(Received for publication, September 18, 1995; and in revised form, October 20, 1995)
From the
The B subunit of the vacuolar (H)-ATPase
(V-ATPase) has previously been shown to participate in nucleotide
binding and to possess significant sequence homology with the
subunit of the mitochondrial F-ATPase, which forms the major portion of
the noncatalytic nucleotide binding sites and contributes several
residues to the catalytic sites of this complex. Based upon the recent
x-ray structure of the mitochondrial F
ATPase (Abrahams, J.
P., Leslie, A. G., Lutter, R., and Walker, J. E.(1994) Nature 370, 621-628), site-directed mutagenesis of the yeast VMA2 gene has been carried out in a strain containing a
deletion of this gene. VMA2 encodes the yeast V-ATPase B
subunit (Vma2p). Mutations at two residues postulated to be contributed
by Vma2p to the catalytic site (R381S and Y352S) resulted in a complete
loss of ATPase activity and proton transport, with the former having a
partial effect on V-ATPase assembly. Interestingly, substitution of Phe
for Tyr-352 had only minor effects on activity (15-30%
inhibition), suggesting the requirement for an aromatic ring at this
position. Alteration of Tyr-370, which is postulated to be near the
adenine binding pocket at the noncatalytic sites, to Arg, Phe, or Ser
caused a 30-50% inhibition of proton transport and ATPase
activity, suggesting that an aromatic ring is not essential at this
position. Finally, mutagenesis of residues in the region corresponding
to the P-loop of the
subunit (H180K, H180G, H180D, N181V) also
inhibited proton transport and ATPase activity by approximately
30-50%. None of the mutations in either the putative adenine
binding pocket nor the P-loop region had any effect on the ability of
Vma2p to correctly fold nor on the V-ATPase to correctly assemble. The
significance of these results for the structure and function of the
nucleotide binding sites on the B subunit is discussed.
Vacuolar acidification plays an important role in a number of
cellular processes, including receptor-mediated endocytosis,
intracellular targeting of lysosomal enzymes, macromolecular
processing, and degradation and the coupled transport of small
molecules (for review, see Forgac(1989)). Vacuolar acidification is
carried out by the vacuolar family of (H)-ATPases (or
V-ATPases), (
)which have been purified from a variety of
sources, including clathrin-coated vesicles (Arai et al.,
1987), chromaffin granules (Moriyama and Nelson, 1987), renal
microsomes (Gluck and Caldwell, 1987), the central vacuoles of yeast
(Uchida et al., 1985; Kane et al., 1989), Neurospora (Bowman et al., 1989), and plants (Parry et al., 1989; Ward and Sze, 1991), and the apical membranes of
insect midgut cells (Schweikl et al., 1989).
The V-ATPase
complex from clathrin-coated vesicles, like other members of this
class, is composed of two functional domains (for review, see
Forgac(1992)). The V domain, which has the structure
A
B
40
34
33
(Arai et al. 1988), is a 500-kDa peripheral complex that
is responsible for ATP hydrolysis, with both the 73-kDa A subunit and
58-kDa B subunit participating in nucleotide binding. The integral V
domain, which has the structure
100
38
19
c
(Zhang et
al., 1992), is a 250-kDa complex responsible for proton
translocation. The structure of the yeast V-ATPase (Kane et
al., 1989) is very similar to that of the bovine coated vesicle
enzyme, with the exception that several subunits of the yeast V-ATPase,
including the 54-kDa product of the VMA13 gene (Ho et al. 1993), the 14-kDa product of the VMA7 gene (Graham et
al., 1994; Nelson et al., 1994), and the 13-kDa product
of the VMA10 gene (Supekova et al., 1995) have not
yet been identified in the mammalian enzyme.
Both structural
analyses (Arai et al., 1988; Adachi et al., 1990a,
1990b) and sequence homology (Zimniak et al., 1988; Bowman et al., 1988a, 1988b; Manolson et al., 1988; Mandel et al., 1988; Hirata et al., 1990; Puopolo et
al., 1991, 1992) indicate an evolutionary relationship between the
V-ATPases and the F-ATPases of mitochondria, chloroplasts, and bacteria
(Senior, 1990; Penefsky and Cross, 1991; Pedersen and Amzel, 1993).
Thus the A and B subunits of the V-ATPases are homologous to the
and
subunits of the F-ATPases, respectively, indicating that
these proteins are derived from an ancestral nucleotide binding
protein. The recent x-ray crystal structure of the F
domain
of the mitochondrial F-ATPase (Abrahams et al., 1994) shows
the nucleotide binding sites located near the interfaces of the
and
subunits, with the catalytic sites located principally on the
subunits and the noncatalytic sites located principally on the
subunits, in agreement with extensive mutagenesis and chemical
modification data (Senior, 1990; Penefsky and Cross, 1991; Futai et
al., 1994).
Several lines of evidence suggest that the
catalytic sites of the V-ATPase are located on the A subunit. First,
ATP protectable labeling of this subunit is observed using the
inhibitors N-ethylmaleimide and
7-chloro-4-nitrobenz-2-oxa-1,3-diazole (see Forgac et
al.(1989) for references), and modification of Cys-254 of the A
subunit with sulfhydryl reagents has been shown to lead to inactivation
(Feng and Forgac, 1992). Second, labeling of the A subunit by
2-azido-[P]ATP correlates well with inhibition
of ATPase activity, with complete inhibition observed upon modification
of a single A subunit per complex (Zhang et al., 1995).
Finally, the A subunit possesses all of the consensus sequences,
including the glycine-rich loop region, which appear critical for ATP
hydrolysis by the F-ATPase
subunit (Zimniak et al.,
1988; Bowman et al., 1988b; Hirata et al., 1990;
Puopolo et al., 1991).
The V-ATPase B subunit has also been
shown to participate in nucleotide binding. Thus, the B subunit is
modified by the photoaffinity analog
3-O-(4-benzoyl)benzoyl-ATP (Manolson et al., 1985)
and by 2-azido-[P]ATP (Zhang et al.,
1995). Interestingly, modification of the B subunit by
2-azido-[
P]ATP occurs only at rapidly
exchangeable sites, under which conditions the A subunit is also
modified. These results suggest that, as with the F-ATPases, the
catalytic sites are also at the interface between the A and B subunits.
Unlike the
,
and A subunits, the B subunit lacks the
glycine-rich loop consensus sequence which, from the crystal structure
of F
, appears to be in close proximity to the triphosphates
of ATP (Abrahams et al., 1994). The importance of this
consensus sequence for ATP hydrolysis (Senior, 1990; Penefsky and
Cross, 1991; Futai et al., 1994) supports the idea that the
nucleotide binding site on the B subunit is noncatalytic.
To determine the effect of modifying specific residues in the B subunit on activity of the V-ATPase, we have carried out site-directed mutagenesis of the yeast VMA2 gene in a strain lacking this gene. The VMA2 gene encodes the yeast V-ATPase B subunit (Vma2p). Our results reveal that changes in residues predicted to be contributed by the B subunit to the catalytic nucleotide binding sites dramatically decrease activity, whereas those predicted to affect the noncatalytic site on the B subunit itself have significant but less marked effects on activity.
Yeast VMA2 deleted strain,
SF838-5AV1(vat2-1::LEU2), and pCY41 (VMA2 in
pBluescript) were as described in Yamashiro et al.(1990) and
were a kind gift from Dr. Tom Stevens, University of Oregon.
Mutagenesis was conducted following the manufacturer's protocol, and was confirmed by DNA sequencing using the method of Sanger et al.(1977). All mutants as well as wild-type VMA2 were subcloned in the shuttle vector pRS316 through KpnI and SacI sites. No other mutations were detected in the final product.
Transformation
of yeast and selection of transformants on Ura plates
was carried out as described previously (Ausubel et al.,
1992). The mutants were then tested for growth on pH 7.5 or pH 5.5 YPD
plates buffered with 50 mM KH
PO
, 50
mM succinic acid (Yamashiro et al., 1990).
Deletion of genes encoding the yeast V-ATPase subunits
results in a conditional lethal phenotype (Nelson and Nelson, 1990).
Such strains are unable to grow at neutral pH but are able to grow at
acidic pH. As demonstrated previously (Nelson and Nelson, 1990), we
observe that a strain lacking functional Vma2p is able to grow at pH
5.5 but grows very poorly relative to the wild-type strain at pH 7.5.
Of the Vma2p mutations tested, only R381S and Y352S showed defective
growth at neutral pH, whereas the remaining mutations grew normally
under these conditions (results not shown). It has previously been
observed that strains containing as little as 20% of the wild-type
V-ATPase activity show relatively normal growth at pH 7.5. ()It is thus necessary to directly test the activity of the
V-ATPase in the mutant strains to determine whether these mutations
caused loss of less than 80% of the wild-type activity.
Fig. 1A shows ATP-dependent proton transport activity in
isolated vacuoles from the mutant and wild-type strains, while Fig. 1B shows bafilomycin-sensitive ATPase activity in isolated
vacuoles. As can be seen, both R381S and Y352S completely eliminated
proton transport activity and reduced V-ATPase activity in the vacuole
by greater than 90%. This is consistent with the growth phenotype
observed for these mutants described above. Both of these residues
correspond to subunit residues contributed to the catalytic site
of the F-ATPases (Abrahams et al., 1994). Interestingly,
substitution of Phe for Tyr-352 had a relatively small effect on
activity (15-20%), suggesting that the aromatic ring rather than
the hydroxyl group is important at this position.
Figure 1: Effect of Vma2p mutations on bafilomycin-sensitive ATP-dependent proton pumping and ATPase activity in purified vacuoles. Proton pumping activity was measured in aliquots of isolated yeast vacuoles containing 1 µg of protein (a), and ATPase activity was tested in aliquots of EDTA-washed vacuoles containing 2.5 µg protein (b) as described under ``Experimental Procedures.'' Proton transport and ATPase activities are expressed relative to the wild-type control. The specific activity of the V-ATPase in EDTA-washed vacuoles from the strain expressing the wild-type Vma2p on a plasmid was 2.6 µmol ATP/min/mg of protein at 30 °C. This value is in good agreement with the values reported for the specific activity of the V-ATPase in washed wild-type vacuoles (1.3-3.3 µmol ATP/min/mg of protein) (Uchida et al., 1985; Kane et al., 1989). As a negative control, the V-ATPase activity on washed vacuoles from the vma2-deleted strain was 0.05 µmol ATP/min/mg of protein. No proton transport was observed in vacuoles isolated from the deleted strain. Each bar represents the average of three (a) or five (b) determinations from two different vacuolar preparations, with the error shown corresponding to the average deviation from the mean.
By contrast,
mutations at other positions had significant but much less dramatic
effects on proton transport and V-ATPase activity. Thus, mutation of
Tyr-370, to Arg, Phe, or Ser generally inhibited activity by
40-50%, although the Y370F mutation showed only a 20% inhibition
of ATPase activity. Tyr-370 corresponds to an Arg residue located in
the adenine binding pocket of the noncatalytic nucleotide binding site
of F (Abrahams et al., 1994). Similarly, mutation
of residues that correspond to the glycine-rich loop region of the
F-ATPase
subunit, including H180K, H180G, H180D, and N181V,
inhibited ATPase and proton transport activity by 30-50%,
although the H180D mutation again gave only a 20% inhibition of ATP
hydrolysis. It is possible in the cases where proton transport is
inhibited to a greater extent than ATPase activity (as with Y370F and
H180D) that the mutations are causing some uncoupling of proton
transport from ATP hydrolysis.
Because mutations in the homologous
subunit of F
have in some cases been shown to effect
assembly and/or stability of the F-ATPase complex (Maggio et
al., 1987; Soga et al., 1989; Weber et al.,
1993; Jounouchi et al., 1993), it is necessary to determine
the effect of each of the Vma2p mutations constructed on the assembly
of the yeast V-ATPase. Vacuoles were isolated from each strain, and the
V-ATPase was solubilized with detergent and isolated by density
gradient sedimentation as described under ``Experimental
Procedures.'' Fig. 2shows the SDS-PAGE profile for the
fractions containing the peak of V-ATPase activity, and Fig. 3shows the V-ATPase activity in the peak fraction. As can
be seen, nearly normal assembly of the V-ATPase was observed in all of
the mutants with the exception of R381S, where a partial loss of
assembly is observed. In addition, for most of the mutations, there is
generally good agreement between the level of V-ATPase activity
observed for the isolated enzyme and the level of bafilomycin-sensitive
ATPae activity in the native vacuole, with the following exceptions.
For the Y370F mutation, only 53% of control activity was observed for
the isolated V-ATPase as compared with 80% in the native vacuole.
Conversely, for H180K, nearly normal activity was observed for the
isolated V-ATPase as compared with 40% inhibition in the vacuole. In
these cases, the observed differences are unlikely to be due to a
change in stability of the isolated V-ATPase since nearly normal
assembly was observed for these mutants by SDS-PAGE (Fig. 2). In
fact, of the mutations tested, only R381S had any significant effect on
assembly or stability of the V-ATPase complex.
Figure 2:
Effect of Vma2p mutations on V-ATPase
assembly. Yeast vacuoles containing 2 mg of protein were washed 3
times with EDTA, solubilized, and purified by sedimentation on
20-50% glycerol density gradients as described under
``Experimental Procedures.'' Fractions were tested for ATPase
activity, and the peak fractions (
2 µg of protein) were
subjected to SDS-PAGE on 12% acrylamide gels. Lane 1 is the
wild-type; lanes 2-11 show the mutants Y370R, Y370F,
Y370S, R381S, Y352S, Y352F, H180K, H180G, H180D, and N181V,
respectively; lane 12 is the negative
control.
Figure 3: Effect of Vma2p mutations on ATPase activity of purified V-ATPase. Glycerol density gradient fractions containing the peak of V-ATPase activity were prepared and assayed as described under ``Experimental Procedures.'' Approximately 0.6 mg of protein was assayed in each case, and the ATPase activity is expressed relative to the wild-type control, which had a specific activity of approximately 18 µmol/min/mg of protein. The values shown represent the average of two determinations, with the error shown corresponding to the average deviation from the mean.
While photochemical labeling studies indicate that the
V-ATPase B subunit participates in nucleotide binding (Manolson et
al., 1985; Zhang et al., 1995), the structure and
function of the nucleotide binding sites on the B subunit remain
uncertain. In the case of the homologous F-ATPases, the subunit
forms the major portion of the noncatalytic nucleotide binding sites
and contributes several residues to the catalytic sites, which are
located principally on the
subunits (Abrahams et al.,
1994). The effects of mutagenesis of the Escherichia coli
subunit residues on F-ATPase activity together with the data
on mutagenesis of yeast Vma2p residues presented in this paper are
summarized in Table 1.
The x-ray crystal structure of
mitochondrial F reveals two residues, Arg-373 and Ser-344,
which are contributed by the
subunit to the catalytic nucleotide
binding sites, with both residues near the terminal phosphate of ATP
(Abrahams et al., 1994). The positively charged guanidinium
group of Arg-373 is postulated to stabilize the negative charge, which
develops on the
phosphate during the transition state of ATP
hydrolysis, and mutation of this Arg to Cys in the E. coli enzyme results in a total loss of ATPase activity (Soga et
al., 1989). Mutation of the corresponding Arg residue to Ser in
Vma2p has a similarly dramatic effect on activity, suggesting that this
residue also plays a critical role in the V-ATPases. The partial loss
of assembly may indicate that this residue also forms an important
contact between the A and B subunits.
Mutagenesis of the E. coli residue corresponding to Ser-344 (Ser-347), has not been reported, but this residue has not been conserved as a serine residue in the V-ATPase B subunit. It is, instead, a tyrosine residue in the B subunits from Neurospora (Bowman et al., 1988a), plants (Manolson et al., 1988), yeast (Nelson et al., 1989), and bovine brain (Puopolo et al., 1992), as well as the archaebacterial ATPases from Sulfolobus (Denda et al., 1988) and methanobacteria (Inatomi et al., 1989). Interestingly, it is a phenylalanine residue in the B subunit from bovine kidney (Nelson et al., 1992). Mutagenesis of this residue to Ser in yeast totally abolishes V-ATPase activity, whereas substitution of Phe at this position has a relatively minor effect (Table 1). This result, consistent with the presence of a phenylalanine at this position in the bovine kidney sequence, suggests that an aromatic residue rather than a hydroxyl group is important at this position, making a direct interaction between this residue and the terminal phosphates of ATP unlikely in the V-ATPases.
Combined
mutagenesis, photochemical labeling, and fluorescence energy transfer
studies indicate that Arg-365 is near the adenine ring of nucleotides
bound at the noncatalytic sites of the E. coli subunit
(Weber et al., 1993). Thus, substitution of Tyr at this
position results in significant fluorescence quenching of lin-benzo-ATP
bound at the noncatalytic site. This mutation resulted in a 30%
decrease in ATPase activity, while substitution of Phe at this position
caused approximately 40% loss of activity (Weber et al.,
1993). By contrast, the corresponding residue in the V-ATPase B subunit
from all species (Bowman et al., 1988a; Manolson et
al., 1988; Nelson et al., 1989; Puopolo et al.,
1992), as well as the
subunit of the archaebacterial ATPases
(Denda et al., 1988; Inatomi et al., 1989) is a
tyrosine. Consitent with the results obtained for the E. coli
subunit, replacement of Tyr-370 with Arg, Phe, or Ser
inhibited proton transport and ATPase activity by only 30-50% (Table 1), suggesting that tyrosine is not absolutely required at
this position. Other mutations in the vicinity of Arg-
365,
including S373F, G351D, and S375F, have been shown to impair catalysis
(Maggio et al., 1987), and the x-ray crystal structure of
F
confirms that this arginine residue is near the adenine
binding pocket on
.
Considerable mutagenesis has been carried
out on residues in the P-loop region of the E. coli
subunit (Jounouchi et al., 1993), which from the crystal
structure lies in close proximity to the triphosphates of ATP bound to
the noncatalytic sites of F1 (Abrahams et al., 1994). Thus
replacement of Lys-
175 with either His or Gly results in 42 or 92%
inhibition of activity, respectively, while replacement with Phe or Trp
abolishes assembly (Jounouchi et al., 1993). By contrast,
replacement of the corresponding Vma2p residue (His-180) with Lys, Gly,
or Asp had somewhat less dramatic effects on activity, inhibiting
20-50% of the activity. Similarly, for the E. coli
subunit, T176V and T176L showed 23 and 31% of wild-type activity,
respectively, whereas replacement of the corresponding Vma2p residue
(Asn-181) with Val gave 50-70% of wild-type activity (Table 1). In fact, the glycine-rich loop region has not been
conserved in the V-ATPase B subunit sequence, although this region
(Ser-174 to Asn-181 in yeast) is nearly perfectly conserved in all the
available V-ATPase B subunits. Thus, despite extensive amino acid
substitution between the
and B subunit sequences, this region has
retained an important role in maintaining maximal ATPase activity.
Interestingly, a positively charged residue at position 180 of the
yeast B Vma2p does not appear to be essential, suggesting that this
residue may not be interacting directly with the triphosphates of ATP
at the noncatalytic sites or that binding of ATP to the noncatalytic
sites of the V-ATPase is not absolutely required for activity.
These studies thus represent a first step in the use of site-directed mutagenesis for the molecular dissection of the structure and function of the V-ATPase B subunit.