(Received for publication, May 30, 1995; and in revised form, October 4, 1995)
From the
A high-yield yeast expression system for site-directed
mutagenesis of the Neurospora crassa plasma membrane
H-ATPase has recently been reported (Mahanty, S. K.,
Rao, U. S., Nicholas, R. A., and Scarborough, G. A.(1994) J. Biol.
Chem. 269, 17705-17712). Using this system, each of the
eight cysteine residues in the ATPase was changed to a serine or an
alanine residue, producing strains C148S and C148A, C376S and C376A,
C409S and C409A, C472S and C472A, C532S and C532A, C545S and C545A,
C840S and C840A, and C869S and C869A, respectively. With the exception
of C376S and C532S, all of the mutant ATPases are able to support the
growth of yeast cells to different extents, indicating that they are
functional. The C376S and C532S enzymes appear to be non-functional.
After solubilization of the functional mutant ATPase molecules from
isolated membranes with lysolecithin, all behaved similar to the native
enzyme when subjected to glycerol density gradient centrifugation,
indicating that they fold in a natural manner. The kinetic properties
of these mutant enzymes were also similar to the native ATPase with the
exception of C409A, which has a substantially higher K
. These results clearly indicate that
none of the eight cysteine residues in the H
-ATPase
molecule are essential for ATPase activity, but that Cys
,
Cys
, and Cys
may be in or near important
sites. They also demonstrate that the previously described disulfide
bridge between Cys
and Cys
or Cys
plays no obvious role in the structure or function of this
membrane transport enzyme.
The plasma membrane H-ATPase of Neurospora
crassa is an electrogenic (1) proton pump(2) ,
that belongs to the P-type family of ion translocating ATPases that
form an aspartyl phosphate intermediate in their reaction
cycle(3, 4) . This ATPase is closely related to the
yeast and plant plasma membrane H
-ATPases and
homologous with numerous mammalian cation translocating ATPases
including the Na
/K
-,
H
/K
-, and
Ca
-ATPases(5, 6, 7, 8, 9) .
Many studies involving chemical modification of the cysteine
residues in the Neurospora H-ATPase have been
reported(10, 11, 12, 13, 14, 15, 16, 17) .
The sulfhydryl reagent, N-ethylmaleimide was originally shown
to have no effect on the H
-ATPase
activity(10) , but subsequent studies reported inhibition by N-ethylmaleimide when treatments were carried out at elevated
pH
values(11, 12, 13, 14, 15, 16) .
More recent studies of the effect of N-ethylmaleimide on the
ATPase have been interpreted to indicate that Cys
and
Cys
residues are near important
sites(15, 16) , but similar studies with methyl
methanethiosulfonate have shown that neither of these cysteines nor
cysteines at positions 376, 409, and 472 play a covalent role in the
reaction cycle of the enzyme. In another study(17) , the
chemical states of the eight cysteine residues in the
H
-ATPase molecule were determined, establishing the
presence of a disulfide bridge between Cys
and either
Cys
or Cys
. These studies provided valuable
information as to the structure of the ATPase molecule but could not
address the possible function of the disulfide bridge.
Site-directed
mutagenesis is a powerful tool for exploring structure-function
relationships in the H-ATPase by identifying
individual amino acid residues that are important for its structure and
activity. We have recently developed a yeast expression system for such
studies of the Neurospora H
-ATPase(18) , and in this article, this
system is used to further probe the roles of the eight cysteine
residues in its structure and molecular mechanism.
For densitometric analysis of the amounts of
mutant Neurospora ATPases present in the glycerol gradient
fractions, aliquots of the pooled peak activity fractions (numbers 9,
10, 11, and 12 from the top) were first subjected to SDS-PAGE as
described above, and the gels were silver-stained and photographed. The
photographs were then scanned using a Bio-Rad Image Densitometer (Model
GS-670) and the ATPase region was integrated using the Molecular
Analyst TM/PC Software (Image Analysis Software Version 1.1.1). The
amounts of the mutant ATPases were calculated by comparision with 5,
10, and 20 ng samples of purified native Neurospora H-ATPase (27) present in the same gel.
As described previously(18) , in the yeast expression
system used for these studies, both the wild type yeast
H-ATPase and the plasmid-encoded Neurospora H
-ATPase are produced when the cells are grown in
medium containing galactose as the carbon source. When the cells are
transferred to glucose medium, synthesis of the yeast ATPase ceases,
which limits growth to only a few doublings unless a functional Neurospora H
-ATPase is present. Fig. 1shows these features with several control yeast strains
used in this investigation. Growth of the parent strain SKM9 with an
unmutated Neurospora H
-ATPase is shown along
with the growth of strain SKM3, which contains the expression plasmid
with no ATPase cDNA, and strain D378A, which contains a cDNA in which
the codon for the active site Asp
of the Neurospora enzyme has been changed to an Ala codon. The parent strain grows
well, whereas both of the other control strains grow for only a few
doublings and then stop growing as expected in glucose medium. Growth
in glucose medium is thus a convenient screening assay for the
functionality of the Neurospora H
-ATPase
mutants we produce.
Figure 1:
Growth of several control yeast
strains. Shown are the growth characteristics of several control yeast
transformants in glucose medium assessed as described under
``Experimental Procedures.'' Unmutated Neurospora H-ATPase producing strain, SKM9 (
); no
ATPase, plasmid control strain SKM3 (
); active site aspartate
ATPase mutant D378A (
).
Table 1shows the growth rates of the control strains and the various cysteine mutant ATPase strains calculated from growth curves similar to those shown in Fig. 1. Both strain C148S and strain C148A grow at about the same rate as the unmutated ATPase control strain. Strain C376S does not grow any more than the negative control strains and strain 376A grows only very slowly. Strain C409S grows about the same as the unmutated ATPase strain, and significantly better than strain C409A. On the other hand, strain C472S grows substantially slower than strain C472A. Interestingly, strain C532S does not grow any more than the negative controls, whereas strain C532A grows well. Strains C545S and C840S both grow well whereas both strains C545A and C840A grow somewhat more slowly than their serine counterparts. Strain C869S grows at an intermediate rate whereas strain C869A grows at a rate about the same as that of the wild type strain SKM9.
To ascertain the amounts of the ATPase systhesized in the various strains, membranes were isolated from each of the mutants grown in glucose medium and analyzed by SDS-PAGE and silver staining as described under ``Experimental Procedures.'' The results indicated the presence of substantial amounts of the mutant ATPases in the membranes of all of the mutants except C376S, C532S, and D378A (data not shown). The levels of mutant Neurospora ATPase present in the membranes of these three strains were substantially lower, which precluded further analysis of these enzymes.
When solubilized with the detergent, lysolecithin,
the native Neurospora H-ATPase is a hexamer (28) that migrates to the bottom third of a 20-40%
glycerol gradient(29) . This sedimentation behavior provides a
simple but effective assay for proper folding of the recombinant Neurospora H
-ATPase molecules produced in
yeast. To assess the extent to which the various cysteine mutants fold
properly, each of the mutant ATPases was solubilized from the membranes
with lysolecithin and the solubilized extract was subjected to glycerol
density gradient centrifugation as described(18) . The results
indicated that all Cys
Ala and the six remaining Cys
Ser
mutant H
-ATPase molecules migrated quantitatively to a
position identical to that of the SKM9 recombinant ATPase and the
native enzyme (data not shown), providing strong evidence that each of
these mutant ATPases folds correctly in theoretical yields.
In
addition to providing a simple folding assay, lysolecithin
solubilization and glycerol density gradient centrifugation produces
substantially purified preparations of the mutant ATPases useful for
further analysis. Fig. 2shows the H-ATPase
region of SDS-PAGE analyses of the pooled peak glycerol gradient
fractions from two different membrane preparations from each of the
various Cys
Ser and Cys
Ala mutants and the control
strains SKM9 and SKM3. In the region between the 97.4 and 116.2 kDa
standards, three bands are routinely seen. The uppermost of these bands
is the yeast H
-ATPase, the middle one is the
recombinant Neurospora H
-ATPase, and the
lower one is an unidentified band of no interest in these studies. The
yeast ATPase band represents residual amounts of this enzyme present
when the cells are transferred to glucose medium. The analyses in Fig. 2show that the amounts of the Neurospora ATPase
synthesized are generally reproducible for each strain. To quantitate
the amount of each mutant H
-ATPase present in the
pooled peak glycerol gradient fractions, the gels of Fig. 2were
subjected to densitometric analysis as described under
``Experimental Procedures.'' The amounts of the mutant
ATPases produced in the various strains are listed in Table 2.
The amounts were variable from strain to strain in the range of roughly
half to 1.6 times that of strain SKM9. The analyses also showed that
none of the partially purified ATPase preparations contained more
residual yeast H
-ATPase than that of the control
strain SKM3, which contained only the yeast ATPase (Fig. 2, lanes 2).
Figure 2:
SDS-PAGE analysis of the pooled peak
glycerol gradient fractions from strains SKM9, SKM3, and the various
Cys Ser and Cys
Ala H
-ATPase mutants. Panels A and B show silver-stained SDS-PAGE gel
analyses of the pooled peak glycerol gradient fractions from two
different experiments carried out on different days. Three mg of
membrane protein from each strain was solubilized with lysolecithin (5
mg of lysolecithin/mg of membrane protein) and the solubilized extracts
were layered on the top of 20-40% (w/v) linear glycerol gradients
and centrifuged overnight as described under ``Experimental
Procedures.'' One-ml fractions were collected and aliquots were
assayed for ATPase activity. The peak fractions (9-12 from the
top) were pooled and analyzed by SDS-PAGE. Lane 1 contained 10
µl of SKM9 pooled peak glycerol gradient fractions, lane 2 contained 10 µl of SKM3 pooled glycerol gradient fractions
9-12, and lanes 3-16 contained 10 µl of the
pooled peak glycerol gradient fractions from mutants C148S, C409S,
C472S, C545S, C840S, C869S, C148A, C376A, C409A, C472A, C532A, C545A,
C840A, and C869A, respectively. Lanes 17-19 contained 5,
10, and 20 ng of purified native N. crassa H
-ATPase, respectively. The numbers to
the left of panels A and B indicate the
molecular masses of the SDS-PAGE standards used in kDa. Only the
approximate 100-kDa H
-ATPase regions of the gels are
shown.
With this information, the kinetic
characteristics of the various partially purified Cys Ser and
Cys
Ala mutant H
-ATPases could be determined.
Importantly, the pooled gradient fractions from the control strain SKM3
showed no ATPase activity in the assay system used. Therefore, since
none of the other preparations contained more yeast ATPase than strain
SKM3, the ATPase activities measured reflect only that of the Neurospora enzymes. Table 2shows the kinetic constants
determined for the ATPases from all of the viable mutant and control
strains. For all mutant ATPases analyzed, the K
and V
values are not substantially altered
except in the case of mutant C409A, which shows a greater than 10-fold
increase in its K
with no difference in V
. Although not shown, the vanadate
sensitivities of all of the mutant enzymes were comparable to that in
the wild type ATPase strain SKM9 (I
about 1
µM).
The results obtained in these studies allow several important
conclusions regarding the functions of the eight cysteine residues in
the Neurospora H-ATPase molecule. First, it
is clear that none of the cysteines is essential for the catalytic
mechanism of the enzyme. Replacement of each cysteine with an alanine
residue produces functional ATPase molecules in strains C148A, C376A,
C409A, C472A, C532A, C545A, C840A, and C869A, since all of these mutant
ATPases are able to support the growth of yeast cells in glucose medium
to some degree. Moreover, except for the C409A enzyme all of these
mutant ATPases exhibit kinetic properties similar to that of the wild
type strain SKM9.
Second, although none of the cysteines is
essential, several appear to be in or near important sites of action in
the H-ATPase molecule. Cys
is clearly
near the active site of the ATPase as it is only one residue removed
from the active site aspartate at position 378, and the mutagenesis
results reported here suggest that it may serve an important function.
Although the C376A enzyme folds correctly and exhibits kinetic
characteristics nearly the same as those of the wild type ATPase, it
only weakly supports the growth of the cells. This suggests the
possibility that the C376A ATPase may be uncoupled with respect to ATP
hydrolysis and proton translocation. Alternatively, the rate of
synthesis of this mutant ATPase may be somehow slowed. Obviously, a
more detailed investigation of the C376A ATPase will be necessary
before the defect in this interesting mutant will be understood.
Cys is also important and appears to be a part of the
MgATP binding site. Replacement of this cysteine with an alanine causes
a 10-fold increase in the K
without alteration of
the V
or vanadate sensitivity (Table 2).
On the other hand, replacement of Cys
with a serine does
not change the K
near as drastically. This
suggests that the -SH group of Cys
may be involved in
binding with some part of the substrate, MgATP, in the native enzyme.
Cys may also be in or near an important site, since
the mutant C532S ATPase totally failed to support growth on glucose
medium (Table 1). However, mutant C532A supported cell growth and
displayed normal kinetic characteristics. Cys
has been
reported to be protected against reaction with N-ethylmaleimide by MgADP and may thus be in or near the
nucleotide binding site(15) . If so, our results suggest that
some aspect of the substrate binding and phosphoryl transfer reactions
occurs less efficiently when the cysteine -SH is replaced with an -OH
by conversion to serine than when it is removed entirely by replacement
with an alanine.
The third conclusion that can be drawn from these
experiments is that the previously detected disulfide bridge linking
Cys and either Cys
or Cys
(17) plays no obvious role in the structure or function
of the H
-ATPase molecule. Replacement of
Cys
, Cys
, or Cys
with a
serine or an alanine residue does not affect the ability of the ATPase
to support cell growth in any major way, and the folding and kinetic
properties of these mutant ATPases are normal. These results preclude
an important structural role for the disulfide bridge in the Neurospora H
-ATPase as made in yeast and
assayed in these experiments.
In our original description of the
yeast expression system used in these studies, it was pointed out that
in certain cases measuring the activities of expressed Neurospora H-ATPase molecules in the presence of the
endogenous yeast H
-ATPase may be problematic. However,
from the studies reported here, it is clear that the yeast
H
-ATPase is inactive in the ATPase assay procedure
employed, because no ATPase activity could be detected in the
appropriate glycerol gradient fractions produced with the plasmid
control strain SKM3. Thus, contamination by the endogenous yeast
H
-ATPase does not appear to be a problem for future
mutagenesis studies of the Neurospora H
-ATPase using this expression system. However,
as can be seen from our results with mutants C376S and C532S, a problem
does remain for mutants that are totally unable to support cell growth.
Whereas a negative growth test by a particular mutant is a strong
indication that an important residue has been altered, the low amounts
of the mutant ATPases produced makes further analysis of such
interesting mutants more difficult than those that support growth.
Encouragingly, preliminary results of membrane fractionation studies
with mutants D378A, C376S, and C532S indicate that these ATPases are
enriched in a light membrane fraction distinct from the plasma
membrane. Thus, although more work will be required to study such
ATPase mutants, detailed analyses of their activities and partial
reactions should neverthless be possible.