Structure
–
function relationships in membrane segment 6 of the yeast plasma
membrane Pma1 H
+
-ATPase
Manuel Miranda
a,⁎
, Juan Pablo Pardo
b, Valery V. Petrov
ca
Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, 500 West University Avenue, El Paso, TX 79968, USA
bDepartamento de Bioquimica, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Ap. Postal 70159, Mexico D.F. 04510, Mexico c
Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Russia
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 18 September 2010
Received in revised form 24 November 2010 Accepted 30 November 2010
Available online 13 December 2010
Keywords:
PMA1 H+-ATPase
Plasma membrane ATPase P2-ATPases
Ion pump
Transmembrane segment 6
The crystal structures of the Ca2+- and H+-ATPases shed light into the membrane embedded domains involved in binding and ion translocation. Consistent with site-directed mutagenesis, these structures provided additional evidence that membrane-spanning segments M4, M5, M6 and M8 are the core through which cations are pumped. In the present study, we have used alanine/serine scanning mutagenesis to study the structure–function relationships within M6 (Leu-721-Pro-742) of the yeast plasma membrane ATPase. Of the 22 mutants expressed and analyzed in secretory vesicles, alanine substitutions at two well conserved residues (Asp-730 and Asp-739) led to a complete block in biogenesis; in the mammalian P-ATPases, residues corresponding to Asp-730 are part of the cation-binding domain. Two other mutants (V723A and I736A) displayed a dramatic 20-fold increase in the IC50for inorganic orthovanadate compared to the wild-type control, accompanied by a significant reduction in theKmfor Mg-ATP, and an alkaline shift in the pH optimum for ATP hydrolysis. This behavior is apparently due to a shift in equilibrium from the E2conformation of the ATPase towards the E1conformation. By contrast, the most striking mutants lying toward the extracellular side in a helical structure (L721A, I722A, F724A, I725A, I727A and F728A) were expressed in secretory vesicles but had a severe reduction of ATPase activity. Moreover, all of these mutants but one (F728A) were unable to support yeast growth when the wild-type chromosomalPMA1gene was replaced by the mutant allele. Surprisingly, in contrast to M8 where mutations S800A and E803Q (Guerra et al., Biochim. Biophys. Acta 1768: 2383–2392, 2007) led to a dramatic increase in the apparent stoichiometry of H+ transport, three substitutions (A726S, A732S and T733A) in M6 showed a reduction in the apparent coupling ratio. Taken together, these results suggest that M6 residues play an important role in protein stability and function, and probably are responsible for cation binding and stoichiometry of ion transport as suggested by homology modeling.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
P-type ATPases, which are found throughout prokaryotic and eukaryotic cells, use the energy from ATP hydrolysis to pump different cations and some other substrates across biological membranes[1–3]. They have a single large catalytic subunit, embedded in the lipid bilayer by 8–10 hydrophobic α-helices, and a common reaction mechanism in which theγ-phosphoryl group of ATP is transferred to a conserved aspartate residue to form a covalent phosphorylated intermediate[1,2]. This P-ATPase family contains several subfamilies (P1 to P5); the P2 pumps, one of the largest subfamilies, include the physiologically important mammalian Ca2+-, Na+,K+-, and H+,K+
-ATPases and H+-ATPases of fungi and plants. The yeast Pma1 H+
-ATPase, being the primary pump of the cell, belongs to this subfamily,
is encoded byPMA1gene, and it has been shown to be essential for growth.[4].
Within the last decade, crystal structures of the mammalian Ca2+ -[5,6]and Na+,K+-ATPases[7–9]and fungal and plant H+-ATPases [10,11]have appeared, providing a valuable framework for studying the molecular mechanism of P-type ATPases. The structure includes a cytoplasmic headpiece that is folded into three discrete domains: one (N) binds ATP; another (P) contains the phosphorylated aspartyl residue; and a third (A) may function as an“actuator”or anchor domain. A thick stalk-like region connects the cytoplasmic headpiece to the membrane domain (M), which consists of 10α-helices (M1 to M10) with varying lengths and inclinations. Earlier site-directed mutagenesis studies of the sarcoplasmic reticulum Ca2+- ATPase
located residues essential for Ca2+transport in 4 of the 10α-helices:
M4 (Glu-309), M5 (Asn-768 and Glu-771), M6 (Asn-796, Thr-799, and Asp-800), and M8 (Glu-908)[12–14]. As expected, the crystal structure showed that side-chain oxygen atoms from these residues contribute to two Ca2+-binding sites (I and II), situated in a pocket
⁎Corresponding author. Tel.: +1 915 747 6645; fax: +1 915 747 5808.
E-mail address:[email protected](M. Miranda).
0005-2736/$–see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2010.11.034
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j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m e mnear the middle of the membrane; three additional residues from M4 (Val-304, Ala-305, and Ile-307) also furnish main-chain carbonyl oxygens to site II[5]. Similarly, the crystallographic structures of the Na+,K+-ATPases from pig kidney [7] and shark rectal glands [9]
revealed the amino acid residues involved in the formation of two Na+/K+binding sites. In the pig kidney enzyme, the binding site I is
structured with three amino acid residues from M5 (main-chain oxygen of Thr-772 and the side-chains of Ser-775 and Asn-776) and two residues from M6 (Asp-804 and Asp-808, via a water molecule). Oxygens from main-chain of Val-322, Ala-323, Val-325 in M4, and side-chain of Asn-776 and Glu-779 in M5, Asp-804 in M6, and probably Glu-327 in M4 participate in the construction of binding site II[7,9,15].
The P-ATPase family is noteworthy for its ability to pump a wide range of cations, including H+, Na+, K+, Mg2+, Ca2+, Cu2+, Cd2+, Mn2+.
There are also major differences in cation stoichiometry from one member of the family to the next, ranging from 1H+/ATP for the plasma
membrane H+-ATPase of fungi and plants[16–18]to 3 Na+/2K+/ATP in
the Na+,K+-ATPase of animal cells (reviewed in [19]). Based on
sequence alignments, supplemented by cryo-electron microscopy images of theNeurosporaH+-ATPase[11]and the crystal structures of
theArabidopsisplasma membrane H+-ATPase[10]and the mammalian Ca2+and Na+, K+-ATPases[5–9], it is evident that the cation specificity
and stoichiometry determinants lie in the core of membrane-spanning segments M4, M5, M6, and M8 of these cation pumps. Indeed, site-directed mutagenesis of the Na+,K+-ATPase identified at least eight
residues in M4, M5, and M6 that are essential for cation occlusion and/or transport [20–22]. Site-directed mutagenesis of the yeast plasma membrane H+-ATPase has located positions in M5 and M8 at which
amino acid substitutions alter the coupling between ATP hydrolysis and H+transport[23,24].
The results described in this paper extend a systematic study of the yeast H+-ATPase by focusing on M6 of that enzyme. As described
above, M6 is part of the Ca2+-binding pocket in the sarcoplasmic
reticulum ATPase, contributing one residue to site I (Thr-799), a second to site II (Asn-796), and a third to both (Asp-800) [5,6]. Likewise, M6 of the Na+,K+-ATPase participates with two residues
(Asp-804 and Asp808) in the organization of Na+binding sites I and II
[7–9]. In Pma1, these residues correspond to Ala-729, Ala-726 and Asp-730, respectively. It therefore seemed worthwhile to carry out alanine-scanning mutagenesis along the complete length of M6 of the yeast H+-ATPase, searching for residues that may play a role in H+
transport and in any other aspect of the reaction cycle or the biogenesis and stability of the enzyme.
2. Materials and methods
2.1. Yeast strains and growth conditions
Two strains ofSaccharomyces cerevisiaewere used in this study: SY4 (MATa, ura3-52, leu2-3,112, his4-619, sec6-4ts GAL2, pma1:: YIpGAL-PMA1) and NY13 (MATaura3-52). In SY4, the chromosomal copy of thePMA1gene has been placed under control of theGAL1
promotor by gene disruption [25] using the integrating plasmid, YIpGAL-PMA1[26]. SY4 also carries the temperature-sensitivesec6-4
mutation which, upon incubation at 37 °C, blocks the last step in plasma membrane biogenesis and leads to the accumulation of secretory vesicles in the cell[27].
2.2. Site-directed mutagenesis
Mutations were introduced by polymerase chain reaction into a 519 bp BglII–SalI restriction fragment of the PMA1 gene that had previously been subcloned into a modified Bluescript plasmid[28]. Each fragment was sequenced, and then moved into the full-length
PMA1gene in plasmid pPMA1.2 for study in secretory vesicles[26]or
plasmid pVP3, a wild-type version of pGW201[24,29], for study in plasma membrane. To express the ATPase in secretory vesicles, a 3.7-kb HindIII to SacI piece of pPMA1.2 containing the entire coding sequence of the gene was transferred to the centromeric plasmid YCp2HSE, placing the mutant pma1 allele under control of two tandem arranged heat-shock elements; the resulting plasmid was transformed into strain SY4[26]. To introduce the mutation into the chromosomal copy of the PMA1 gene for expression in plasma membranes, a 6.1-kb HindIII fragment containing the mutant allele linked to URA3 was excised from plasmid pVP3 and integrated into strain NY13 using the Alkali-Cation Yeast transformation kit (Bio 101). In all cases, PCR amplification from genomic DNA and automated DNA sequencing was repeated to confirm the identity of the mutant allele.
2.3. Cell fractionation and measurement of expressed ATPase
For studies in secretory vesicles, transformed SY4 cells were grown to mid-exponential phase (A600~ 1) at 23 °C in supplemented minimal
medium containing 2% galactose, shifted to medium containing 2% glucose for 3 h, and then heat-shocked at 37 °C for an additional 2 h. The cells were harvested, washed, and secretory vesicles were isolated by differential centrifugation, further purified by gradient centrifugation[30], and suspended in 0.8 M sorbitol, 1 mM EDTA, 10 mM triethanolamine–acetic acid, pH 7.2, containing 1 mM diiso-propylfluorophosphates (DFP), chymostatin (2μg/ml) and leupeptin, pepstatin, and aprotinin (each 1μg/ml) as previously described[24]. For studies on plasma membranes, NY 13-derived strains were grown to mid-exponential phase in supplemented minimal medium (YNB) containing 2% glucose, and a plasma membrane-enriched fraction was obtained by the method of Perlin et al.[31], followed by washing in 1 mM EGTA–Tris, pH 7.5, and resuspension in the same buffer containing all protease inhibitors except DFP. All preparative procedures were carried out at 0–4 °C.
The amount of expressed ATPase was measured by SDS-polyacrylamide gel electrophoresis and Western blotting, as described elsewhere[32]. Blots were incubated with affinity-purified polyclonal antibody against the closely related plasma membrane H+-ATPase of Neurospora crassa[33]and then with125I-protein A (ICN, Irvine, CA), and
assayed by means of a PhosphorImager equipped with ImageQuant software version 5.0 (Molecular Dynamics). The expression level of mutant ATPase relative to a wild-type control was calculated from the average of three or more determinations.
2.4. ATP hydrolysis
Unless otherwise noted, ATP hydrolysis was assayed at 30 °C in 0.5 ml of 50 mM MES–Tris, pH 5.7, 5 mM KN3, 5 mM Na2ATP, 10 mM
MgCl2, and an ATP regenerating system (5 mM phosphoenolpyruvate
and 50μg/ml pyruvate kinase) in the presence and absence of 100μM Na-orthovanadate. The reaction was terminated after 20–40 min and the release of inorganic phosphate from ATP was measured by the method of Fiske and Subbarow[34]. The IC50for vanadate inhibition
was determined by measuring ATP hydrolysis in the presence of increasing concentrations of vanadate. For determination of Km
values, concentration of Na2ATP was varied between 0.15 and
7.5 mM with 5 mM MgCl2excess over ATP; actual concentrations of
Mg-ATP were calculated by the method of Fabiato and Fabiato[35]. To determine the pH optimum for ATP hydrolysis, the pH of the assay medium was adjusted to values between 5.2 and 7.5 with Tris base.
2.5. H+pumping; coupling between proton pumping and ATP hydrolysis
H+ pumping into the secretory vesicles was monitored by
fluorescence quenching of the pH-sensitive dye acridine orange as described previously[24,30]. Assays were carried out at 29 °C with
continuous stirring on a Hitachi F2000 fluorescence spectropho-tometer (excitation, 430 nm; emission, 530 nm) equipped with Intracellular Cation Measurement System software. Freshly prepared vesicles (50μg) were suspended in 1.5 ml of 0.6 M sorbitol, 20 mM HEPES-KOH, pH 6.7, 100 mM KCl, 20 mM KNO3, 2μM acridine
orange, and ATP (0.3–2.0 mM). After stabilization of baseline
fluorescence (120–150 s), proton pumping was initiated by the addition of MgCl2 (5.3–7.0 mM). Parallel measurement of ATPase
activity under the same conditions was performed in 100μl of 0.6 M sorbitol, 20 mM HEPES–KOH, pH 6.7, 100 mM KCl, 20 mM KNO3,
containing ATP (0.3–2.0 mM) and MgCl2(5.3–7.0 mM) at 29 °C for
20–40 min as described previously [23,24]. The reaction was stopped with 1 ml of 1.25% trichloroacetic acid and inorganic phosphate was measured as above.
2.6. Protein determination
Protein concentrations were assayed by the method of Lowry et al.
[36]or Bensadoun and Weinstein[37], with bovine serum albumin as standard. An equal volume of secretory vesicles or plasma membrane
resuspension buffers were added to the standards to compensate for changes in absorbance due to the presence of interfering compounds.
2.7. Homology modeling
A three-dimensional model of the yeast Pma1 H+-ATPase was built with the program SwissModel[38], using the atomic coordinates of the crystallographic structure of the Arabidopsis thaliana H+
-ATPase (PDB code 3B8C) in the E1conformation[10], and Clustal X for
sequence alignment[39]. Since the local identity between the two proteins can reach up to 50% in some regions and the global identity is close to 35%, the number and size of the gaps in the alignment were small, facilitating the construction of the model. The yeast Pma1 H+
-ATPase model was evaluated with PROCHECK[40]and WHAT_CHECK
[41] to assess the stereochemistry of the main- and side-chain residues, the packing quality and planarity of rings. ERRAT[42]was used to examine non-bonded interactions between different atom types, PROVE[43]to check atomic volumes and PROSAII to evaluate model accuracy using knowledge based potentials of mean force[44]. Accessible surface area of the amino acid residues was calculated with
Fig. 1.Sequence alignment of transmembrane segment 6. The 22-amino acid stretch from the beginning of M6 has been aligned for 33 representative P2-ATPases. Accession numbers
(top to bottom) are as follows: P05030, P49380, O94195, A3LP36, P28877, Q6FXU5, EDO17118, P24545, P09627, Q750N5, Q6BYK8, P07038, CAG83458, Q07421, Q92446, CAK43734, O74202, A1CS32, A1D509, Q4WKH3, Q96VD3, O74242, CAP70082, O14437, Q7Z8B7, Q6VAU4, P54211, O04956, P19456, P04191, P11505, P54707, and P04074.
Gerstein's algorithm[45]. The results indicate a medium quality for both the yeast model and the crystallographic structure of the plant H+-ATPase: Ramachandran plots with 76.2% and 52.7% of the
residues in the most favored regions (model and template, respectively); overall quality factors of 46.6 and 42.3 in the ERRAT analysis; average z-scores of 1.65 and 1.98 in PROVE: and z-scores of
−7.32 and−7.51 in PROSAII. Interestingly, the model built for theS. cerevisiaeH+-ATPase obtained better scores than the plant ATPase
template. Another homology model obtained for the yeast Pma1 H+ -ATPase based on the crystal structure of the Ca2+-ATPase has been
previously described[24].
3. Results
3.1. Choice of residues to be studied 3.1.1. Alignment and conservation
To explore structure–function relationships of the membrane segment 6 of the yeast plasma membrane H+-ATPase, amino acid
residues along this segment were subjected to Ala/Ser scanning mutagenesis. Based on an earlier hydropathy analysis and sequence alignments, 22 residues from Leu-721 through Pro-742 were included in this study (Fig. 1). Sixteen of these residues were replaced by alanine, while the six remaining alanine residues (Ala-726, Ala-729, Ala-732, Ala-735, Ala-737, and Ala-741) were replaced by serine. Each mutant allele was cloned into the expression vector YCp-2HSE and expressed in yeast secretory vesicles, as previously described[26]. As shown inFig. 1, it is significant the high conservation of Asp-730 in all cation motive ATPases, as well as Asp-739 and Ala-726 in the proton pumps. Asp-739 is substituted with either Asn or Glu and shifted by one position towards the C-terminus in the Na+,K+-, and H+,K+
-ATPases (Fig. 1). The alignment also shows the large divergence in amino acid sequence when ascomycetes are compared with three basidiomycetes (U. maydis, U. fabae, and C. neoformans), one glomeromycete (G. mosseae), and one fungus-like oomycete (P. infestans). In general, substitutions at variable positions are rather conservative: Ile→Leu, Val; Val→Leu, Ile; Leu→Ile; Ala→Val (Fig. 1). Nissen and coworkers have recently presented a crystal structure for the close relative AHA2 H+-ATPase ofArabidopsis thalianaobtained at
3.6 Å resolution[10]. The AHA2 model shows M6 extending from Ala-673 to Asp-693 (Ile-719 to Asp-739 inS. cerevisiaePma1 ATPase), with thefirst 7 amino acid residues structured inα-helix (Ala-673 to Ile 681), and the next 12 residues in either turn or coil conformations (Leu-682 to Asp 693,Fig. 2).
3.2. Expression and activity of the M6 mutant ATPases in secretory vesicles
Of the 22 mutations studied, only two (D730A and D739A) led to complete block in membrane biogenesis that prevented the ATPase from reaching the secretory vesicles (Table 1). Other amino acid substitutions of the same two residues (D730N, D730V, D739N, and D739V) gave similar results in our earlier study[23,46]. Typically, this kind of behavior can be traced to a severe defect in protein folding, causing the abnormal ATPase to be retained by quality control mechanisms in the endoplasmic reticulum. Moreover, altered protein folding of the mutant Pma1 was indicated by increased rate of trypsinolysis of the metabolically labeled D730N, D730V, and D739V ATPases carried out in total membrane fractions[23,46].
The remaining 20 mutations were expressed at 18–100% of the wild-type level and had ATPase activities ranging from 3 to 101% of the control value. It should be mentioned that there was no direct correlation between the expression level and the activity of a mutant: poorly expressed (18%) strains like I725A, or reasonably expressed (29–46%) like L721A, I722A, L727A, and F728A or well expressed (76–
90%) like F724A, L734A, and Y738A) retained very low ATPase
activities (3–9% of the wild-type) which were equal or only slightly above the negative control (Table 1). Worth noting is the stretch of seven almost successive positions (Leu-721, Ile-722, Phe-724, Ile-725, Ile-727, Phe-728, and Asp-730), starting from the extracellular end of M6, at which Ala substitutions interfered markedly with ATPase activity, biogenesis, or both; by contrast, only 3 (Leu-734, Tyr-732, and Asp-739) of the 12 Ala substitutions towards the cytoplasmic end of M6 led to pronounced effects on biogenesis and/or activity (Table 1, depicted inFig. 2).
3.3. Kinetic properties
Previous work has shown that measurements of kinetic para-meters including theKmfor Mg-ATP, IC50for orthovanadate, and pH
optimum can give useful information about the reaction mechanism of mutant H+-ATPases[47,48]. When such measurements were made
for the 12 M6 mutants with sufficient activity to be characterized in detail, two mutants stood out from the rest: V723A and I736A. Both were strikingly resistant to orthovanadate, with IC50values 20-fold
higher compared to the wild-type control (Table 2). They also displayed 15- to 2.5-fold decreases in Km for Mg-ATP (to 0.1 and 0.6 mM, respectively) and, in the case of V723A, an alkaline shift in pH optimum (to 6.6). As discussed previously, such changes can be accounted for by a shift in equilibrium from the vanadate-sensitive E2
conformational state towards E1, which has a much lower affinity for
orthovanadate but a higher affinity for Mg-ATP (Fig. 4). Mutations leading to similar kinetic changes have been described in the Na+,K+
-ATPase and have been interpreted in the same way[49,50].
Fig. 2. Amino acid residues important for biogenesis, stability, and activity in transmembrane segment 6 of the yeast H+-ATPase. The homology model of the yeast
proton pump was built using the atomic coordinates of the E1 conformation of the
A. thalianaH+
-ATPase as described inMaterials and methods. Thefigure was prepared with PyMol version 0.97[59]. For clarity, the lower half of M6 helix is represented in green.
3.4. H+pumping by the M6 mutants
Given the known contribution of M6 to the transport pathway of sarcoplasmic reticulum Ca2+-ATPase and Na+,K+-ATPase, it was of
particular interest to ask whether any of the mutations in the present study affected proton pumping by the yeast plasma membrane ATPase. For this purpose, the initial rate of acridine orange
fluorescence quenching was measured over a range of Mg-ATP concentrations, and the rate of quenching was plotted as a function of the rate of ATP hydrolysis, measured in parallel under the same conditions.
Plots for 12 M6 mutants are shown inFig. 3. In every case, there was a roughly linear relationship between the rates of quenching and hydrolysis, as one would expect if the stoichiometry of proton transport remained constant over the entire range of pump velocities. For most of the mutants, including V723A and A729S towards the extracellular end of M6 and V731A, A735S, I736A, A737S, N740A, A741S, and P742A located towards the cytoplasmic end (Fig. 3), the apparent coupling between transport and ATP hydrolysis (pumping slope) was close to that seen in the wild-type control (Table 2). Only minor changes were accounted for V723A, V731A, and N740A mutant pumps. Three mutants, however, gave slopes significantly lower than the wild-type value (1.00): A726S (0.44), A732S (0.46), and T733A (0.45), consistent with a partial uncoupling between ATP hydrolysis and H+transport.
3.5. Expression and activity of the M6 mutant ATPases at the plasma membrane
To look further at the role of M6 in biogenesis and function, the eight Ala mutations (L721A, I722A, F724A, I725A, I727A, F728A, L734A, and Y738A) expressed in secretory vesicles at 18–90% but lacking ATPase activity were integrated into the chromosomal copy of thePMA1 gene. The goal of this experiment was to ask whether any of the mutant ATPases might regain activity at the plasma membrane in the absence of heat-shock step (23 to 37 °C), an integral part of the secretory vesicle expression system. All but one (F728A) alleles were unable to support growth at temperatures between 23 and 30 °C, suggesting that these positions are important for the proper functioning and/or folding of the yeast Pma1 ATPase. The wild-type-like expressed F728A mutant grew well and showed ATPase activity
Table 1
Effect of mutations in transmembrane segment 6 on expression, ATP hydrolysis and H+pumping.
Strain Expressiona ATP hydrolysisb H+pumpingc
Uncorrected Corrected Uncorrected Corrected
% U/mg % % %Q/mg % % Wild-typed 100 4.53 100 100 1258 100 100 Vectore 3 0.09 2 * * * * L721A 35 0.26 6 * * * * I722A 29 0.30 7 * * * * V723A 88 0.71 16 18 811 65 87 F724A 76 0.36 8 * * * * I725A 18 0.12 3 * * * * A726S 100 1.00 22 22 320 25 25 I727A 29 0.15 3 * * * * F728A 46 0.30 7 * * * * A729S 90 3.46 76 84 1406 112 124 D730A 2 0.06 1 * * * * V731A 98 2.81 62 63 823 66 67 A732S 76 1.13 25 33 288 23 30 T733A 70 2.26 50 71 647 52 74 L734A 78 0.18 4 * * * * A735S 85 1.58 35 41 591 47 55 I736A 86 1.18 26 30 332 26 30 A737S 89 1.57 35 39 524 42 47 Y738A 90 0.39 9 * * * * D739A 6 0.09 2 * * * * N740A 93 4.14 91 98 817 65 70 A741S 99 4.58 101 102 1006 80 81 P742A 93 2.88 64 69 672 54 60
aThe specific expression of 100-kDa ATPase protein was calculated by quantitative immunoblotting as described inMaterials and methods. b
ATP hydrolysis was measured as outlined inMaterials and methods. This series of experiments included 31 wild-type preparations with an average ATPase activity of 4.53 ± 0.26μmol Pi/min.mg. Data for mutant ATPases are the average of 3–12 determinations; each mutant value was corrected for expression relative to a wild-type control run in parallel
on the same day.
c
Fluorescence quenching of acridine orange was used to monitor pumping of protons into secretory vesicles as described underMaterials and methods. Wild-type quenching averaged 1258% Q/min.mg; each value was corrected for expression relative to its parallel wild-type control.
d Secretory vesicles were isolated from cells containing the expression plasmid YCp2HSE with the wild-typePMA1gene (positive control).
e Secretory vesicles came from cells carrying the plasmid with noPMA1gene (negative control). Asterisks indicate that no corrections were made for preparations with low
expression and/or ATPase activity.
Table 2
Effect of mutations in transmembrane segment 6 on ATP hydrolysis kinetics and H+ pumping (%)a
.
Strain Km IC50 pH optimum Coupling ratio
Wild-type 1.5 2.5 5.7 1.00 V723A 0.1 49.1 6.6 0.73 A726S 0.7 5.9 6.0 0.44 A729S 1.3 9.8 5.7 0.97 V731A 1.3 2.4 5.7 0.72 A732S 0.6 6.7 5.7 0.46 T733A 2.5 3.1 5.4 0.45 A735S 0.9 3.0 5.7 0.80 I736A 0.6 53.5 5.7 0.79 A737S 1.4 5.9 5.4 0.82 N740A 1.6 1.4 5.7 0.71 A741S 1.1 2.0 5.4 0.95 P742A 1.2 2.3 5.4 0.93
aATP hydrolysis kinetics and coupling ratio between ATP hydrolysis and proton
pumping were measured as outlined inMaterials and methods.Kmcorresponded to
mM ATP; IC50, toμM orthovanadate. Fluorescence quenching of acridine orange was
used to monitor H+
similar to non-mutated enzyme (72% of the wild-type control, not shown).
4. Discussion
The plasma membrane H+-ATPase ofS. cerevisiaebelongs to the
family of P-type ATPases, which contain enzymes that catalyze the translocation of a large variety of cations across the membrane[1–3]. Some members of this family also participate in the transport of phospholipids and the maintenance of the lipid bilayer asymmetry
[3]. P-type ATPases are highly distributed along the branches of the life tree, from eubacteria and archaea to all eukaryotes[3]. During their catalytic cycle, the cation motive ATPases alternate between two major conformations (E1 and E2) with different affinities for the substrate Mg-ATP and different orientations of the cation-binding sites, features that are important for the coupling of the hydrolysis of ATP with the translocation of cations across the membrane (Fig. 4). A distinctive characteristic of these enzymes is the ATP-dependent phosphorylation of a strictly conserved aspartate residue during the catalytic cycle[1,2]. Amino acid sequence alignments, cryo-electron
Fig. 3.H+
transport as a function of ATP hydrolysis in secretory vesicles for mutant ATPases. Proton translocation was measured as the initial rate of acridine orangefluorescence quenching at different concentrations of Mg-ATP (0–2.0 mM) and plotted against the rate of ATP hydrolysis obtained under the same conditions for wild-type and several mutants. WT, wild-type.
microscopy and the recent crystal structures of the Ca2+-, Na+,K+
-and H+pumps, point to a common folding pattern for the P-ATPases [8,10,11], with the determinants of cation specificity and cation stoichiometry situated in a core of membrane-spanningα-helices corresponding to M4, M5, M6, and M8.
Given the known contribution of M6 to the transport pathway of Ca2+- and Na+/K+-ATPases, it was of particular interest to ask
whether any of the mutations affected H+pumping by the Pma1
ATPase. For this purpose, the initial rate of acridine orangefl uores-cence quenching was measured over a range of Mg-ATP concentra-tions, and the rate of quenching was plotted as a function of the rate of ATP hydrolysis, measured in parallel. As illustrated inTable 1and
Fig. 2, there is a stretch of seven almost successive positions (Leu-721, Ile-722, Phe-724, Ile-725, Ile-727, Phe-728, and Asp-730), starting from the extracellular portion of M6, at which Ala substitutions interfered markedly with ATPase activity, biogenesis, or both. By contrast, only 3 (Leu-734, Tyr-732, and Asp-739) of the 12 Ala sub-stitutions toward the cytoplasmic end of M6 led to pronounced effects on biogenesis and/or activity (Table 1,Fig. 2). Based on these results, we suggest that the correct structure of the extracellular face of the M6 helix is critical for proper folding and functioning of the enzyme, including coupling between H+transport and ATP hydrolysis. Apparently, improper folding should also affect interaction between transmembrane segments and/or between them and lipid microenvi-ronment, causing malfunction of the enzyme.
For most of the active mutants, including V723A and A729S towards the extracellular side of M6 or V731A, A735S, I736A, and A737S towards the cytoplasmic side, the apparent coupling ratio, measured as the pumping slope, was close to that seen in the wild-type control (Table 2). The mutants A726S (Ala-726 corresponded to Asn-796 of SERCA1a Ca2+-ATPase), A732S, and T733A, however, gave
slopes significantly lower than the wild-type value, indicating a twofold decrease in the apparent coupling ratio between ATP hydrolysis and H+transport, most likely due to decreased coupling
efficiency.
Two mutants (D730A and D739A) led to complete block in membrane trafficking that prevented the ATPase from reaching the secretory vesicles (Table 1). The presence of Asp-730 in all plasma membrane H+-ATPases and site-directed mutagenesis experiments
suggest that this residue plays an essential role in H+binding and
transport, and biogenesis of the pump. In our earlier study, conservative substitution D730E was reasonably expressed in
secretory vesicles (48% of the wild-type level). This mutant retained low ATP hydrolyzing activity (15%) but undetectable H+transport [23,51]. Although other mutations at Asp-730 in theS. cerevisiaeH+
-ATPase led to unfolding of the enzyme and retention in the endoplasmic reticulum, the essential role of Asp-730 in H+-binding
and transport is obvious and comes from several lines of evidence including: (i) expressed in yeast, theA. thalianaH+-ATPase mutants
D684N, D684A, and D684R reached the plasma membrane and retained the ability to hydrolyze ATP; however, these mutant ATPases were unable to pump protons, suggesting a defect in the transition from E1P to E2P[52,53]; (ii) Asp-730 in M6 is strictly conserved in all plant, yeast and protozoa H+-ATPases; in the mammalian Ca2+ -ATPases (Asp800 in SERCA1), Na+,K+-ATPases (Asp808, in the pig
enzyme) and the H+,K+-ATPase (Asp824 in the human enzyme);
(iii) similarly, in the yeast Pmr1 Ca2+,Mn2+-ATPase corresponding
residue (Asp-778) is responsible for cation transport[54]; andfinally, (iv) based on the high resolution crystal structure for the SERCA1 ATPase in the E1 conformation, Asp-800 clearly participates in binding of Ca2+[5].
The structural model of theS. cerevisiaeH+-ATPase obtained by
homology modeling using the plant ATPase shows that Asp-730 is close to the well conserved Asn-154 in M2 (at 2.9 Å), compatible with the occurrence of a hydrogen bond between the two residues (Fig. 2). In the crystal structure of theA. thalianaH+-ATPase, it was proposed
that proton specificity of these enzymes depends on the Asp-730-Asn-154 couple, which acts as the gate keeper along the transport pathway
[10]. However, replacement of Asp-730 by alanine inS. cerevisiaeH+
-ATPase led to a block in the biogenesis of the -ATPase, indicating an additional role of this residue in the folding or stability of the protein (Fig. 4). The other conserved residue, Asp-739, is outside the membrane domain, in the cytoplasmic portion of the enzyme and situated in a coiled region of the homology model (Fig. 2). Although these residues are in coil conformation, Asp-739 is not fully exposed to the solvent, showing an accessible surface area of 40.8 Å2. Of all the
amino acid residues in close proximity to Asp-739, Arg-687 seems to be the most relevant. This residue is located at 5.0 Å from Asp-739 and deeply buried from the solvent (accessible surface area of 11.4 Å2).
Since Asp-739 is the only acidic residue close to Arg-687, we can speculate that it is important for the neutralization of the positive charge, and replacement of Asp-739 by alanine will have a negative impact on the stability of the protein.
Three other M6 mutations (I722A, I725A and I727A) were ex-pressed rather poorly in secretory vesicles and, accordingly, displayed ATPase activities that were not significantly different from negative control (empty plasmid,Table 1). Since substitutions by large residues are observed at these three positions in H+-ATPases (I→L, M, V, Y, Fig. 1), it is likely that the replacement of isoleucine (169 Å3) by the
smaller alanine residue (92 Å3) will induce rearrangements of both the main- and side-chains of the protein with the consequent alteration in the folding of the protein.
The remaining 17 mutations were expressed at 35–100% of the wild-type level, with 12 mutants displaying ATPase activities ranging from 18 to 101% of the control value, and 8 mutants with ATPase activities comparable to the negative control (empty plasmid,
Table 1). Since the mutations were located in the membrane domain and the expression of these proteins was satisfactory, indicating insignificant changes in the correct folding of mutant ATPases, it is reasonably the assumption that only the H+binding site (but not the
ATP binding site) was altered, and therefore that the low ATPase activity was due to the tight coupling between the hydrolysis of ATP and the binding/translocation of protons (Fig. 4). On the other hand, lower coupling efficiency in mutants A726S, A732S, and T733A can be explained by the instability of the E1-P intermediate (Fig. 4).
Interestingly, site-directed mutagenesis of M6 residues provided different results when compared to our previous study on Pma1 M8 segment[24]. In M8, of the 21 substitutions made, four were found to
Fig. 4.Simplified Post–Albers scheme of the catalytic cycle of plasma membrane H+
-ATPase. In the E1 conformation, the ATPase binds nH+(n, one or two) and ATP from the
cytoplasm triggering the phosphorylation of the fully conserved catalytic aspartate residue. In the E2 conformation, the enzyme has high affinity for phosphate and its transition state analog vanadate.
be not expressed (1–9%); two others were poorly expressed (18–19%) and inactive. By contrast, of the 22 Ala substitutions studied in M6 only two mutants (D730A and D739A) were not expressed; three (I722A, I725A and I727A) were expressed rather poorly; and the rest was reasonably expressed (Table 1). At the same time it should be noted that even if the mutant is expressed poorly, it may be active enough for detailed studies (e.g., L797A mutant in M8 with expression of 19% and activity of 20% of the wild-type level) [24]. However, among 20 expressed M6 mutants only 12 were active enough to measure the ATPase activity and perform detailed studies (Tables 1, 2andFig. 3); 2 of them have kinetics significantly altered and 3 M6 mutants showed a reduced H+/ATP coupling efficiency (Table 2). However, the
differ-ences were not as dramatic as in M8 where substitutions at 5 positions led to strong decrease in coupling efficiency (0.16–0.20, compared to 1.0 for the wild-type), while two others caused significant increase of this ratio (1.80–2.60), indicating probably that 2 H+are pumped per
ATP molecule [24]. Therefore, one can suggest that M6 plays an important role in H+-ATPase functioning, being probably responsible
for cation binding and selectivity, similarly to pmr1 ATPase[54], while M8 is mostly responsible for stoichiometry[24]. Since the crystal structure of the plant H+-ATPase contains a cavity of 380 Å3, with
capacity for 12 water molecules[10], we built a model of the yeast Pma1 H+-ATPase with the proton binding pocket containing two
hydronium ions (Fig. 5B) which may undergo fast deprotonation as suggested by molecular dynamic simulations of the H+-binding site of
ATP-synthase[55]. However, more efficient H+transport by E803Q
mutant ATPase suggests that hydronium ions may be the ionic species to be transported, as it was proposed previously for the gastric H+,K+
-ATPase[56,57]. Homology modeling of the yeast pump with the Ca2+
-ATPase also showed that two hydronium ions could be bound and transported by the yeast enzyme (Fig. 5A)[24]. It is important to highlight the role in cation binding of the strictly conserved aspartate residue (Asp-730 in the yeast enzyme) in the calcium (Asp-800) and sodium (Asp-804) pumps as compared to the H+-ATPases. As pointed
before, the side-chain of this residue is in front of M5 in the Ca2+- and
Na+,K+-ATPases, and participates in the configuration of binding sites
I and II. Early site-directed mutagenesis experiments showed the importance of this residue for Ca2+and Na+transport, supporting its
role in cation binding. However, in the plant enzyme Asp-684 is oriented towards M2, away from M5 and the prototypic binding site pocket (Fig. 5B). Given the low resolution of the crystal structure of the plant H+-ATPase, further work is required to define the orientation of
this residue in the proton ATPases. On the other hand, one might assume that an increase in H+/ATP stoichiometry is an abnormal
property of these particular mutations on M8, without any physio-logical relevance. However, it has been shown that the stoichiometry of the plasma membrane H+-ATPase ofN. crassachanges from 1 H+/
ATP to 2 H+/ATP under chronic energy restriction[58], suggesting that
adjustments in H+/ATP stoichiometry might play a role in the
adaptation of fungal cells to different environmental conditions.
Acknowledgement
The authors are grateful to Prof. C.W. Slayman who was a scientific adviser of this project and to K.E. Allen for the expert technical assistance (Yale University School of Medicine). This study was supported in part by the grant 5G12RR008124 to the Border Biomedical Research Center (BBRC)/University of Texas at El Paso) from the National Center for Research Resources (NCRR, NIH) and NIMH 5SC1MH 086070-02 to M.M.A., and by Program P-24“Origin of Biosphere and Evolution of Geo-Biological Systems”of the Presidium of the Russian Academy of Sciences to V.V.P.
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