ABSTRACT
Structural features of the putative helical hairpin region
comprising transmembrane segments 1 (TM1) and 2 (TM2) of the yeast
plasma membrane H
-ATPase were probed by site-directed
mutagenesis. The importance of phenylalanine residues Phe-116, Phe-119,
Phe-120, Phe-126, Phe-144, Phe-159, and Phe-163 was explored by alanine
replacement mutagenesis. It was found that substitutions at all
positions, except Phe-120 and Phe-144, produced viable enzymes,
although a range of cellular growth phenotypes were observed like
hygromycin B resistance and low pH sensitivity, which are linked to in vivo action of the H
-ATPase. Lethal
positions Phe-120 and Phe-144, could be replaced with tryptophan to
produce viable enzyme, although the F144W mutant was highly perturbed.
ATP hydrolysis measurements showed that K
was not significantly altered for most mutant enzymes,
whereas V
was moderately reduced with two
mutants, F144W and F163A, showing less than 50% of the normal activity.
Double Phe
Ala mutations in TM1 and TM2 were constructed to
examine whether such substitutions would result in a higher degree of
enzyme destabilization. Mutant F116A/F119A was viable and gave a normal
phenotype, while F159A/F163A was not viable. Other double mutants,
F116A/F159A and F119AF/159A, which are predicted to lie juxtaposed on
TM1 and TM2, produced non-functional enzymes. However, a viable
F119V/F159A mutant was isolated and showed hygromycin B resistance.
These results suggest that double mutations eliminating 2 phenylalanine
residues strongly destabilize the enzyme. A putative proline kink at
Gly-122/Pro-123 in TM1 is not essential for enzyme action since these
residues could be variously substituted (G122A or G122N; P123A, P123G,
or P123F) producing viable enzymes with moderate effects on in
vitro ATP hydrolysis or proton transport. However, several
substitutions produced prominent growth phenotypes, suggesting that
local perturbations were occurring. The location of Pro-123 is
important because Gly-122 and Pro-123 could not be exchanged. In
addition, a double Pro-Pro created by a G122P mutation was lethal,
suggesting that maintenance of an
-helical structure is important.
Other mutations in the hairpin, including modification of a buried
charged residue, E129A, were not critical for enzyme action. These data
are consistent with the view that the helical hairpin comprising TM1
and TM2 has important structural determinants that contribute to its
overall stability and flexibility.
The yeast plasma membrane H
-ATPase is a typical
P-type ion translocation ATPase that is related to the family of
enzymes, which includes the mammalian
Na
,K
-ATPase,
Ca
-ATPase, and
H
,K
-ATPase; the plant
H
-ATPase; and the bacterial K
-ATPase,
Mg
-ATPase, and
Cu
-ATPases(1, 2, 3) . These
enzymes couple ATP hydrolysis to ion transport and cycle between two
principal conformational states during catalysis. They
characteristically form an acylphosphate intermediate during catalysis
and are sensitive to inhibition by vanadate(4) . The plasma
membrane H
-ATPase from yeast is essential for
growth(5) , where it plays a critical role in the maintenance
of electrochemical proton gradients and the regulation of intracellular
pH(6) . Significant sequence similarity exists between the
various family members, with the greatest degree of sequence homology
found within the cytoplasmic domain catalyzing ATP
hydrolysis(7, 8) . The topology of these enzymes is
similar, with the N and C termini residing in the
cytosol(9, 10) , and most recent data are consistent
with the presence of 10 transmembrane
segments(11, 12) . There is general agreement on the
identity and orientation of the first four transmembrane segments, with
discrepancies occurring in the remaining C-terminal transmembrane
elements.
The mechanistic nature of how the
H
-ATPase couples energy from ATP binding and
hydrolysis within the cytosolic-domain to transport of ions within the
membrane sector is not understood. Diverse studies involving drug
interactions and immunological probing of higher eukaryotic
enzymes(13, 14, 15, 16, 17, 18) ,
as well as genetic modifications of the yeast H
-ATPase (19, 20, 21) , support the involvement of
long range conformational interactions. There is growing evidence that
transmembrane segments 1 (TM1) (
)and 2 (TM2) are
conformationally linked to the catalytic ATP hydrolysis
domain(19, 20, 22) .
Recently, we proposed
a detailed structural model for TM1 and TM2 and used molecular dynamic
simulations to assess potential conformational determinants in this
region that help account for its functional role (23) . TM1 and
TM2 are predicted to form a helical hairpin structure that has a number
of prominent structural features including a short 4-6-amino acid
turn linking the
-helices, a tightly packed head region, an N-cap
structure stabilizing the turn region, a cluster of phenylalanine
residues near the cytoplasmic face of the hairpin structure, and a
flexible region consisting of Gly-122/Pro-123 that may kink TM1. The
hairpin structure is hydrophobic, and only one charged amino acid,
Glu-129, is present in TM1. The short turn region linking TM1 and TM2
was extensively probed by mutagenesis and found to be highly
conformationally active with perturbations being manifested as
alterations in catalytic function(21) . In this report, we have
examined the effects of amino acid substitutions on the 7
phenylalanines, the flexible proline kink region, and other putatively
important residues within the hairpin region of TM1 and TM2. We provide
evidence that the hairpin region is conformationally sensitive since
viable mutations in this region yield hygromycin B-resistant and low
pH-sensitive cellular phenotypes, and many of the mutant enzymes show
altered catalytic properties. We further provide evidence that the
cluster of phenylalanine residues near the cytoplasmic face of the
bilayer may be important for structural stability.
MATERIALS AND METHODS
Yeast Strains and Cultures
All yeast strains
utilized in this study are isogenic derivatives of Y55 (HO
gal3 MAL1 SUC1)(24) . Wild type control strain GW201 (HO ade6-1 trp5-1 leu2-1 lys1-1
ura3-1 PMA1::URA3) was constructed by transplacing a 6.1-kb HindIII fragment containing intact PMA1 linked 3` to URA3 into SH122 (HO ade6-1 trp5-1
leu2-1 lys1-1 ura3-1 pmal
::LEU2/PMA1), as
described by Harris et al.(19) . Wild type strain SN236 (HO ade6-1 trp5-1 leu2-1
lys1-1 ura3-1PMA1) is a derivative of SN236 (20) in which the URA 3 marker has been lost. All
yeast cultures were grown to early log-phase in YEPD medium (1% yeast
extract, 2% peptone, and 2% dextrose, pH 5.7) at 22 °C to an A
3. Growth sensitivity to hygromycin B
was monitored in YEPD agar plates containing 0, 100, 150, 200, and 300
µg/ml hygromycin B. Growth sensitivity to low pH medium was
determined at pH 2.5 in YEPD plates in the presence of 5 and 10 mM potassium acetate. Temperature sensitivity of growth was assessed
by comparing growth at 30 and 40 °C. (The wild type yeast strain,
Y55-background, used in this study grows normally at 40 °C unlike
other wild type strains of Saccharomyces. It is important that
the 40 °C incubator should be saturated with water during cell
growth.)
Site-directed Mutagenesis
Site-directed pma1 mutants were constructed essentially, as described
previously(20, 21) . PMA1 mutants F119A,
F126A, M128A, E129A, F159A, G122N, M128C, and M128S were initially
prepared in phagemid vector pSN54, which consists of a 2.1-kb Asp718 fragment from PMA1 subcloned into
pGEM-3zf(20) . Mutants prepared in pSN54 were first excised as
part of a 0.7-kb BstEII-EcoRV fragment, and then
purified by agarose gel electrophoresis. The purified fragment was
reconstituted in PMA1 by subcloning into identical sites in
alkaline phosphatase-treated pIV100 (derived from pSN57 containing an
additional BamHI site in the BstEII-EcoRV
exchange region of PMA1)(21) . Mutants G122A and P123A
were prepared in phagemid vector pDP100, which carried a new AvaII restriction site, also in BstEII-EcoRV
exchange region of PMA1. These mutants were first screened by
restriction digest analysis and then sequenced. A purified 0.7-kb BstEII-EcoRV fragment was transplaced into pSN57 to
reconstitute intact PMA1, as described above. Other mutants
were prepared in phagemid vector pGW201, which consists of a 6.1-kb HindIII fragment containing PMA1 marked with URA3 at the 3` non-coding end(19) . The entire 6.1-kb region
was excised and transplaced into yeast directly. All vectors containing
reconstituted pma1 genes were sequenced prior to
transplacement into yeast to confirm the primary site mutation and to
eliminate potential secondary mutations in the target region. Isogenic pma1 mutants were prepared as described by Harris et
al.(19) . All pma1 mutations were reconfirmed
after isolating meiotic segregants by polymerase chain reaction
amplification of chromosomal DNA and sequence analysis, as described
previously(21) .
Plasma Membrane Isolation and ATP Hydrolysis
Measurement
Plasma membranes were purified from wild type and pma1 mutant strains by centrifugation on a sucrose step
gradient, as described previously(25) . ATP hydrolysis
measurements were performed in a microplate assay in a 100-µl
volume containing 10 mM Mes/Tris (pH 6.5), 25 mM
NH
Cl, 5 mM ATP, 5 mM MgCl
,
0.5 mM NaN
, and 1 µg of membrane protein, as
described by Monk et al.(26) .
Reconstitution and Proton Transport Measurements
A
microsomal membrane fraction was prepared essentially by the method of
Perlin and Brown(27) , except that a lower centrifugal force
(75,000
g for 30 min) was used for membrane recovery.
The membrane vesicles were extracted with 0.5% (w/v) deoxycholate in
Solubilization Buffer, consisting of 10 mM Hepes-KOH (pH 7.0),
0.1 M KCl, 45% glycerol, 0.2 mM EDTA, 1 mM
dithiothreitol, and 1 mg/ml asolectin, as described
previously(25) . The detergent-extracted membranes were washed
with an equal volume of 0.3 M KCl-Solubilization Buffer by
resuspension and centrifugation, as above. The KCl-washed membranes
(350 µg) were resuspended in a 800-µl volume containing 10
mg/ml asolectin, Solubilization Buffer, and 0.5% (w/v) deoxycholate
(added dropwise with gentle stirring). The mixture was placed on ice
for 5 min and then rapidly diluted into 25 ml of ice-cold Dilution
Buffer containing 10 mM Hepes-KOH (pH 7.0), 100 mM KCl, and 1 mM dithiothreitol. The reconstituted vesicles
were recovered by centrifugation at 250,000
g for 1 h.
The pellet was resuspended in 400 µl of the dilution buffer. Proton
transport measurements were made according to the method of Perlin et al.(28) . A fluorescence quenching reaction volume
consisted of 1 ml of 10 mM Hepes-KOH (pH 7.0), 50 mM KCl, 5 mM ATP, 1 µg/ml valinomycin, 1.5 µM acridine orange, and 50 µg of reconstituted vesicles. The
reaction was initiated by the addition of 5 mM MgCl
. Fluorescence intensity was monitored on a
Perkin-Elmer LS-5 spectrofluorometer.
H
-ATPase Abundance
Measurements
SDS-polyacrylamide gel electrophoresis and semidry
electroblotting of plasma membrane proteins were performed, as
described previously(26) . Western blot analysis was performed
with a polyclonal anti-H
-ATPase antibody, described by
Seto-Young et al.(21) . Western blots were scanned
with a UMAX color scanner (UMAX Data Systems, Inc.), and Adobe
Photoshop and NIH Image software were used to quantitate the level of
the intact H
-ATPase (molecular mass
100 kDa).
Standard default settings were used for all measurements, and all
mutant enzymes were compared to an internal wild type control on the
same gel.
Other Procedures
Protein was determined by a
modified Lowry method(28) . Yeast transformants were prepared
by the lithium acetate treatment method, as described in the
alkali-cation kit (Bio 101, Inc.). DNA sequencing of plasmid DNA was
performed either with Sequenase (version 2.0, United States Biochemical
Corp.) or by polymerase chain reaction amplification of the target
region and sequencing with the fmol sequencing system
(Promega). Transmembrane helices 1 and 2 of the yeast
H
-ATPase were constructed with the molecular modeling
program Insight II (version 2.2.1; Biosym Technologies) on a Silicon
Graphics IRIS computer (model 4D/70GT).
RESULTS
Phenylalanine Mutagenesis of Hairpin
Region
Aromatic residues are suggested to play an important role
in the folding and structural stability of many proteins(29) .
Seven Phe residues are contained within the TM1 and TM2 hairpin region
of the yeast plasma membrane H
-ATPase (Fig. 1).
Five of the 7 residues are located toward the cytoplasmic end. They are
predicted to be nested and overlapping(23) , and are
anticipated to be important for structural stability. The aromatic
amino acids in this region are conserved among the fungal ATPases,
apart from a single F163Y change in the Candida albicans enzyme(26) . Scanning Ala mutagenesis was used to replace
all of the Phe residues in the hairpin region with Ala by site-directed
mutagenesis. Alanine was chosen because it is frequently found in
helical structures, both inside and outside of the
bilayer(30, 31) . Table 1shows that Ala
substitution of Phe at positions 116, 119, 126, 159, and 163 produced
viable cells, indicating expression of a functional
H
-ATPase. (The H
-ATPase is essential
for growth and non-functional enzymes give a lethal phenotype.) F116A
and F163A produced growth phenotypes that were essentially wild type;
both mutations are predicted to lie at the base of TM1 and TM2,
respectively, near the cytoplasmic interface of the membrane. Mutant
strains carrying mutations F119A, F126A, and F159A showed growth
resistance to hygromycin B, which has been correlated with a defect in
membrane potential formation and high capacity proton
pumping(32) . Mutations F120A or F144A produced recessive
and/or dominant lethal phenotypes, respectively. To determine whether
an aromatic group was important, Trp was used in place of Ala. Both
F120W and F144W produced viable mutants, with F120W showing a normal
phenotype and F144W showing hygromycin B resistance and temperature
sensitivity at 40 °C. (It should be noted that the wild type yeast
strain used in this study, which carries a Y55 background, grows
normally at 40 °C unlike other wild type strains of Saccharomyces.) It was previously shown that Trp was the only
residue that could substitute for Phe at this position(21) .
Figure 1:
Molecular model for TM1 and TM2. A
molecular structure model for TM1 and TM2, as described by Monk et
al.(23) , showing the proposed membrane organization of
the hairpin region and the amino acid residues studied in this
report.
Plasma membranes were purified from wild type and all viable pma1 mutants. The abundance of intact
H
-ATPase (molecular mass of
100 kDa) in the
mutant membranes was assessed by SDS-polyacrylamide gel electrophoresis
and Western blot analysis; it was found to exceed 70% of the wild type
level (Table 2). ATP hydrolysis measurements (Table 2)
showed that the K
for ATP was not significantly
altered for most mutant enzymes; only enzymes from mutants F159A and
F144W showed slightly lower K
values. In contrast, V
(adjusted for enzyme abundance) was
significantly reduced in mutants F144W and F163A to 39 and 49%,
respectively, of the wild type level of activity, while other mutants
were reduced from 57 to 74%. The F144W mutation produced an enzyme with
lower activity (39%) than the F120W mutation (68%).
The vanadate
inhibition profiles (I
) for most of the
Phe-pma1 mutant enzymes were comparable to the wild type (Table 2). The pH dependence of ATP hydrolysis was assessed at pH
5.5, 6.5, and 7.5, and the hydrolysis activities at pH 5.5 and 7.5 were
expressed as a function of activity at the normal pH optimum pH 6.5.
All the Phe-pma1 mutant enzymes showed a near wild type-like
activity ratio, which at most, was 20% less than wild type (data not
show). The H
transport properties of mutants showing
hygromycin B-resistant phenotypes were examined in a reconstituted
vesicle system. The mutations had no significant effect on ATP-mediated
proton transport when equivalent amounts of ATP hydrolysis units were
assayed. The F144W enzyme showed a somewhat lower initial rate of
pumping, although the steady state pH gradient reached the same level
as wild type (Table 2).
Structural stability was assessed by
measuring ATPase activity in sucrose gradient-purified plasma membranes
at increasing temperature (30, 35, 40, 50, and 55 °C). Mutant
enzymes showed the same relative heat inactivation profile as wild type
(data not show). However, when mutant and wild type enzymes were heated
for 15 min at 45 °C in the presence of increasing concentrations of
urea (0-2.67 M), mutant enzymes F163A and F144W showed
enhanced heat inactivation (Fig. 2). Mutant enzymes F119A,
F126A, and F159A were comparable to wild type (data not shown).
Figure 2:
Urea/heat inactivation profile for mutant
enzymes. Plasma membranes (3 µg) were resuspended in 20 µl of
buffer consisting of 10 mM Tris/HCl (pH 7.0), 1 mM EGTA, 20% glycerol, and different concentrations of urea, as
indicated. The mixtures were incubated at 45 °C for 15 min and
diluted with 200 µl of ATP hydrolysis reaction buffer. ATP
hydrolysis was assessed for 1 h at 30 °C. The ATP hydrolysis
activities were expressed as a function of the control activity at 30
°C without urea.
Double Phe Mutants in the Hairpin Region
Double
Phe mutants were constructed to examine whether two Phe residues could
be substituted, which would result in a higher degree of enzyme
destabilization. Double mutant F116A/F119A, lying nearby on TM1, was
viable and gave normal growth phenotypes and enzymatic properties (Table 2). This was a somewhat curious result because the
individual mutant, F119A, showed abnormal growth and enzymatic
properties ( Table 1and Table 2). It appears that the F116A
mutation relieved the stress created by the F119A mutation. In
contrast, double mutant F159A/F163A, lying nearby on TM2, was not
viable. Other double mutants, F116A/F159A and F119AF/159A, which are
expected to be juxtaposed on TM1 and TM2, also produced non-functional
enzymes. The results suggest that a double substitution with Ala on TM1
and TM2 is not tolerated. However, a viable double mutant, F119V/F159A,
was isolated. This double mutant with Val on TM1 at position 119 showed
hygromycin B-resistant and low pH-sensitive growth phenotypes,
indicative of a perturbed enzyme. However, the mutant enzyme showed
rates of ATP hydrolysis (Table 2) and proton transport that were
wild type in behavior, indicating that the in vivo perturbation was relieved upon plasma membrane purification. Overall, these results suggest that eliminating two Phe from the
base of each transmembrane segment is highly destabilizing. The ability
of F119V to stabilize a second mutation at F159A may result from the
side chain partially filling a cavity created by the loss of the
aromatic residues from each helix. This would imply that, at the very
least, a space-filling role is indicated for the phenylalanines.
Mutagenesis of Proline Kink Region
TM1 contains a
Gly-122/Pro-123 combination that may form a highly flexible and helix
distorting region. Pro-123 is conserved among all fungal
H
-ATPases(8, 23) . Mutagenesis was
used to investigate whether a potential Pro kink involving Pro-123
might play an important role in the function of the
H
-ATPase. A P123A mutation produced viable cells that
showed growth phenotypes with hygromycin B resistance and low pH
sensitivity (Table 1). An additional substitution with a small
Gly residue or a bulkier Phe residue resulted in hygromycin B-resistant
phenotypes (Table 1). The P123G and P123A mutations showed 62%
and 71%, respectively, of the normal abundance of enzyme in the
membrane, although they showed only a moderate reduction in ATP
hydrolysis rates (Table 2). Gly-122, which precedes Pro-123, was
substituted with Asn, Ala, and Pro. The G122N and G122A mutations
showed growth resistance to hygromycin B, but only G122N showed low pH
sensitivity (Table 1). However, the G122N mutation showed normal
biochemical properties, while the G122A mutation was significantly
reduced in activity. A G122P mutation, which produced double Pro-Pro
residues at positions 122 and 123, was not permissible. Doublets of
Pro-Pro are rarely, if ever, found in
-helices(33) . A
double mutant, G122P/P123G, was constructed to shift the position of
the Pro down the helix, but it resulted in a nonfunctional enzyme (Table 1). A Pro was also introduced into TM2 by substituting Pro
for Ala at position 155. The mutant was highly perturbed, showing
mutant growth phenotypes (Table 1) and ATP hydrolysis rates that
were 20% of the wild type level (Table 2). This result suggests
that a Pro in TM2, potentially kinking or altering the helical
structure, was not favorable. Enzymes from mutants G122A, G122N, and
P123A showed small perturbations of H
transport, with
initial rates at 64-76% of the wild type. In each case, steady
state proton pumping levels approached that of wild type (Table 2). These results suggested that a Pro kink, if it exists,
in helix 1 is not required for H
transport. On the
other hand, the lethality of G122P, which creates a helix-destabilizing
Pro-122/Pro-123 cluster, along with the prominent mutant growth
phenotypes observed for viable mutations in this region, suggests that
maintenance of an
-helix is important.
Other Mutants in the Hairpin Region
Glu-129 is
predicted to lie in the middle of transmembrane segment 1. In the Neurospora H
-ATPase, this residue can be
modified by DCCD(34) , which inhibits enzyme activity. A
previous study indicated that E129Q or E129L mutations had little
effect on enzyme activity. Unfortunately, more subtle growth effects
could not be examined in the expression system utilized(35) .
We explored whether Glu-129 plays a role in function by substituting a
small Ala at this position. The E129A mutant had a weak effect on
phenotype, producing only mild hygromycin B resistance (Table 1).
As previously proposed (35) , this result suggests that the
bilayer-buried charged moiety Glu-129 is not important for catalytic
function. The mutation has some effect on the initial rate of the
H
transport, but steady state pH gradient formation
was comparable to the wild type (Table 2). It is puzzling that a
membrane-embedded charged moiety would be so highly conserved among the
fungal enzymes(8) , if it plays no apparent role in function. According to the molecular structure model, Met-128 on helix 1 is
predicted to lie within close proximity to Cys-148 on helix 2. An M128C
mutation was created to examine whether a disulfide linkage could be
established between the 2 residues. The M128C mutant yielded hygromycin
B-resistant and low pH-sensitive phenotypes, and the mutant enzyme was
significantly reduced in ATP hydrolysis and initial rate of
H
transport (Table 2). However, there was no
effect of sulfhydryl reagents on enzyme activity, which precluded an
affirmation of a disulfide linkage. In addition, Ala and Ser
substitutions at Met-128 were not viable, which further complicates the
analysis, since it is not possible to distinguish between perturbations
created by the mutation in helix 1 and a potential cross-linking of the
hairpin structure.
DISCUSSION
A Role for TM1 and TM2
It has been suggested for
P-type ATPases that the primary transported ion binds to a
cytoplasmically-exposed site on the membrane surface of the enzyme,
which is a considerable distance (50-60 Å) (36, 37) away from the catalytic center engaged in ATP
hydrolysis. Thus, long range structural interactions are necessary for
coupling to occur. Emerging evidence suggests that local interactions
in TM1 and TM2 provide important clues to the nature of such long
distant energy coupling. In the yeast H
-ATPase, the
notion that the hairpin region encompassing TM1 and TM2 is
conformationally linked to the catalytic ATP hydrolysis domain has
arisen from genetic studies of the extracellular turn
region(21) , and from studies identifying second site
suppressor mutations, which either partially or fully complement
phenotypes produced by a primary site mutation in PMA1. For
example, it was shown that the phenotype induced by a primary site
mutation, S368F, near the site of phosphorylation (Asp-378), could be
suppressed by second-site mutations in TM1 and TM2(19) .
Conversely, the phenotypes induced by a mutation, A135V, near the
extracytoplasmic face of TM1, could be suppressed by secondary site
mutations within the catalytic core of the ATP binding
domain(20) . Additional evidence linking the TM1 and TM2 to the
catalytic ATP hydrolysis domain in the yeast enzyme was provided by
showing that modification of Cys-148 in TM2 with omeprazole was closely
correlated with enzyme inhibition(38) . In addition, a G158D
mutation in TM2 was found to produce a partially uncoupled enzyme when
assayed in vitro(28) . Diverse studies on higher
eukaryotic enzymes also support a potential linkage between TM1, TM2
and the catalytic ATP hydrolysis domain. Genetic modification of
residues in TM1 and TM2, which alter ouabain sensitivity, was also
observed to alter catalysis by the
Na
,K
-ATPase(39) . A
monoclonal antibody that recognizes an epitope in the extracellular
turn region between TM1 and TM2 of the
Na
,K
-ATPase inhibited
catalysis(16) . Finally, the
H
,K
-ATPase antagonist SCH28080, which
blocks ATP hydrolysis, appears to bind within the loop region linking
TM1 and TM2(13) .TM1 and TM2 are predicted to form a
helical hairpin structure(13, 23) . We have used
molecular dynamic simulations to predict how perturbations in the
hairpin head region could be propagated throughout the structure (23) . In addition, we used a detailed genetic analysis to
explore limited conformational flexibility and tight packing in the
head region (21) , as predicted from the model studies. In this
study, we systematically investigated residues comprising putatively
important features of this structural region.
Importance of Aromatics
Interacting aromatic
residues in proteins are believed to be important for structural
stability and assembly of
proteins(29, 40, 41) . In addition, clustered
aromatics may be important for translocation across the bilayer as has
been recently proposed for sugar transport through the porin
channel(42) . The TM1/TM2 hairpin region contains seven Phe
residues, with five residues predicted to form a clustered grouping at
the cytoplasmic interface. These residues are predicted to be important
for structural stability through the involvement of potential
-
interactions(23) . In fact, the sequence
arrangement of Phe-116 and Phe-120 on TM1 and Phe-159 and Phe-163 on
TM2 should place these residues on the same face of their respective
-helical segments. A similar cluster of aromatics is found in the
-subunit of the Na
,K
-ATPase and
in the Ca
-ATPase(43, 44) . We
substituted each of the seven Phe residues with Ala and found only two
positions, Phe-120 and Phe-144 (described previously; (21) ),
which could not support the loss of side chain mass (Table 1). A
Trp substitution at these positions produced viable enzymes, suggesting
that bulky aromatic character was required. Of the remaining Phe
residues that could be substituted with Ala (Phe-116, Phe-119, Phe-126,
Phe-159, and Phe-163), the viable mutants showed varying growth
phenotypes ranging from wild type to strongly hygromycin B-resistant
and low pH-sensitive (Table 1). Two mutants, F163A and F144W,
showed enhanced thermal/chaotropic denaturation (Fig. 2)
indicative of a stability defect. More severe effects were observed
with double mutants constructed to remove a single Phe residue each
from TM1 and TM2. Double mutants F116A/F159A and F119A/F159A were
recessive lethal, as was a double mutant F159A/F163A on TM2 (Table 1). However, an F116A/F119A on TM1 produced a viable
enzyme that appeared wild type in behavior. Unless aromatic residues on
TM2 are interacting with Phe-120 on TM1, it appears that potential
interactions between the helical segments are not as important as the
presence of aromatic character on TM2. Of course, interactions with
other elements or lipid cannot be ruled out. The bulkiness of the Phe
side group is likely to be important. Deletion of these two residues
from TM1 and TM2 could create a cavity that could be highly
destabilizing, as has been observed with T4 lysozyme(45) . The
fact that a F119V/F159A mutation was viable (Table 1) suggests
that Val may partially substitute for the bulky Phe in this position.
Overall, these data suggest that aromatic residues are important for
the stability and/or folding the TM1,TM2 hairpin structure.In many
cases, reduced enzyme activity is correlated with growth phenotype, as
previously observed(19, 20, 28) . However, in
some cases (e.g. F119V/F159A), cells showing prominent mutant
growth phenotypes produced mutant enzymes displaying normal enzymatic
properties when examined in vitro (Table 2). One
possible explanation may be that altering phenylalanines in TM1,TM2
influences the assembly efficiency of this region, which could result
in an apparent growth irregularity. However, once the enzyme is
assembled, it behaves normally. This suggestion is intriguing in view
of the recently proposed role of TM1/TM2 as a catalyst in the membrane
assembly of the H
-ATPase in Neurospora(46) . Alternatively, since the in vivo enzyme is displaced from equilibrium and experiences numerous
potential constraints on catalytic activity such as pH gradients,
membrane voltage, turgor pressure, and regulation due to
phosphorylation, it may be that small perturbations are amplified and
show more pronounced affects on cell physiology. In contrast, the in vitro enzyme operating at V
capacity
would not be subject to these constraints, and would not be expected to
show significant differences from the wild type enzyme under the same
conditions. This latter explanation would be pertinent to all subtly
perturbing mutant enzymes which show differential in vivo and in vitro properties.
Is a Proline Kink Important in TM1?
The helix in
TM1 was predicted to kink toward TM2 about one third into the bilayer
due to the presence of a flexible Gly-122 and a helix-breaking
Pro-123(23) . Pro is an unusual amino acid in which its side
chain is cyclized back on the backbone amide position and backbone
conformation is restricted leading to a kinked structure(47) .
From a structural point of view, a Pro kink provides a way to make
curved helices that could be packed into a funnel-like structure or
into a cage-like structure(48) . Such packing is important for
membrane proteins such as bacteriorhodopsin in which structural
interactions between helices can be maximized(49) . In fact,
Pro is frequently found in bilayer-associated structures of membrane
proteins(50) . To examine the role of Pro-123, substitutions
were made with Ala, Gly, or Phe. The mutations produced viable enzymes
with moderate affects on growth phenotype and ATP hydrolysis ( Table 1and II), but a minor effect on the initial rate of
H
transport. The substitution of the preceding Gly-122
residue with either Ala or Asn produced very prominent hygromycin B
resistance, and the G122N substitution also gave a low pH-sensitive
phenotype. The G122A mutant enzyme was diminished in ATP hydrolysis,
although surprisingly, the G122N mutant enzyme appeared wild type in
ATP hydrolysis and H
transport. Swapping the Gly-122
and Pro-123 positions was not tolerated, and the introduction of a
double Pro-122/Pro-123 sequence, which should disrupt the helix, was
also lethal. These results suggest that neither Gly-122 nor Pro-123 are
essential for catalytic activity and the formation of a Pro kink, if it
exists, is not critical. However, since helix-forming residues like Ala
induce growth phenotypes indicative of a perturbed enzyme, it may be
that a Pro kink is important for efficient assembly. The results do
clearly suggest that TM1 is required to be helical because the double
Pro insertion was lethal. In addition, the introduction of Pro at
position 155 in TM2 was also lethal, suggesting that TM2 could not
sustain a kinked structure.
Other Features of Hairpin Region
Glu-129 is
predicted to lie in the middle of transmembrane segment 1. In the Neurospora H
-ATPase, this residue can be
modified by DCCD (34) , which inhibits enzyme activity. In view
of the role bilayer-associated DCCD-reactive Glu or Asp residues play
in H
transport by F
F
-type
H
-ATPases, we explored whether Glu-129 might play a
similar role. The substitution of Ala for Glu at this position had a
weak effect on phenotype producing only mild hygromycin B resistance (Table 1). The mutant enzyme showed somewhat lower levels of ATP
hydrolysis and initial rate of H
transport, but the
steady state pH gradient formation was comparable to wild type.A
prediction of the molecular structure model is that Met-128 on helix 1
should lie within close proximity to Cys-148 on helix 2(23) .
An M128C mutation was created to examine whether a disulfide linkage
could be established between the 2 residues. The M128C mutant showed
perturbed growth phenotypes, and it was significantly reduced in
catalytic activity (Table 2). However, there was no effect of
sulfhydryl reagents on this enzyme, which would be consistent with
disulfide bond formation. This analysis was further complicated by the
observation that Ala and Ser substitutions at Met-128 were not viable.
Thus, it was not possible to distinguish between perturbations created
by the mutation in helix 1, and a potential cross-linking of the
hairpin structure.
Conclusion
The most significant finding in this
study is that the aromatic side chains on TM2 appear most important for
the structural stability and viability of the
H
-ATPase. In addition, the presence of aromatic side
groups at positions 120 and 144 are essential for enzyme function. The
Pro-123 on TM1 is not critical to the enzyme, but an intact
-helix
is important. Overall, these results suggest that the helical hairpin
model provides a reasonable description of the TM1/TM2 region of the
H
-ATPase. They provide additional evidence that
perturbations within the helical hairpin, which are most significantly
manifested as changes in cellular growth phenotypes, alter the
efficiency of enzyme action.