全 文 ：Received 30 Aug. 2003 Accepted 10 Oct. 2003
Supported by the State Key Basic Research and Development Plan of China (G1999011700, 2003CB114300), the National Natural Science
Foundation of China (30170088, 30370120) and the Doctoral Program Foundation of the Educational Ministry of China (20020019030).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (4): 428-435
Identification of the Phosphorylation Site of the V-ATPase
Subunit A in Maize Roots
LIU Guan-Shan, CHEN Shuo, CHEN Jia*, WANG Xue-Chen
(College of Biological Sciences, China Agricultural University, State Key Laboratory
of Plant Physiology and Biochemistry, Beijing 100094, China)
Abstract : In the present study our investigation shows that phosphorylation of tonoplast proteins
purified from maize (Zea mays L.) roots increases obviously V-type H+-ATPase (V-ATPase) activities of ATP
hydrolysis and H+ transport. Further research indicates that some of the purified tonoplast proteins can be
thiophosphorylated and one band about 69 kD is identified as subunit A with antibody against subunit A of V-
ATPase. In order to determine the phosphorylation site(s) in subunit A of V-ATPase, the subunit A band at
69 kD was isolated from thiophosphorylated gel and then completely digested by trypsin. After purification
of these enzymatic lysis fragments with RP-HPLC, the molecular weight of phosphorylated peptide
fragment was determined as 573.83 Da with mass spectrometry. Data search indicates that subunit A can
generate 61 peptide fragments after tryptic digestion, of which only F56 with molecular weight of 573.66
Da is close to that of the identified fragment, and F56 can only be phosphorylated at Ser525. Therefore our
research suggests that Ser525 is the potential phosphorylation site of V-ATPase subunit A in maize roots.
To our knowledge, this is the first time to determine the phosphorylation site of V-ATPase subunit A in
plants.
Key words : V-ATPase; subunit A; phosphorylation site; tonoplast; maize
V-type H+-ATPas e (V-ATPas e) is widely distributed
within the eukaryotic endomembrane system including the
endoplasmic reticulum, the Golg i apparatus , membrane
vesicles, the tonoplast and even the plasma membrane. At
the plant tonoplast the V-ATPase is highly abundant, mak-
ing up 6.5%-35.0% of the total tonoplast protein in differ-
ent plant species (Klink et al., 1990; Fischer-Schliebs et al.,
1997). The V-ATPase can prov ide the driv ing fo rce fo r a
variety of secondary active trans port even ts of ions and
metabolites. When plants are subjected to environmental
stress, the V-ATPase exhibits a high degree of adjustability,
which is of important significance to plant adaptations and
survival under conditions of environmental stress (Rockel
et al., 1998; Xia et al., 2000;Yu et al., 2001).
The V-ATPase is a multisubunit protein complex, com-
posed of up to ten subunits, of which subunits A, B and c
exist in all species and are responsible for catalyzing ATP
hydrolysis , regu lating enzyme activity and transport ing
proton from cytoplast to vacuole respectively (Ratajczak,
2000). The V-ATPase holoenzyme consists of two domains,
a membrane peripheral domain (V1) hydrolyzing ATP and a
membrane integral domain (V0) trans porting proton. Sub-
units A and B are located in V1 sector and subunit c in V0
sector. Subunit A is the most conserved, its molecular mass
ranging from 67 to 73 kD in different plant species. In maize
(Zea mays), it has been reported that the V-ATPase subunit
A is composed of 561 amino acids with molecular mass of
69 kD (GenBank accession P49087). Protein phosphoryla-
tion and dephosphorylation exhibit regulatory roles to the
activity of a lot of cellular proteins and enzymes and are an
important regulatory manner in cellular signal transduction.
Recently it has been approved that a C-terminal penultimate
threonine residue can be phosphorylated by application of
P-type H+-ATPas e iso lated from s p inach (Spinacia
oleracea) leaf tis sue and Nicot iana plumbaginifo lia P-
type H+-ATPase isoform PMA2 expressed in Saccharomy-
ces cerevisiae and purified (Olsson et al., 1998; Maudoux
et al., 2000). There are some hin ts that phosphorylat ion
also plays important roles in V-ATPase activity regulation
(Ratajczak, 2000). However, relatively little is known about
molecular mechanisms by which phosphorylation regulates
V-ATPase ATP hydrolysis and proton transport activities
because of the very complex structure of V-ATPase. More-
over phosphorylation sites of V-ATPase subunits have not
yet been reported.
In this study, based on the fact that the activation of
LIU Guan-Shan et al.: Identification of the Phosphorylation Site of the V-ATPase Subunit A in Maize Roots 429
calcium-dependen t phosphorylation to V-ATPase ATP-
hydrolytic and proton-transport activities was observed at
tonoplast vesicles, one phosphorylation site of subunit A
was identified by substitution of fluorophore-labeled ATP-
g-S for ATP, providing direct evidence at molecular level for
elucidating V-ATPase regulatory mechanisms.
1 Materials and Methods
1.1 Materials
Maize (Zea mays L.) seeds were soaked in aerated water
for 36 h at room temperature and geminated on a net for 5 d
at 27 °C in darkness, rinsed twice with water daily. Root tips
(1-2 cm) of seedlings were harvested to prepare for tono-
plast vesicles. The fluorescent reagent 5-{[((2-iodoacetyl)
amino)ethyl]amino} naphthelene-1-sulfonic acid (1,5-
IAEDANS) was obtained from Molecular Probes and calf-
intestine alkaline phosphatase from Boehringer Mannheim.
All other reagents were of analytical grade and purchased
from Sigma.
1.2 Isolation and purification of maize root tonoplast-
enriched vesicles and assay of ATP-hydrolytic and H+-
transport activities of H+-ATPase
Tonoplast-enriched vesicles were isolated by the method
of Xia et al . (2000). The method of Yu e t a l. (2001) was
followed to identify the purity of the isolated tonoplast and
determine ATP-hydrolytic and H+-transport activities.
1.3 Phosphorylation and dephosphorylation of tonoplast
proteins
Phosphorylation reaction mixture (1 mL) contained 250
mmol/L s orbito l, 20 mmol/L Tris/Mes (pH 6.5), 2 mmol/L
MgCl2, 10 mmol/L free Ca2+ (calculated by the program Max-
chelator), 1 mmol/L NaF and 1-2 mg tonoplast p roteins
with (treatment phosphorylation a) and without (treatment
phosphorylation b) addition of the antibody of rabbit anti-
subunit A of oat V-ATPase (kindly supplied by Dr. H Sze),
respectively. The reaction mixtures were incubated at 30 °C
for 2 min and then added 1 mmol/L ATP for another 30 min;
control was the same as treatment phosphorylation b only
by substitution of red istilled water for ATP. Pre-cooled
buffer (250 mmol/L sorbitol, 2.5 mmol/L Tris/Mes, pH 7.2) in
10-fold volume was added into the above reaction and cen-
trifugation was carried out at 80 000g for 30 min. After re-
suspended with a sus pens ion (300 mmol/L sucrose, 10
mmol/L Hepes/Tris (pH 7.2), 1 mmol/L dithiothreitol), some
of the pellet was used to determine enzyme activity and the
other to carry out dephosphorylation reaction at 30 °C for
15 min in 1 mL mixture containing 250 mmol/L sorbitol, 10
mmol/L Tris/Mes (pH 7.5), 2 mmol/L MgCl2, 1-2 mg phos-
phorylated tonoplast p ro teins and 10 mg/mL alkaline-
phosphatase (treatment dephosphorylation).
1.4 Thiophosphorylation and fluorescent labeling of tono-
plast proteins
Iodoacetic acid was used to block surface cysteine resi-
dues of tonoplast proteins (1 mg each, with and without
addit ion of the antibody against subunit A of V-ATPase,
respectively), as described by Fadden and Haystead (1995).
Then the acetylated proteins were dialyzed against the buffer
(20 mmol/L Tris/Mes (pH 6.5), 2 mmol/L MgCl2, 1 mmol/L
NaF, 0.2 mmol/L EGTA and 10 mmol/L Ca2+) to remove ex-
cessive iodoacetic acid. Thiophosphorylation reaction was
carried out in the dialysate containing 500 mmol/L ATP-g-S
and 5 mmol/L dithiothreitol, as described by Jeong and
Nik ifo r ov ( 1999) and Che and Xia (2000). The
thiophosphorylated tonoplast proteins were dissolved in 1
mL of 50 mmol/L Tris -HCl (pH 7.0) containing 1.6 mmol/L
1,5-IAEDANS at room temperature for 5 h to carry out fluo-
rescent labeling. The reactions were stopped by addition
of 2× SDS-PAGE sample buffer.
1.5 Tryptic digestion of V-ATPase subunit A
A 69 kD fluorescent band was cut from SDS-PAGE gel
according to standard molecular weight and Western blot-
ting band. The gel band was cut up and washed twice at 30
°C with 150 mL of 50% acetonitrile in 200 mmol/L NH4HCO3
(pH 8.9) for 20 min each time. After dried at room temperature,
the gel band was digested at 30 °C overnight by addition of
50 mL of 0.02% Tween-20, 200 mmol/L NH4HCO3 (pH 8.9)
and 10 mL of tryptic mother solutio n (250 mg/mL in
NH4HCO3, pH 8.9). The reaction was stopped with 100%
trichloroacetic acid (TCA). The supernatant was extracted
twice with 100 mL of 60% acetonitrile in 0.1% TCA and then
freeze-dried.
1.6 Separation of tryptic digest fragments of subunit A by
RP-HPLC
The freeze-dried s ample was dis s olved in 0.1%
trifluoroacetic acid (TFA) and separated by reverse phase
high performance liquid chromatography (RP-HPLC), us-
ing a Nova-Pak (Waters) C18 column (3.9 mm× 150 mm)
with an acetonitrile/0.1% TFA gradient (1.0 mL/min) reach-
ing 50% for 35 min and with 50% acetonitrile/0.1% TFA for
5 min. All elution peaks were collected and monitored using
absorbance at 214 nm. The fluorescent peak was chosen
under ultraviolet ray and separated again by RP-HPLC un-
der the same conditions as above except 5%-25% acetoni-
trile/0.1% TFA. The fluorescent elute was freeze-dried.
1.7 Determination of the molecular weight of the fluores-
cent peptide by mass spectrometry
The fluorescent dried peptide was dissolved in the so-
lution of 1:1 acetonitrile/0.1% formic acid and its accurate
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004430
molecu lar weight determined by elect ros pray mas s
spectrometer, Quattro (Micromass, UK). Spray voltage was
4.25 kV, capillary entrance temperature 180 °C, accelerating
voltage 13V.
1.8 Protein electrophoresis and Western blotting
Protein electrophoresis and Western blotting were car-
ried out according to the methods of Laemmli (1970) and
Towbin (1979), respectively.
1.9 Protein determination
Protein was determined by the method of Peterson (1977)
with BSA as the standard.
2 Results
2.1 Effects of phosphorylation and dephosphorylation on
V-ATPase activities in maize roots
Tonoplas t-enriched ves icles can phosphory late
V-ATPase under existence of 10 mmol/L free Ca2+, the ATP-
hydrolytic activity increasing by 47% and the initial veloc-
ity and maximum of H+ transport increasing by 65% and
61% respectively (treatment phosphorylation b compared
with control in Table 1); otherwise dephos phorylation by
add ition of alkaline phosphatase in the phosphorylated
tonoplast proteins leads to a decrease in V-ATPase activi-
ties of ATP hydrolysis and H+ transport (treatment dephos-
phorylation compared with treatment phosphorylation b in
Table 1). The ATP-hydrolytic and H+-transport activities
can be partially res tored by treating the phosphorylated
tonoplast proteins with dephosphorylation. These results
indicate that the ATP-hydrolytic and H+-transport activi-
ties of V-ATPase at the tonoplast can be regulated by phos-
phorylation and dephosphorylation.
2.2 Thiophosphorylation of tonoplast proteins and West-
ern blotting of subunit A
Because of the increased activities of V-ATPase in phos-
phorylated tonop last p roteins, in order to determine the
phosphorylation site(s), we used the antibody against sub-
unit A of oat V-ATPase to determine whether the subunit A
can be phosphorylated. Our result clearly shows that there
are many proteins thiophosphorylated in (Fig.1, lanes 1
and 2). Among these proteins only one with 69 kD could be
recognized by the antibody against subunit A (Fig.1, lanes
2 and 3), its molecular weight was consisten t with that re-
ported in GenBank (accession number P49087) and there
was no corresponding band in lane 1 of Fig.1. In addition,
there are obvious decreases about the ATP-hydrolytic and
H+-transport activities after addition of the antibody against
subunit A, indicating that V-ATPase subuit A at maize root
tonoplast was inhibited (treatment phosphorylation a com-
pared with control in Table 1). All these together showed
that subunit A could be phosphorylated.
2.3 Identification and mass spectrometry of the fluores-
cent peptide from the tryptic digest fragments of subunit A
After it was confirmed that subun it A cou ld be phos-
phorylated in maize root tonoplast proteins, the following
assays were conducted to determine the phosphorylation
site(s) combined with data search. The band with 69 kD in
thiophosphorylated gel was d igested with trypsin. After
separation with RP-HPLC, the chromatogram of the tryptic
diges t of subunit A showed a strong fluorescence in the
elution peak with an asterisk and no fluorescence in other
elu tion peaks (Fig.2). Then the fluorescent pept ide was
Table 1 Effects of phosphorylation and dephosphorylation on the activities of H＋ -ATPase at maize root tonoplast
Treatment Relative hydrolytic activity (%)
Relative H+-transport activity (%)
Initial velocity Maximum
Control 100 100 100
Phosphorylation a 50± 3 60± 4 56± 4
Phosphorylation b 147± 9 165± 11 161± 10
Dephosphorylation 108± 6 130± 7 119± 6
The data are mean values in four separate experiments.
Fig.1. Ident ification of the phosp horylated subunit A of V-
ATPase at maize root tonoplast. 1, the fluorop hore-labeled and
thiophosphorylated tonoplast proteins after addition of the anti-
body against subunit A of oat V-ATPase under ultraviolet light; 2,
the fluorophore-labeled and thiophosphorylated tonoplast pro-
teins under ultraviolet light; 3, the West ern blott ing using the
antibody against subunit A of oat V-ATPase as first antibody.
LIU Guan-Shan et al.: Identification of the Phosphorylation Site of the V-ATPase Subunit A in Maize Roots 431
collected and freeze-dried for mass spectrometry to ana-
lyze its molecular weight. The analysis of electrospray mass
spectrometry with the above peptide fragment indicated
that there was a strong signal m/z 489 representing a double-
charged part icle peak [M+2H]2+of the pep tide by
biprotonation, m/z 500 [M+H+Na]2+ peak, m/z 511 [M+2Na]2+
peak, m/z 977 [M+H]+ peak and m/z 999 [M+Na]+ peak (Fig.
3). Mass spect rum information confirmed that a pept ide
fragment after trypt ic diges tion was thiophosphorylated
and fluorophore-labeled indeed , and also indicated that
under the electros pray s oft ion izat ion cond it ion, the
thiophosphorylated and fluorophore-labeled peptide frag-
ment was steady. Molecular weight o f the peptide frag-
ment was accurately determined as 976.00 Da by mass
spectrometry.
2.4 Analys is of the phosphorylation site of V-ATPase
subunit A
It has been reported that trypsin recognizes specifically
and hydrolyzes peptide bonds formed through arginine or
lysine carboxyl and there are 60 tryptic cut sites on maize V-
ATPase subunit A (GenBank accession P49087). After the
thorough tryptic digestion of subunit A, 61 peptide frag-
ments should be generated. The detailed information about
these fragment’s position, length, sequence and molecular
weight is listed in Table 2. In above investigations we have
confirmed that the molecu lar weight of the fluorophore-
labeled peptide fragment was 976.00 Da by mass spectrom-
etry (Fig.3). After subtraction of increased parts due to ad-
d itional modificat ion by th iophos phory lat ion and
fluorophore reagents, the original molecular weight of the
peptide fragment is 573.83 Da. The molecular weight is most
close to that of F56 (573.66 Da) and differs obviously from
those of other peptide fragments in Table 2. Data analysis
indicates that there is only one phosphorylated serine
namely Ser52 5 on F56, thus it could be considered that
thiophosphorylation took place at Ser525. The phosphory-
lation at Ser525 of subunit A analyzed by NetPhos2.0 soft-
ware (Blom et al., 1999) also indicates that there is a high
probability with the score equal to 0.996.
3 Discussion
3.1 Regulation of phosphorylation on V-ATPase activi-
ties in maize roots
The results in this article indicate that phosphorylation
of tonoplast proteins in maize roots increases significantly
the V-ATPase activities of both the ATP-hydrolysis and
H+-transport (treatment phosphorylation b compared with
control in Tab le 1) and dephosphorylation leads to a de-
crease in V-ATPase activ ities of ATP hydrolysis and H+
transport (treatment dephosphory lation compared with
treatment phosphorylation b in Table 1). Thus the activity
regulation manner of phosphorylation/dephosphorylation
exists in maize root V-ATPase. It was found that the V-
ATPase activities of ATP hydrolysis and H+ transport were
higher in plants under a given extent of environmental
stresses than in control plants in our previous work (Xia et
al., 2000; Yu et al., 2001) and other laboratory res earches
(Reuveni et al., 1990; Reuveni, 1992; Zhang and Liu, 2002).
Furthermore, cytosolic Ca2+ concentrations increase in en-
vironmental stresses (Reddy, 2001), so it can be speculated
that an increase in V-ATPase activity is made due to Ca2+-
mediated phosphorylation.
3.2 V-ATPase subunit A in maize roots can be phosphory-
lated
The roles of Ca2+ in plant cell signal transduction are
versatile . It is difficult to conclude that V-ATPase is the
protein kinase subs trate only from the fact that Ca2+ de-
pendent phosphorylation can regulate V-ATPase activity.
As shown in lane 2 of Fig.1, a multitude of proteins were
Fig.2. Purification of the tryptic digest of fluorophore-labeled
and thiophosphorylated maize V-ATPase subunit A by RP-HPLC.
An asterisk means the fluorescent fraction. A broken line means
the elution manner.
Fig.3. Electrospray mass spectrum to determine the molecular
weight of the thiophosphorylated and fluorophore-labeled pep-
tide fragment.
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004432
Table 2 The theoretic tryptic digest fragments of maize V-ATPase subunit A
Fragment Position Length Amino acid sequence MW
F1 1-2 2 AR 245.26
F2 3-22 20 ATIQVYEETAGLMVNDPVLR 2 219.43
F3 23-24 2 TR 275.29
F4 25-48 24 KPLSVELGPGILGNIFDGIQRPLK 2 562.03
F5 49-53 5 TIAIK 544.67
F6 54-61 8 SGDVYIPR 905.99
F7 62-70 9 GVSVPALDK 885.00
F8 71-80 10 DVLWEFQPTK 1 262.41
F9 81-115 35 LGVGDVITGGDLYATVFENTLMQHHVALPPGSMGK 3 626.14
F10 116-140 25 ISYIAPAGQYNLQDTVLELEFQGIK 2 811.18
F11 141 1 K 146.16
F12 142 1 K 146.16
F13 143-152 10 FTMLQTWPVR 1 278.52
F14 153-155 3 SPR 358.38
F15 156-160 5 PVASK 500.57
F16 161-172 12 LAADTPLLTGQR 1 255.43
F17 173-196 24 VLDALFPSVLGGTCAIPGAFGCGK 2 293.71
F18 197-205 9 TVISQALSK? 946.09
F19 206-220 15 YSNSEAVVYVGCGER 1 632.75
F20 221-242 22 GNEMAEVLMDFPQLTMTLPDGR 2 465.83
F21 243-248 6 EESVMK 721.80
F22 249 1 R 174.18
F23 250-264 15 TTLVANTSNMPVAAR 1 545.76
F24 265-279 15 EASIYTGITIAEYFR 1 733.95
F25 280-294 15 DMGYNVSMMADSTSR 1 664.82
F26 295-300 6 WAEALR 744.83
F27 301-305 5 EISGR 560.59
F28 306-322 17 LAEMPADSGYPAYLAAR 1 797.03
F29 323-329 7 LASFYER 884.98
F30 330-332 3 AGK 274.29
F31 333-334 2 VK 245.29
F32 335-341 7 CLGSPDR 746.82
F33 342-378 37 NGSVTIVGAVSPPGGDFSDPVTSATLSIVQVFWGLDK 3 719.15
F34 379 1 K 146.16
F35 380-383 4 LAQR 486.55
F36 384 1 K 146.16
F37 385-397 13 HFPSVNWLISYSK 1 577.78
F38 398-400 3 YSK 396.42
F39 401-408 8 ALESFYEK 986.08
F40 409-417 9 FDPDFIDIR 1 137.25
F41 418-419 2 TK 247.27
F42 420-421 2 AR 245.26
F43 422-426 5 EVLQR 643.72
F44 427-439 13 EDDLNEIVQLVGK 1 471.60
F45 440-447 8 DALAESDK 847.86
F46 448-454 7 ITLETAK 774.90
F47 455-457 3 LLR 400.50
F48 458-471 14 EDYLAQNAFTPYDK 1 674.78
F49 472-477 6 FCPFYK 803.79
F50 478-483 6 SVWMMR 808.98
F51 484-498 15 NIIHFNTLANQAVER 1 739.93
F52 499-506 8 AAGTDGHK 755.76
F53 507-513 7 ITYSVIK 822.98
F54 514-515 2 HR 311.32
F55 516-522 7 LGDLFYR 883.00
F56 523-527 5 LVSQK 573.66*
F57 528-541 14 FEDPAEGEEALVGK 1 490.58
F58 542-543 2 FK 293.34
F59 544 1 K 146.16
F60 545-554 10 LYDDLTTGFR 1 200.31
F61 555-561 7 NLEDEAR 845.85
LIU Guan-Shan et al.: Identification of the Phosphorylation Site of the V-ATPase Subunit A in Maize Roots 433
phosphorylated at tonoplast in such a Ca2+ dependent man-
ner and the fluorescent band with 69 kD was subunit A,
indicating that subunit A was also one of phosphorylated
substrates in the same Ca2+ dependent manner. Furthermore,
there are a large number of pro teins phosphorylated at
tonoplast, which indicates in a way that phosphorylation
plays an important role in post-translational regulations of
tonoplast proteins.
3.3 The phosphorylated si te of V-ATPase subunit A in
maize roots is at C-terminal
It can be inferred that there are many serine or threonine
sites possibly phosphorylated according to the amino acid
sequence of V-ATPase subunit A in maize roots. V-ATPase
subunit A was thoroughly in-gel digested with trypsin af-
ter thiophosphorylation and fluores cent labeling and the
digest fragments were separated by RP-HPLC in order to
determine which amino acid (s) was phosphorylated in vitro.
As a result, only one fluorescent fragment was obtained by
purification, so all phosphorylated sites should be included
in the fragment. The molecular weight, which was deter-
mined and calculated by mass spectrum, is very close to
that of predicted tryptic fragment F56. Moreover, there is
only one phosphorylated amino acid residue (Ser525) on
F56, so it can be judged that Ser525 is the potential phos-
phorylation site of V-ATPase subunit A in maize roots. Be-
cause the phos phorylation site and adjacent sequence at
C-terminal are all conserved in each V-ATPase subunit A of
many p lants (Table 3), phosphorylation at this site might
be ubiquitous in plants. To our knowledge, this is the first
identification of the phosphorylation site of V-ATPase sub-
unit A in plants. The amino acid sequence adjacent to Ser525
(R-L-V-S525) completely satisfies the need of plant CDPKs
to the substrate phosphorylation site, namely R/K-X-X-S/
T, where X is any amino acid (Roberts, 1989). Sze (1985)
pointed out that V-ATPase could be regulated by a protein
kinase, whose structure is different from that of PKC in a
certain extent. There is a calcium dependent protein kinase
(CDPK) confirmed at maize root tonoplast (Chen et al., 2002).
Thus it can be deduced that CDPKs might phosphorylate
V-ATPase subunit A in maize roots at the serine site and
consequently regulate its activity.
The addition of a charged phos phate group to the C-
terminal of subunit A might regulate V-ATPase activity by
changing subunit A conformation, activity and interaction
with some unknown intracellular factors such as 14-3-3 pro-
teins that bind the target protein at the phosphorylated
serine (Yaffe et al., 1997). Moreover, P-loop s equence
(GXXXXGKT/S, where X refers to any amino acid) is the
ATP/GTP-binding site in subunit A (Saras te et al., 1990)
and phosphorylation at Ser525 might affect the binding of
P-loop sequence to ATP through some way and sequen-
tially V-ATPase activity. Therefore, further studies should
be carried out to elucidate how phosphorylation at Ser52 5
affects V-ATPase activity.
Acknowledgements: The authors wish to thank Dr. H. Sze
for his kindly supply of polycloned antibody against sub-
unit A of V-ATPase from oat.
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Species Residue Sequence Accession number
Acetabularia acetabulum 569-586 GDLLYKVSSQKFEDPSDG Q38676
Arabidopsis thaliana 579-596 GDLFYRLVSQKFEDPAEG O23654
Brassica napus 579-596 GDLFYRLVSQKFEDPAEG Q39291
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(Managing editor: HE Ping)