at the indicated pH. ATPase activity is expressed as a
percentage of the activity at pH 6.5. Upper panel, -
- -, wild type; , A165V;
, K474R;
, P536L. Lower panel, - - -, wild type;
, V484I;
,
V484I/E485K;
, E485K/E486K.
-ATPase has been used to
identify interacting domains within the protein. The Lys-474 residue is
located within the KGAP sequence which is fully conserved among all the
eukaryotic P-ATPases. Mutation at this amino acid was chosen for
revertant analysis as previous studies with the yeast ATPase suggested
that it was involved in the phosphorylated intermediate formation
(Portillo and Serrano, 1988, 1989). Using this genetic approach, we
have isolated revertants located within three functional domains: (i)
the stalk region, (ii) the phosphorylation domain, and (iii) the ATP
binding domain. This suggests the functional coupling between the KGAP
motif and these regions; nevertheless, if there are direct interactions
between these regions, then the suppressor mutations might be expected
to be allele-specific. The fact that only the V396I revertant
specifically suppresses the K474R mutation suggests that only Val-396
and Lys-474 residues may interact directly. The location of V484I,
V484I/E485K, and E485K/E486K near Lys-474 and their pattern of
intragenic suppression support the idea that the suppressor effect of
these mutations is probably due to compensatory structural alterations.
The A165V, V169I/D170N, and P536L mutations were isolated previously as
suppressors of a mutation (S911A/T912A) located in the regulatory
domain. The fact that no other S911A/T912A revertants were able to
suppress the K474R mutation suggests suppression specificity. Although
no conclusions can yet be drawn about the mechanism of this
allele-nonspecific suppression, it can be argued that interaction
between Lys-474 and these regions, if it occurs, is probably indirect
and mediated by the conformational coupling between the stalk sector
and either the ATP binding or the phosphorylation domains described
(Harris et al., 1991; Na et al., 1993; Perlin et
al., 1992). An alternative possibility is that the suppression
patterns of A165V, V169I/D170N, and P536L result from an effect of the
mutations on protein stability. Global intragenic suppressors which
appear to affect protein stability have been isolated in other systems
(Shortle and Lin, 1985; Thomas et al., 1991). Consistent with
this idea, A165V, V169I/D170N, and P536L mutants exhibited
significantly increased amounts of plasma membrane ATPase.
higher than
that of the wild-type enzyme. The higher V
correlated with an increased amount of plasma membrane enzyme;
the reason for this increase remains to be elucidated. In addition, the
P536L mutation led to an enzyme resistant to FITC and erythrosine B
inhibition, suggesting that this mutation directly or indirectly
affects the binding sites of both inhibitors.
relative to wild-type enzyme, as would be expected if this
mutation caused a structural alteration in the KGAP motif (see above).
In addition, these mutant enzymes show a K
for ATP lower than that of the wild type. The fact that the
sensitivity of the mutant enzymes to ATP binding inhibitors was also
altered reinforces the notion that these mutations affect ATP binding.
Interestingly, V484I, V484I/E485K, and E485K/E486K second-site
mutations render an ATPase with a pH optimum displaced to the neutral
range, suggesting that residues implicated in these amino acid
substitutions may play an essential role, direct or indirect, in the pH
dependence of the ATP binding and/or hydrolysis rates. It has been
shown that the equilibrium between the E
and
E
conformations of the cation-transporting ATPases
is influenced by the pH (Pick and Karlish, 1982; Blanpain et
al., 1992). Alkaline pH stabilizes the E
conformation, which is the high ATP affinity and low vanadate
affinity conformation (Goffeau and Slayman, 1981; Serrano, 1985).
Considering these findings, it is reasonable to think that V484I,
V484I/E485K, and E485K/E486K mutations could mimic the pH effect and
displace the conformational equilibrium toward the E
conformation, thus accounting for the higher ATP affinity
observed (I). Future research involving intrinsic
fluorescence studies should confirm the alteration of the steady-state
distribution of E
and E
catalytic intermediates in the mutant enzymes.
Table: Suppression spectrum of K474R and
S911A/T912A revertants
Table: ATP hydrolysis activity and FITC
sensitivity of enzymes from intragenic suppressors of the K474R mutant
Table: Kinetic properties of mutant ATPases
containing only the second-site mutations
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.