(Received for publication, August 24, 1995; and in revised form, September 19, 1995)
From the
The yeast plasma-membrane H-ATPase contains
nine cysteines, three in presumed transmembrane segments (Cys-148,
Cys-312, and Cys-867) and the rest in hydrophilic regions thought to be
exposed at the cytoplasmic surface (Cys-221, Cys-376, Cys-409, Cys-472,
Cys-532, and Cys-569). To gather new functional and structural
information, we have studied the yeast ATPase by cysteine mutagenesis.
It proved possible to replace seven of the nine cysteines by alanine,
one at a time, without any significant decrease in ATP hydrolysis or
ATP-dependent proton pumping. In the remaining two cases (Cys-409 and
Cys-472), there were small but reproducible effects; the results
clearly indicated, however, that no single Cys is required for activity
and that, if a disulfide bridge is formed in the yeast ATPase, it does
not play an obligatory structural or functional role.
Next, multiple
mutants were constructed to ask how many Cys residues could be replaced
simultaneously while leaving a fully functional enzyme. After
substitution of all ``membrane'' Cys (Cys-148, Cys-312, and
Cys-867) together with two non-conserved Cys located in hydrophilic
regions (Cys-221 and Cys-569), there were no significant abnormalities
in expression (87%) or activity (89% ATP hydrolysis/93% H pumping) of the mutant protein. Replacement of two additional
cysteines (Cys-376 near the phosphorylation site and Cys-532, in or
near the ATP-binding site) caused a drop in expression (to 54%),
although the corrected hydrolytic and H
pumping
activities were still normal. When Cys-472 was also mutated, the
corrected activity fell to 44% hydrolysis/47% pumping; finally,
substitution of Cys-409 to give a ``cysteine-free'' ATPase
led to a very poorly expressed and poorly active enzyme. Brief exposure
of the ``one-cysteine'' and ``two-cysteine''
ATPases to trypsin revealed a normal pattern of degradation, but there
was a slight impairment in the ability of vanadate to protect against
proteolysis. Thus, although single Cys replacements are tolerated well
by the yeast ATPase, multiple replacements are progressively more
harmful, suggesting that they cause small but additive perturbations of
protein folding.
The P-ATPases are a widespread family of cation transporters,
ranging from bacterial K-, Mg
-,
Ca
-, Cd
-, and
Cu
-ATPases to the
Na
,K
-,
H
,K
-, Ca
-, and
Cu
ATPases of mammalian cells (Serrano, 1988; Green,
1992; Fagan and Saier, 1994; Bull and Cox, 1994; Solioz et
al., 1994). In spite of their physiological diversity, there is
clear evidence for a common evolutionary origin. At least 70 P-ATPase
genes have been cloned since 1986, and hydropathy analysis of the
corresponding protein sequences has revealed a shared topological plan
in which the large catalytic polypeptide is anchored in the membrane by
hydrophobic segments at both N- and C-terminal ends (reviewed by
Serrano(1988) and Nakamoto et al.(1989)). Within the central
hydrophilic portion of the polypeptide are well conserved regions that
bind ATP and form the essential
-aspartyl phosphate reaction
intermediate. During the past several years, site-directed mutagenesis
has made it possible to pinpoint functionally important residues (see, e.g., MacLennan et al.(1992)) and to identify, at
least provisionally, the transmembrane segments that form the actual
transport pathway (MacLennan et al., 1992; Inesi et al., 1994). In parallel, useful structural information has emerged from
cryo-electron microscopy (Green and Stokes, 1992; Toyoshima et al., 1993; Stokes et al., 1994). Much remains to be done,
however, before the transport mechanism of the P-ATPases can be
completely understood.
With the long range goal of gathering new
structural and functional information, we have undertaken to study the
yeast plasma-membrane H-ATPase by cysteine
mutagenesis. The yeast ATPase, which is encoded by the PMA1 gene (Ulaszewski et al., 1983; Serrano et al.,
1986) and serves as the primary ion pump in the plasma membrane, lends
itself well to analysis by both genetic and molecular biological
strategies (Cid et al., 1987; McCusker et al., 1987;
Nakamoto et al., 1991; Perlin et al., 1992).
Furthermore, the yeast enzyme has only nine cysteine residues, unlike
its mammalian counterparts which contain 15-33 cysteines (Solioz et al., 1994). Thus, it seemed feasible to start by replacing
each of the cysteines in turn with alanine to see which (if any) are
required for biogenesis, stability, ATP hydrolysis, and/or
ATP-dependent proton pumping. With such information in hand, we could
proceed to construct multiple mutants, winding up with a
``minimal'' Cys ATPase that could be used in a variety of
ways: for example, to introduce new Cys residues at structurally or
functionally interesting positions where they could be probed with
radioactive, fluorescent, or spin-labeled SH reagents. In principle, as
shown by work on other membrane proteins, this would pave the way to
examine the location, accessibility, and conformational role of
individual residues (Falke and Koshland, 1987; Falke et al.,
1988; Flitsch and Khorana, 1989; Careaga and Falke, 1992; Pakula and
Simon, 1992; Dunten et al., 1993; Frillingos et al.,
1994; Lee et al., 1994) and to measure distances between
residues in the three-dimensional structure (Wang et al.,
1992; Corbalan-Garcia et al., 1993; Baker et al.,
1994).
Secretory vesicles
containing newly synthesized mutant ATPase were isolated by
differential centrifugation and gel filtration (Nakamoto et
al., 1991). To increase the yield of vesicles in the present
study, spheroplasts were lysed in 0.4 M sorbitol, 10
mM triethanolamine-acetic acid, pH 7.2, 1 mM EDTA.
Protease inhibitors were included in the lysis buffer and in all
subsequent steps at the following concentrations: diisopropyl
fluorophosphate, 1 mM; chymostatin, 2 µg/ml; and
leupeptin, pepstatin, and aprotinin, 1 µg/ml each. After
sedimentation at 14,500 g for 10 min, crude secretory
vesicles were resuspended for gel filtration in 0.8 M sorbitol, 10 mM triethanolamine-acetic acid, pH 7.2, with
protease inhibitors. Fractions containing secretory vesicles were
collected and centrifuged at 100,000
g for 45 min, and
the vesicles were resuspended at a protein concentration of
0.8-3.0 mg/ml in the same buffer excluding diisopropyl
fluorophosphate. The average yield of secretory vesicles was
0.7-0.9 mg of protein per 1000 OD of yeast culture. All
preparative procedures were performed at 0-4 °C.
Figure 1:
Predicted topology of the yeast
plasma-membrane H-ATPase. Positions of cysteines are
indicated by solid circles and numbers; the underlined numbers indicate cysteines conserved in all known
fungal PMA ATPases. Abbreviations are as follows: P,
phosphorylation site (Asp-378); FITC, fluorescein
isothiocyanate-reactive lysine (Lys-474); NEM,
ADP-protectable, N-ethylmaleimide-reactive cysteine
(Cys-532).
For reasons outlined in an earlier section, the
first step in this project was to replace each of the Cys residues, one
at a time, with Ala. In every case, the mutated pma1 gene was
cloned downstream of two tandemly arranged heat-shock elements in the
expression plasmid YCp2HSE and transformed into yeast strain SY4, in
which the resident copy of the PMA1 gene has been placed under
control of the GAL1 promoter (Nakamoto et al., 1991).
Thus, when the cells were incubated in galactose medium at 23 °C,
the wild-type ATPase was produced and could support growth; by
contrast, when the cells were transferred to glucose medium at 37
°C, the mutant ATPase was produced for study. Because strain SY4
carries a temperature-sensitive sec6-4 mutation that
blocks the last step in plasma membrane biogenesis (Schekman and
Novick, 1982), the shift to 37 °C also led to the accumulation of
secretory vesicles containing the newly synthesized mutant ATPase. The
secretory vesicles were isolated by differential centrifugation
followed by size fractionation on a Sephacryl column (Walworth and
Novick, 1987). They were relatively free of contamination by plasma
membrane fragments containing pre-existing wild-type ATPase (Nakamoto et al. 1991; see also Table 1), and could readily be
assayed for ATP hydrolysis and ATP-dependent H pumping
(the latter, by acridine orange fluorescence quenching; Nakamoto et
al., 1991).
Results for the nine Cys Ala mutants are
summarized in Table 1. In every case, the measured expression
level was 80% or greater of the wild-type control, indicating that all
nine mutant ATPases were synthesized properly, moved from the
endoplasmic reticulum to the secretory vesicles, and remained stable
over the time course of the experiment. Similarly, all of the mutant
proteins had relatively high ATPase activities and ATP-dependent
H
pumping rates. After correction for the expression
level, values ranged from 55% hydrolysis/53% pumping in the C472A
mutant and 74% hydrolysis/81% pumping in the C409A mutant to 113%
hydrolysis/119% pumping in C312A. No significant abnormalities were
detected in the kinetic parameters of ATP hydrolysis, including the K
for ATP (0.6-1.8 mM), K
for orthovanadate (1.1-3.1
µM), and pH optimum (5.6-5.9). Thus, it appears that
none of the nine Cys residues plays an essential role in the biogenesis
or functioning of the yeast PMA1 H
-ATPase.
Because
Ser is sometimes better than Ala at substituting for Cys, especially in
relatively hydrophilic regions of proteins, Cys Ser replacements
were also constructed for Cys-409 and Cys-472 (Table 1). Indeed,
the C409S ATPase had marginally higher hydrolytic and pumping
activities (94%/99% after correction for expression levels, compared
with 74%/81% for the C409A ATPase). The same was true for the C472S
enzyme (74%/78% compared with 55%/53% for the C472A enzyme), although
in the latter case the Ser replacement gave a noticeably lower
expression level (75%) than the Ala replacement (134%). In any event,
the Ser substitutions provided an added option for the design and
characterization of multiple mutants (see below).
A parallel set of constructs explored the consequences of combining mutations within the central hydrophilic region. The double mutant C409A/C472A was expressed normally (93%) but had reduced rates of ATP hydrolysis and ATP-dependent proton pumping (44% and 46%, respectively), somewhat below the values seen with the corresponding single mutants (C409A, 74% and 81%; C472A, 55% and 53%; Table 1). Serine replacements at the same positions (in C409S/C472S) were less successful, giving corrected hydrolytic and pumping rates of 50% and 58% but lowering expression to 48% (Table 3). When mutations were added at two neighboring sites to give constructs with four alanines (C376A/C409A/C472A/C532A), three alanines and one serine (C376A/C472A/C532A/C409S), or two alanines and two serines (C376A/C532A/C409S/C472S), expression occurred at 43%, 51%, and 25% of the wild-type level, respectively. Even after correction, hydrolytic rates were quite low (21%, 30%, and 32%); ATP-dependent proton pumping was detectable only in the middle case (45%; Table 3).
The last step was to combine mutations throughout
the ATPase. In a ``two-cysteine'' strain containing only
Cys-409 and Cys-472, expression was acceptable (54%) and the corrected
rates of ATP hydrolysis and ATP-dependent proton pumping were
essentially normal (103% and 97%). When Cys-472 was replaced with Ala,
leaving only Cys-409 (``one-cysteine'' strain), expression
remained at 55% but hydrolysis and pumping dropped to 44% and 47%. And
finally, when Cys-409 was replaced with Ser to give a
``cysteine-free'' enzyme, expression fell to 20%. ATP was now
hydrolyzed at a barely measurable rate (6% before correction; 33% after
correction), and no ATP-dependent proton pumping was seen. Taken
together, the results point to additive effects of multiple cysteine
substitutions, even though the replacement of any single Cys can be
tolerated quite well. Indeed, it appears impossible to achieve a
properly functioning H-ATPase that is entirely
cysteine-free, even though such an enzyme is capable of splitting ATP
at a very slow rate. By contrast, mutant enzymes with either two
cysteines (Cys-409 and Cys-472) or one cysteine (Cys-409) are active
enough to serve as a useful starting point for further work.
Figure 2:
Enzymatic properties of wild-type (WT) and mutant ATPases. A, dependence of ATP
hydrolysis on MgATP concentration. B, inhibition of ATP
hydrolysis by vanadate; control values in the absence of vanadate were
3.60 (wild type), 1.54 (two-cysteine), and 0.89 (one-cysteine) µmol
of P/mg
min. In both A and B, enzyme
activity was assayed as described under ``Materials and
Methods.''
Figure 3:
H transport by wild-type (WT) and mutant ATPases. Secretory vesicles (50 µg
protein) were suspended in 1.5 ml of 20 mM HEPES-KOH, pH 6.7,
0.6 M sorbitol, 100 mM KCl, 5 mM ATP, and 2
µM acridine orange. The reaction was started by the
addition of 10 mM MgCl
, and fluorescence quenching
was monitored as described under ``Materials and Methods.''
Parallel measurements of ATPase activity under quenching conditions
gave values of 1.55 (wild type), 0.83 (2-cysteine), and 0.39
(one-cysteine) µmol of
P
/min
mg.
Figure 4: Time course of trypsinolysis of wild-type and mutant ATPases. Secretory vesicles (10 µg of protein) containing either wild-type (lanes 1-6), two-cysteine (lanes 7-12), or one-cysteine (lanes 13-18) ATPase were incubated at a trypsin:protein ratio of 1:15 for 0-20 min. Samples were analyzed by SDS-polyacrylamide gel electrophoresis, followed by Western blotting with anti-ATPase antibody and scanning with a PhosphorImager. For experimental details, see ``Materials and Methods.''
Further information came
from digesting with trypsin in the presence of vanadate, which has
previously been shown to protect the yeast PMA1 ATPase against
proteolytic degradation (Perlin and Brown, 1987). With both the
wild-type and mutant enzymes, as little as 1 µM vanadate
was sufficient to give visible protection of the initial 97-kDa
cleavage product (Fig. 5). However, quantitative scanning of the
immunoblot revealed that the extent of protection was noticeably lower
in the mutants (30-40%) than in the wild type (
70%).
Thus, the elimination of seven cysteines or eight cysteines led to
minor changes in conformation, making the ATPase more sensitive (or
more accessible) to the action of proteolytic enzymes.
Figure 5: Effect of vanadate on trypsinolysis of wild-type and mutant ATPases. Secretory vesicles (10 µg of protein) containing wild-type (lanes 1-6), two-cysteine (lanes 7-12), or one-cysteine (lanes 13-18) ATPase were incubated for 7.5 min at a trypsin:protein ratio of 1:15 in the presence of 0-100 µM vanadate. For experimental details, see ``Materials and Methods.''
As shown in this study, it is possible to substitute each of
the nine cysteines of the yeast PMA1 ATPase singly without a major
effect on expression, ATP hydrolysis, or ATP-dependent proton pumping;
we therefore conclude that no individual Cys residue is essential for
the biogenesis or functioning of the enzyme. This is not a surprising
result in the case of the three non-conserved cysteines (Cys-221,
Cys-312, and Cys-569). The remaining six cysteines are found in all
known fungal H-ATPases, however, and at least in
principle might have been predicted to play an important functional
role.
Indeed, working with the closely related Neurospora plasma-membrane H-ATPase, Rao and Scarborough
(1990) have put forward biochemical evidence that two membrane-embedded
cysteines may be linked by a disulfide bridge. Disulfides have also
been reported in the sarcoplasmic reticulum Ca
-ATPase
(Thorley and Green, 1977) and the gastric
H
,K
-ATPase (Sachs et al.,
1992), and the latter enzyme is inhibited by formation of a disulfide
bridge with the anti-ulcer drug omeprazole (Prinz et al.,
1992). Several functions can be imagined for intramolecular disulfide
bridges: for example, to help in the assembly of the nascent ATPase
polypeptide or to stabilize the mature molecule (Rao and Scarborough,
1990). Alternatively, disulfide bridge formation might mediate
reversible activation/deactivation, as has been described for the
vacuolar H
-ATPase from clathrin-coated vesicles (Feng
and Forgac, 1994).
In the case of the yeast PMA1 ATPase, however,
molecular biological evidence now argues quite convincingly against the
presence of an essential disulfide bridge. With respect to
membrane-embedded cysteines, Harris et al.(1991) reported that
Cys-148 can be replaced by Ser with little or no effect on ATPase
activity, and the present study has shown that substitution of Cys-148,
Cys-312, or Cys-867 singly, or even substitution of all three
simultaneously (in strain C148A/C221A/C312A/C569A/C867A; Table 2and Table 3), produces no significant change in
biogenesis or activity. Thus, if a disulfide bridge is formed in the
membranous portion of the yeast ATPase, it cannot play an obligatory
structural or functional role, at least under the conditions of these
experiments. The situation with respect to the six cytoplasmically
located cysteines is a little more complicated, since there were
significant effects of multiple Cys replacements in this region. Once
again, however, no single Cys substitution caused a major impairment of
ATPase biogenesis or function, arguing that no disulfide bond can be
absolutely essential. Consistent with this idea, ATPase activity in
secretory vesicles is not inhibited by the presence of 10 mM dithiothreitol or 28 mM 2-mercaptoethanol during
spheroplast formation. Similarly, Petrov et
al.(1992) found that 2-mercaptoethanol failed to inhibit the yeast
ATPase in isolated plasma membranes. By contrast, the sarcoplasmic
reticulum Ca
-ATPase-which is known to contain
disulfide bonds (see above)-is inactivated by 2-mercaptoethanol
and dithiothreitol (Georgoussi and Sotiroudis, 1985; Mutoh et
al., 1992; Daiho and Kanazawa, 1994).
Of the four conserved
cysteines located in the central catalytic portion of the yeast ATPase,
two (Cys-376 and Cys-409) lie in the phosphorylation domain, while the
other two (Cys-472 and Cys-532) are located in or near the ATP-binding
site. The most strongly conserved among them is Cys-376, found in
nearly all P-ATPases; the only known exceptions are the bacterial
K, Cu
, and
Cd
-ATPases and the human Menkes and Wilson ATPases
(Serrano and Portillo, 1990; Bull and Cox, 1994; Solioz et
al., 1994). In spite of its nearly universal presence, however,
Cys-376 of the yeast H
-ATPase can be replaced by Leu
(Portillo and Serrano, 1989; Serrano and Portillo, 1990) or Ala (this
study) with no significant loss of function. Similarly, in the
sarcoplasmic reticulum Ca
-ATPase, the corresponding
residue (Cys-349) can be replaced by Ala with no measurable change in
the rate of ATP-dependent Ca
transport (Maruyama et al., 1989).
Only in the case of Cys-409 and Cys-472 were there small but reproducible effects of single Ala substitutions in the yeast PMA1 ATPase. The functional importance of these residues was underscored by the finding that they could not be replaced during the stepwise construction of a multiple Cys mutant without sacrificing a substantial fraction of hydrolytic and pumping activity. It will be interesting to see, once a three-dimensional structure is available for the PMA1 ATPase, what role Cys-409 and Cys-472 play in the folded phosphorylation and nucleotide-binding domains. In the meantime, the one-cysteine ATPase (containing only Cys-409) and two-cysteine ATPase (containing Cys-409 and Cys-472) should be useful starting points for a variety of biochemical and biophysical studies.