Plant Cell Physiol. 45(6): 672–683 (2004)
JSPP © 2004
Rapid Paper
Isolation of Intact Vacuoles and Proteomic Analysis of Tonoplast from
Suspension-Cultured Cells of Arabidopsis thaliana
Taise Shimaoka 2, Miwa Ohnishi 1, 3, 8, Takashi Sazuka 4, Naoto Mitsuhashi 1, 8, Ikuko Hara-Nishimura 5,
Ken-Ichiro Shimazaki 6, Masayoshi Maeshima 4, Akiho Yokota 7, Ken-Ichi Tomizawa 2 and Tetsuro
Mimura 1, 3, 8, 9
1
Department of Biological Science, Faculty of Science, Nara-Women’s University, Nara, 630-8506 Japan
Plant Research Group, Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu-cho, Soraku-gun, Kyoto, 619-0292 Japan
3
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Chuou-ku, Tokyo, 113-0027 Japan
4
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan
5
Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502 Japan
6
Department of Biology, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka, 810-8560 Japan
7
Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara, 630-0101 Japan
2
;
A large number of proteins in the tonoplast, including
pumps, carriers, ion channels and receptors support the
various functions of the plant vacuole. To date, few proteins involved in these activities have been identified at the
molecular level. In this study, proteomic analysis was used
to identify new tonoplast proteins. A primary requirement
of any organelle analysis by proteomics is that the purity of
the isolated organelle needs to be high. Using suspensioncultured Arabidopsis cells (Arabidopsis Col-0 cell suspension), a method was developed for the isolation of intact
highly purified vacuoles. No plasma membrane proteins
were detected in Western blots of the isolated vacuole fraction, and only a few proteins from the Golgi and endoplasmic reticulum. The proteomic analysis of the purified
tonoplast involved fractionation of the proteins by SDSPAGE and analysis by LC-MS/MS. Using this approach, it
was possible to identify 163 proteins. These included wellcharacterized tonoplast proteins such as V-type H+-ATPases
and V-type H+-PPases, and others with functions reasonably expected to be related to the tonoplast. There were also
a number of proteins for which a function has not yet been
deduced.
Introduction
The vacuole is one of the most conspicuous organelles in a
plant cell. It contributes to cell growth by occupying most of
the volume of the cell, stores secondary metabolites and excess
nutrients, as well as acting as a repository for substances that
might otherwise interfere with cytoplasmic homeostasis
(Maeshima 2001). It has been well established by recent
research that the vacuole makes an important contribution to
many aspects of the overall physiology of plants (Dietz et al.
1998, Frangne et al. 2002, Massonneau et al. 2000, MatsuuraEndo et al. 1992, Takasu et al. 1997). However, little is known
about most of the activities that occur within the vacuole or on
the tonoplast membrane, especially how these activities are
coordinated and controlled at the molecular level (Maeshima
2001).
Functional mutations of vacuolar function-related genes
have been insufficient to provide useful insights into the
functions of vacuoles. Transformants, which lack the functions
of known vacuolar components (Gogarten et al. 1992,
Schumacher et al. 1999), have revealed some important roles of
vacuoles, but this approach has not been widely employed in
studies to date.
To address these deficiencies in our understanding of vacuolar function, we have attempted a comprehensive analysis of
membrane proteins in the tonoplast, using suspension-cultured
Arabidopsis cells. Membrane-associated proteins carry out a
range of important activities including transport and signaling.
Arabidopsis was chosen because its complete genome has been
sequenced and it was therefore easier to relate molecular information to protein function.
Over the past few years, various proteomic analyses of
plant cells have been reported (Ferro et al. 2003, Ferro et al.
2002, Fukao et al. 2002, Heazlewood et al. 2003, Peltier et al.
Keywords: Arabidopsis — LC-MS/MS — Proteomics —
Tonoplast — Vacuole.
Abbreviations: ACA, autoinhibited Ca2+-ATPase; BBE, berberinebridge-forming enzyme; BiP, immunoglobulin heavy chain-binding
protein; GFP, green fluorescent protein; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MRP, multidrug resistanceassociated protein; Nramp, natural resistance-associated macrophage
protein; SUC1, sucrose transporter 1.
8
9
Present address: Department of Biology, Faculty of Science, Kobe University, 1-1, Rokkodai, Nada-ku, Kobe, 657-8501 Japan.
Corresponding author: E-mail, [email protected], Fax, +81-78-803-5708.
672
Proteome of tonoplast
673
Fig. 1 (a) SDS-PAGE of protein samples from protoplasts, vacuolar sap and tonoplast isolated from suspension-cultured Arabidopsis cells. The
gel was stained with CBB. (b) Western blots of the same samples as those in Fig. 1a. Antibodies for V-type H+-ATPase, BiP and P-type H+ATPase were used for Western staining. Fifty µg tonoplast proteins were loaded in each lane.
2002, Prime et al. 2000, Santoni et al. 1998, Schubert et al.
2002). Chloroplasts have received the most attention, possibly
because of the importance of these organelles to plants, and
also because of the relative ease of isolation pure chloroplasts,
or at least chloroplast envelope (Ferro et al. 2003, Ferro et al.
2002, Peltier et al. 2002, Schubert et al. 2002). These studies
have identified various proteins whose functions have not yet
to be established.
Proteomic analysis of the plasma membrane has been
attempted, but was hampered by the contamination by proteins
derived from organelle membranes (Prime et al. 2000) and it is
therefore difficult to be confident of the origin of the identified
proteins. Very recently, two proteomic analyses of the tonoplast have appeared (Sazuka et al. 2004, Szponarski et al.
2004). Both trials used a membrane fraction isolated by sucrose
density gradient and chromatographic separation. Unfortunately, their results showed significant contamination from
other membranes. In the study reported here, we have developed a protocol for the isolation of intact vacuoles from
suspension-cultured Arabidopsis that yields a tonoplast fraction of sufficient purity for meaningful proteomic analysis.
Results and Discussion
Purification of the tonoplast fraction
Vacuoles isolated by the present method were first confirmed their intactness. Addition of ATP or PPi in the presence
of Mg2+ induced a large accumulation of neutral red (data not
shown). It suggests that activities of H+ pumps and tonoplast
membrane integrity are maintained after isolation. Then the
isolated vacuoles were ruptured by freezing and separated into
the vacuolar sap and tonoplast fractions by centrifugation.
Fig. 1a shows the SDS-PAGE of protoplasts, vacuolar sap
and tonoplast fractions. The protein profile of tonoplast isolated
from intact vacuoles was quite different from that of protoplast
and the vacuolar sap. Purity of the tonoplast fraction was confirmed by the measurements of marker enzymes (Table 1) and
Western blotting of marker proteins (Fig. 1b). V-type H+ATPase was greatly concentrated in the tonoplast fraction as
Table 1 Activities of marker enzymes in the tonoplast fraction
V-type H -ATPase (nmol min µg )
Latent IDPase (nmol min–1 µg–1)
P-type H+-ATPase (nmol min–1 µg–1)
+
–1
–1
Each value is mean of three measurements.
Protoplast
Vacuolar sap
Tonoplast
0.03
0.04
0.05
0
0.06
0.02
0.34
0.06
0.03
674
Proteome of tonoplast
Fig. 2 SDS-PAGE and Western blot of partially fractionated tonoplast samples. The gel was silver-stained. Antibodies for V-type H+-ATPase and H+-PPase were used for
Western staining. H+-PPase was only detected in the transmembrane fraction.
shown both in the enzyme activities and Western blotting. Contamination by Golgi was measured using latent IDPase activity, and that of endoplasmic reticulum was measured using
Western blotting of immunoglobulin heavy chain-binding protein (BiP protein). The levels of IDPase and P-type H+-ATPase
activity were very low (Table 1). However, apparent small
activity of P-type H+-ATPase was remaining, although it was
close to background level. The virtual absence of P-type H+ATPase from the tonoplast sample was further confirmed by
Western blotting (Fig. 1b). Similarly, BiP was barely detectable in Western blots (Fig. 1b). From these tests, it was con-
cluded that the contamination from other organelle membranes
was very low.
Observation of the isolated vacuole fraction under a
microscope showed many small vesicles attached to the main
vacuoles (data not shown). These small vesicles stained with
neutral red like the vacuoles, showing that the intravesicular
environment was also acidic. The vesicles were presumed to be
prevacuolar compartments. The finding of the small vacuoles
suggests that the sample might be slightly contaminated by
proteins in the traffic from the Golgi apparatus and endoplasmic reticulum to the main vacuoles.
Fig. 3 Distribution of proteins detected
in this study according to the number of
membrane spanning domains.
Proteome of tonoplast
Comprehensive analysis of the tonoplast proteins
As shown in Fig. 1, the tonoplast faction of suspensioncultured cells contained a large amount of V-type H+-ATPase,
and this interfered with the identification of other proteins in
this fraction. In the first attempt, only 60 proteins could be
identified with LC-MS/MS measurement. In order to reduce
the influence of V-type H+-ATPase, tightly bound peripheral
proteins like the V1 sector of V-type H+-ATPase were separated from integral proteins such as H+-PPase by washing with
Na2CO3 and KSCN. Fig. 2 shows that the separation of the tonoplast fraction resulted in a large reduction of the V1 sector of
V-type H+-ATPase in the integral fraction, while the H+-PPase
was almost entirely retained in this fraction. Subsequent analysis by LC-MS/MS revealed 91 proteins in the peripheral
fraction and 102 proteins in the integral fraction. Some of
peripheral and membrane spanning proteins were common to
both fractions. In total, 163 proteins were identified in this
study (Table 2–6). The identification of only few plasma membrane proteins in the overall analysis of the tonoplast confirmed the low plasma membrane contamination. Forty-six
proteins were predicted to have more than two transmembrane
domains (Fig. 3). The other 117 proteins were predicted to have
one or no transmembrane domain (Fig. 3).
Classification of the identified proteins
Some of the proteins, which had one or no transmembrane domain, were the subunits of the membrane-integral protein complexes, such as the V1 sector of V-type H+-ATPase.
The Arabidopsis protein database contained annotation for 129
proteins (Table 2–5), but 34 proteins were not annotated
(Table 6). Among the 129 annotated proteins there were wellknown tonoplast proteins, such as V-type H+-ATPase and V-type
H+-PPase, which were the most abundant proteins in the tonoplast. There were also the membrane-integral proteins, which
have not previously been shown to localize to the tonoplast at
the molecular level (Table 3). Some proteins, which are known
to locate to mitochondria and plastids were also detected
(Table 5). If there was significant contamination from these
organelles, it should have been possible to find major proteins
such as F1F0-ATPase, but these were not detected. At present, it
is difficult to judge whether the proteins expected to originate
from other organelles are contamination or whether they are
associated with vacuoles for other reasons, such as being
degraded. Some proteins, which were likely to be derived from
the Golgi and endoplasmic reticulum, were also detected (Table
5). Additionally there were many soluble proteins (Table 5).
Proton pumps
Based on previous studies, it was expected that the major
proteins on the tonoplast would be V-type H+-ATPases, V-type
H+-PPases and aquaporins (Maeshima 2001). While most subunits of V-type H+-ATPase and V-type H+-PPase were found,
aquaporin proteins were not detected (Table 2). This result sug-
675
gests that aquaporins are not strongly expressed in liquid suspension-cultured cells.
The V-type H+-ATPase consists of a V1sector and a V0
sector (Sze et al. 2002). The V1 sector is composed of eight
subunits, A to H, while the V0 sector is composed of five subunits, a, c, c”, d and e. The proposed 28 genes encoding the subunits of the V-type H+-ATPase in the Arabidopsis thaliana
genome are shown on Table 2 (Sze et al. 2002). All subunits of
the V1 sector were identified, but genes encoding AtVHA-E2,
AtVHA-G2 and AtVHA-G3 of the V0 sector were not found.
Only the consensus fragments between AtVHA-B1 and
AtVHA-B3 were detected in AtVHA-B3. A protein (gene
locus: At1g16820) similar to AtVHA-A was detected (Table 5).
The theoretical molecular mass of the protein was 10,426 and
much smaller than AtVHA-A. Although the result indicates
that the gene is expressed in A. thaliana, it is not clear whether
this protein is correlated with AtVHA-A or not.
Two a subunits and one d subunit of the V0 sector were
identified (Table 2). The c subunit was detected, but the gene
locus was not identified, because of the high similarity among
the genes encoding the c subunit. None of c” and e subunits
were identified, possibly because those molecular masses were
too low to extract from the SDS-PAGE gels.
Three genes, AVP1, AVP2 and AVPL1, in the A. thaliana genome were proposed to encode V-type H+-PPase
(Drozdowicz et al. 2000). Recently, Mitsuda et al. (2001)
showed that AVP2/AVPL1 were localized to the Golgi rather
than the tonoplast. The three gene products were detected in
this study, although only the consensus sequence between
AVP2 and AVPL1 was detected in AVPL1. Higher protein content tended to lead to higher sequence coverage, when the proteins, whose molecular masses were similar, were analyzed in
our LC-MS/MS analysis. Therefore, the higher sequence coverage in AVP1 than AVP2/AVPL1 suggest that the sample may
contain more AVP1 than AVP2/AVPL1.
Other transporters
The super family of ABC transporters consists of 115
genes in A. thaliana (Arabidopsis Genome Initiative 2000).
Many ABC transporters are located on the tonoplast or plasma
membranes and contribute to the transport of secondary metabolites across these membranes. In this study, we identified five
ABC transporters (Table 3). Four of them, AtMRP1, AtMRP2,
AtMRP4 and AtMRP10 belong to the multidrug resistanceassociated protein (MRP) subfamily. AtMRP2 was reported to
occur on the tonoplast (Liu et al. 2001) and our results suggest
that AtMRP1, AtMRP4 and AtMRP10 are also located to the
tonoplast. The other identified ABC transporter was AtTAP2.
The homologue of AtTAP2 in barley, fused to GFP (IDI7),
located to the tonoplast when transiently expressed in suspension-cultured tobacco cells (Yamaguchi et al. 2002), which is
consistent with our results.
ACA.c, a calmodulin-binding protein belonging to the
autoinhibited Ca2+/ATPases (ACAs) was also detected (Table 3).
676
Proteome of tonoplast
Table 2
Proton pumps
Subunit names
Gene locus
Sequence coverage (%)
TM a
MW (Da) b
pI c
V-type H+-ATPase
AtVHA-A
AtVHA-B1
AtVHA-B2
AtVHA-B3 d
AtVHA-C
AtVHA-D
AtVHA-E1
AtVHA-E2
AtVHA-E3
AtVHA-F
AtVHA-G1
AtVHA-G2
AtVHA-G3
AtVHA-H
AtVHA-a1
AtVHA-a2
AtVHA-a3
AtVHA-c1 e
AtVHA-c2
AtVHA-c3
AtVHA-c4
AtVHA-c5
AtVHA-c″1
AtVHA-c″2
AtVHA-d1
AtVHA-d2
AtVHA-e1
AtVHA-e2
At1g78900
At1g76030
At4g38510
At1g20260
At1g12840
At3g58730
At4g11150
At3g08560
At1g64200
At4g02620
At3g01390
At4g23710
At4g25950
At3g42050
At2g21410
At4g39080
At2g28520
At4g34720
At1g19910
At4g38920
At1g75630
At2g16510
At4g32530
At2g25610
At3g28710
At3g28715
At5g55290
At4g26710
65
50
44
19
44
29
58
not detected
35
54
32
not detected
not detected
19
17
21
not detected
10
0
0
0
0
0
0
0
0
0
2
0
0
0
0
6
6
6
4
not detected
not detected
46
47
not detected
not detected
4
4
0
0
68795
54090
54288
36341
42602
29041
26042
26835
27067
14242
12379
11724
12098
50267
93089
92817
93398
16554
16625
16554
16668
16554
18357
18201
40774
40770
4.8641
4.7287
4.7766
4.5557
5.202
10.1747
6.3076
9.694
6.1132
6.5306
5.8235
5.2761
4.8192
7.0356
5.2391
5.7743
6.3551
8.6793
8.6793
8.6793
8.6793
8.6793
7.9162
7.9162
4.8156
4.7511
V-type H+-PPase
AVP1
AVP2
AVPL1 d
At1g15690
At1g78920
At1g16780
17
2
1
16
17
17
80803
85116
85332
4.8908
5.4233
5.4202
a
The number of transmembrane domains was shown according to the results of ARAMEMNON database
search.
b
The predicted molecular weights (MW) of the proteins were calculated using the Bioperl 0.7.
c
The predicted isoelectric points (pI) were calculated using the iep program in the EMBOSS 2.0.1.
d
Only consensus fragments among the other isoforms were detected.
e
AtVHA-c was detected, but the gene locus was not identified.
The EST database revealed that ACA.c is expressed in siliques. It has been suggested that ACA4 would occur on the
tonoplast (Geisler et al. 2000) and our results show that ACA.c
is likely to occur on the tonoplast.
A putative Zn transporter was identified (Table 3). It has
previously been reported that Zn-tolerance plants have 2.5
times higher Zn transport activity across the tonoplast than Znsensitive plants (Verkleij et al. 1998). A candidate gene encoding Zn transporter in A. thaliana was identified (Bloss et al.
2002) but its location was not verified. Here we confirm that it
does indeed locate on the tonoplast and is likely to be involved
in the detoxification of Zn.
In addition to the Zn transporter, two natural resistanceassociated macrophage proteins (Nramps), AtNRAMP3 and 4,
were identified (Table 3). Nramps have been implicated in the
transport of a wide range of divalent metal cations (Thomine et
al. 2000). The expression of AtNRAMP3 and 4 can complement the phenotype of yeast strains deficient in uptake of Mn
or Fe (Thomine et al. 2000). Expression of AtNramps 3 and 4
in yeast stimulated Cd2+ accumulation and increases their sen-
Proteome of tonoplast
Table 3
677
Putative Arabidopsis tonoplast transporters
Sequence
TM a MW (Da) b
coverage (%)
pI c
Protein names and AtDatabase annotations
Gene locus
ABC proteins
ABC transporter (AtMRP1)
ABC transporter (AtMRP2) d
ABC transporter (AtMRP4)
ABC transporter-like (AtMRP10)
ABC transporter-like (AtTAP2) d
At1g30400
At2g34660
At2g47800
At3g62700
At5g39040
5
4
5
6
9
15
15
17
17
6
181911
182114
169064
172121
69086
6.0762
6.3606
7.7015
8.7173
8.2802
Pumps
Ca2+-transporting ATPase (ACA.c)
At3g57330
2
10
111928
6.2699
At2g46800
or At3g58810
At2g02040
At2g23150
At5g67330
At5g62890
At2g21160
At5g15090
At3g01280
3
6
7
4
4
5
5
35
29
11
12
12
14
2
0
0
43810
41204
64404
56121
56368
50941
28150
29193
29408
6.6261
6.1699
5.3379
4.8469
4.7232
9.755
4.8432
8.6789
9.2337
12
12
54841
9.1581
Other transporters and channels
Putative zinc transporter
Histidine transport protein (PTR2-B)
Putative metal ion transporter (Nramp)
Nramp metal ion transporter 4 (Nramp4)
Permease 1-like protein
Translocon-associated protein alpha (TRAP alpha) family
Voltage-dependent anion-selective channel protein hsr2
Putative porin similar to outer mitochondrial membrane porin
(voltage-dependent anion-selective channel protein) (VDAC) (POM 34)
Sucrose transport protein SUC1
At1g71880
a
The number of transmembrane domains was obtained by ARAMEMNON database search.
The predicted molecular weights (MW) of the proteins were calculated using Bioperl 0.7.
c
The predicted isoelectric points (pI) were calculated using the iep program in EMBOSS 2.0.1.
d
The proteins were reported the localization to the tonoplast in the published papers: At2g34660, Liu et al. (2001); At5g39040, Yamaguchi et al.
(2002).
b
Table 4
Other suggested Arabidopsis tonoplast integral proteins
Protein names and AtDatabase annotations
Gene locus
Cytochrome b561 family protein (Artb561–1)
Plasma membrane intrinsic protein (SIMIP)
Adenylate translocator
Putative CAAX prenyl protease
Cytochrome c, putative
At4g25570
At4g35100
At3g08580
At4g01320
At3g27240
or At5g40810
At2g07050
At2g07698
At1g65820
Cycloartenol synthase [(S)-2,3-epoxysqualene mutase] (CAS1)
ATP synthase alpha chain, mitochondrial, putative
Microsomal glutathione-S-transferase, putative
Sequence coverage (%) TM a MW (Da) b
4
6
9
6
12
6
6
3
6
2
5
5
15
2
3
3
25847
29725
41458
48465
33633
33673
86016
85916
16568
pI c
7.2913
9.0569
10.3239
8.3549
6.0453
6.1039
6.0093
5.2353
9.3189
a
The number of transmembrane domains was obtained by ARAMEMNON database search.
The predicted molecular weights (MW) of the proteins were calculated using Bioperl 0.7.
c
The predicted isoelectric points (pI) were calculated using the iep program in EMBOSS 2.0.1.
b
sitivity to Cd2+ (Thomine et al. 2000). AtNRAMP3 overexpressing plants also accumulate higher levels of Fe, upon Cd2+
treatment (Thomine et al. 2000). In Arabidopsis, the disruption
of AtNRAMP3 leads to an increase of Cd2+ resistance, whereas
the overexpression of AtNRAMP3 confers increased Cd2+ sensitivity (Thomine et al. 2000). These results indicated that
AtNRAMP3 and 4 play a role in transporting Cd2+, but their
cellular location had not previously been demonstrated. Our
results suggest that these proteins locate on the tonoplast.
There are many reports of amino acid transport across the
tonoplast, mediated by at least three transport systems (Dietz et
al. 1994), the molecular identities of which have not been established. In this study we found a histidine transporter (Table 3).
678
Table 5
Proteome of tonoplast
Arabidopsis proteins, which have one or no transmembrane domain
Protein names and AtDatabase annotations
Proteinases
20S proteasome alpha subunit C (PAC1)
20S proteasome alpha subunit D (PAD1)
20S proteasome alpha subunit G (PAG1)
20S proteasome beta subunit A (PBA1)
20S proteasome beta subunit D (PBD1)
Acylaminoacyl-peptidase like protein
Aminopeptidase-related
Aspartic proteinase-related
Aspartic proteinase-related
Aspartyl aminopeptidase-like
cysteine proteinase RD21A
cysteine proteinase RD21A
Leucine aminopeptidase-related
Leucyl aminopeptidase-like protein PIR2
S-adenosyl-L-homocysteinase-related
Serine carboxypeptidase-related
Serine carboxypeptidase III, putative
Expressed protein tripeptidyl-peptidase II
Acid phosphatase-related
Peptidase family similar to prolyl aminopeptidase
Glutamate hydroxypeptidase ileal peptidase I100
Ribosomal proteins
Putative 60S ribosomal protein L1
60S ribosomal protein L2
60S ribosomal protein L7
Putative ribosomal protein L7
60S ribosomal protein L9 (RPL90B)
60S ribosomal protein L11 (RPL11A)
60S ribosomal protein L14
Putative 60S ribosomal protein L17
Putative 60S ribosomal protein L18
60S ribosomal protein-like
Ribosomal protein
60S ribosomal protein L10 (RPL10A) or 60S ribosomal protein L10 (RPL10C)
Ribosomal protein, putative
Others
FAD-linked oxidoreductase family
Calcineurin-like phosphoesterase family
Cell elongation protein (DWARF1)
Alpha-soluble NSF attachment protein
Glycosyl hydrolase family 17 similar to elicitor inducible chitinase
Glycosyl hydrolase family 19 (basic endochitinase)
Sequence
coverage (%)
TM a
MW
(Da) b
pI c
4
10
0
0
5
4
6
7
3
13
4
15
32
3
7
25
12
2
0
0
0
0
0
0
0
0
0
0
1
1
0
2
4
40
31
10
2
0
0
0
0
0
0
27457
27319
27306
27360
25134
22523
21966
47014
101698
55731
54596
52408
50949
51186
54492
61290
53142
53361
51504
57284
152351
31078
57568
73446
7.142
7.5002
8.9688
6.2566
5.2413
6.3551
6.6778
4.993
6.2599
6.2652
5.2041
6.7635
5.0528
6.1219
5.7231
7.0877
5.4937
5.8281
6.9159
5.0271
5.9292
9.5055
7.3385
5.237
8
9
1
0
13
19
21
0
0
0
21
20
20
21
20
8
0
0
5
0
12
0
8
13
5
4
1
1
0
44685
27842
27931
27922
28153
22000
22000
19727
20844
20844
19757
20844
15489
15488
19879
19836
20908
20949
44704
44542
24900
24912
11.0413
11.5247
11.4548
10.5811
10.5983
10.1367
10.1367
10.4531
10.4839
10.4839
10.4531
10.4839
10.8531
10.7687
10.7918
10.7918
11.6228
11.6107
11.0099
10.8279
11.1477
11.222
At3g25520
20
0
34340
9.7708
At4g20830
At5g34850
At3g19820
At3g56190
At4g31140
At3g12500
8
6
8
13
13
4
0
0
0
0
1
0
63542
54992
65377
32737
52698
34591
10.1073
7.3388
8.1412
4.9771
6.053
7.0713
Gene locus
At3g22110
At3g51260
or At5g66140
At2g27020
At4g31300
At3g22630
or At4g14800
At4g14570
At1g63770
At1g62290
At1g11910
At5g60160
At1g47128
At5g43060
At2g24200
At4g30920
At3g23810
or At4g13940
At2g35780
At3g10410
At4g20850
At1g04040
At3g61540
At5g19740
At3g09630
At2g18020
or At4g36130
At2g44120
At2g01250
At1g33120
or At1g33140
At2g42740,
At3g58700,
At4g18730,
At5g45775
or At5g45775
At2g20450
or At4g27090
At1g27400
or At1g67430
At3g05590
or At5g27850
At5g02870
At1g43170
At1g14320
or At1g66580
Proteome of tonoplast
Table 5
Continued
Gene locus
Sequence
coverage (%)
TM a
MW
(Da) b
pI c
Glycosyl hydrolase family 20 similar to beta-hexosaminidase A
Glycosyl hydrolase family 27 (alpha-galactosidase/melibiase)
Glycosyl hydrolase family 31 similar to alpha-glucosidase precursor
Glycosyl hydrolase family 35 (beta-galactosidase) similar to beta-galactosidase
Glycosyl hydrolase family 38 (alpha-mannosidase) similar to alpha-mannosidase
Glycosyl hydrolase family 38 (alpha-mannosidase) similar to lysosomal alpha-mannosidas
Myrosinase-associated protein, putative
Myrosinase-associated protein, putative
At3g55260
At3g56310
At5g11720
At2g32810
At5g13980
At3g26720
At1g54010
At1g54020
12
5
4
2
6
11
40
10
0
0
1
0
0
0
0
0
61212
48345
101101
99181
115885
115203
43126
32438
Myrosinase-associated protein, putative
N-carbamoylputrescine amidohydrolase
At1g54000
At2g27450
15
9
0
0
Nitrilase 2
Dynamin-like protein 6 (ADL6) or Putative dynamin-like protein (ADL3)
At3g44300
At1g10290
or At1g59610
At1g14830
At2g39700
At3g57030
At1g74010?
At1g74020
At3g02230
At5g15650
At1g24450
At5g42490
At5g19550
At2g30970
At1g51980
At3g02090
At2g28000
At3g23990
At2g33210?
At5g18170
At5g07440
At3g17240
At1g54220
At2g36530
At1g77120
At1g44170
At1g63940
At1g11580
At3g20000
At5g08670,
At5g08680
or At5g08690
At1g35580
At5g40770
At5g44140
At3g27280?
At4g28510
At1g03860
At3g04520
At1g16820
15
3
0
0
1
4
9
7
36
31
30
30
0
9
6
11
12
4
6
3
14
13
5
7
10
26
14
5
16
9
36
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
0
0
0
2
24
8
10
7
8
8
16
0
0
1
0
1
1
0
0
43176
33515
36439
37135
99149
100211
68705
27830
40984
34166
35275
40611
40873
20724
123618
44249
47740
54384
59143
62055
61263
61961
44507
44682
53969
58450
47702
41161
53141
52484
61670
34232
59654
59842
59696
62817
30382
30742
30620
31689
31793
38235
10426
6.2
4.5151
5.7228
8.1099
6.4078
6.701
8.1874
7.837
6.1774
7.1225
5.9276
6.1774
5.2446
9.5736
9.5916
7.635
9.9814
8.0806
8.4188
5.4345
5.7023
5.987
10.1106
4.4791
7.3219
8.3475
6.2635
6.7649
4.8154
5.4153
6.5881
6.8548
6.5103
7.0366
7.9663
5.5147
6.1887
8.652
7.5265
9.077
6.8102
6.5979
6.4527
6.5979
6.8239
7.8895
10.1784
7.8165
9.7318
9.8686
6.4468
6.5052
Protein names and AtDatabase annotations
Dynamin-like protein C (DL1C)
Putative expansin
Putative protein strictosidine synthase
Strictosidine synthase family
Strictosidine synthase family
Reversibly glycosylated polypeptide-1 (RGP1)
Reversibly glycosylated polypeptide-2 (RGP2)
Ribonuclease III-related
Kinesin heavy chain-like protein
Aspartate aminotransferase, cytoplasmic isozyme 1 (transaminase A/Asp2)
Aspartate aminotransferase, mitochondrial (transaminase A/Asp1) I
Mitochondrial processing peptidase alpha subunit, putative
Putative mitochondrial processing peptidase
Chaperonin 60alpha subunit
Chaperonin (CPN60/HSP60)
Chaperonin, putative
Glutamate dehydrogenase 1
Glutamate dehydrogenase 2
Mitochondrial dihydrolipoamide dehydrogenase 2
Dihydrolipoamide S-acetyltransferase-related
Enolase (2-phospho-D-glycerate hydroylase)
Alcohol dehydrogenase (ADH)
Aldehyde dehydrogenase, putative (ALDH)
Monodehydroascorbate reductase, putative
Pectin methylesterase, putative
Membrane import protein, putative
Mitochondrial H+-transporting ATP synthase beta chain-related
Invertase related
Prohibitin
Prohibitin
Prohibitin, putative
Prohibitin-like protein
Putative prohibitin 2
L-allo-threonine aldolase-related
Expressed protein (vacuolar ATP synthase catalytic subunit-related / V-ATPase-related /
vacuolar proton pump-related)
a
679
The number of transmembrane domains was obtained by ARAMEMNON database search.
The predicted molecular weights (MW) of the proteins were calculated using Bioperl 0.7.
c
The predicted isoelectric points (pI) were calculated using the iep program in EMBOSS 2.0.1.
b
680
Proteome of tonoplast
Table 6 Unannotated Arabidopsis proteins
Protein names and AtDatabase annotations
Gene locus
Sequence
MW
TM a
coverage (%)
(Da) b
pI c
Possible subcellular
localization d
Two or more TM domains
Unknown protein (nodulin family protein)
Hypothetical protein (MATE efflux family protein)
Integral membrane protein, putative (MATE efflux family protein)
Putative protein Niemann-Pick C disease protein (patched family protein)
Unknown protein (auxin efflux carrier family protein)
At3g01930
At1g61890
At3g21690
At4g38350
At1g76520
or At1g76530
24 kDa vacuolar protein-like
At5g20660
Putative protein (glycine-rich protein)
At5g11700
Unknown protein (Nuf2 family protein)
At1g61000
Putative protein (mechanosensitive ion channel domain-containing protein) At5g12080
Senescence-associated protein family
At1g32400
Glycine-rich protein
At5g47020
Expressed protein
At4g28770
Unknown protein
At2g20230
Unknown protein
At1g29820
Hypothetical protein (prenylated rab acceptor (PRA1) family protein) At1g55190
Expressed protein
At5g49900
Expressed protein
At5g63910
One or no TM domain
Leucine-rich repeat family protein / extensin family protein
Putative protein embryo-specific protein 3
Unknown protein (outer membrane OMP85 family protein)
Expressed protein (serine carboxypeptidase S28 family protein)
Disease resistance protein-related (LRR)
Expressed protein (meprin and TRAF homology domain-containing
protein / MATH domain-containing protein)
Expressed protein (cathepsin B-like cysteine protease, putative)
Expressed protein (meprin and TRAF homology domain-containing
protein / MATH domain-containing protein)
Unknown protein
Expressed protein (lipid-binding serum glycoprotein family protein)
Hypothetical protein (nicastrin-related)
Expressed protein
Putative protein (band 7 family protein)
Putative protein hypersensitive-induced response protein HIR3 (band 7
family protein)
Unknown protein (band 7 family protein)
Unknown protein (band 7 family protein)
Unknown protein
2
4
6
5
3
2
3
1
0
14
4
1
4
12
8
10
1
15
12
12
12
11
10
8
9
6
6
6
4
6
4
4
2
2
2
2
63500
55116
54934
116033
42615
45568
100077
151531
111643
83015
31538
152296
30959
29704
61051
21054
106692
55281
8.3649
6.5069
5.1902
5.1711
9.3101
7.4275
7.1221
6.4797
5.0072
9.6417
5.1
7.382
4.6481
4.9296
6.2672
7.9101
5.7206
6.1978
other
secreted
other
other
secreted
secreted
secreted
secreted
other
other
other
secreted
At3g24480
At5g62200
At5g05520
At4g36195
At1g33590
At3g20370
4
10
3
20
4
29
0
1
0
0
0
0
54685
21053
58502
54756
51724
43431
6.9274
7.1975
6.4452
6.5035
9.234
6.5366
secreted
secreted
other
secreted
secreted
secreted
At1g02305
At5g26280
8
15
0
0
40016 6.9065 secreted
39428 8.9246 secreted
At2g43950
At1g04970
At3g52640
At3g57990
At5g51570
At5g62740
22
5
20
12
39
29
1
0
0
0
0
0
38818
53476
42353
39874
32361
31413
At1g69840
At3g01290
At5g42330
14
12
6
0
0
0
31388 5.0779 other
31303 5.4979 other
19860 10.6346 other
9.5665
4.4006
6.3042
10.0354
5.0825
5.0697
mitochondria
other
other
other
secreted
chloroplast inner membrane e
secreted
other
other
other
other
a
The number of transmembrane domains was obtained by ARAMEMNON database search.
The predicted molecular weights (MW) of the proteins were calculated using Bioperl 0.7.
c
The predicted isoelectric points (pI) were calculated using the iep program in EMBOSS 2.0.1.
d
The subcellular localizations were predicted using TargetP.
e
The subcellular localization was reported in the published paper: At2g43950; Ferro et al. (2002).
b
Other annotated proteins with two or more transmembrane
domains
Sucrose-H+ symport by sucrose transporter 1 (SUC1) is
known to occur on the plasma membrane (Sauer and Stolz
1994) and it has been predicted that sucrose would be transported into the vacuole by sucrose-H+ antiport. However, our
analysis revealed a SUC1-like transporter in the tonoplast fraction (Table 4). Because we could not identify any other plasma
membrane proteins, such as P-type H+-ATPase in this sample,
it seems probable that there is also sucrose-H+ symport across
the tonoplast, presumably for efflux of sucrose from the vacuole.
There were several other annotated proteins with two or
more transmembrane domains that have previously been
assigned to mitochondria or plastids, which appeared in the
tonoplast (Table 4). The virtual absence of major proteins in
mitochondria and plastids in the tonoplast sample suggests that
the presence of these minor proteins might not be due to simple contamination from those organelles.
Proteome of tonoplast
Annotated proteins with one or no transmembrane domain
We also identified the proteins with one or no transmembrane domain. These proteins were detected in peripheral and/
or integral fractions. The method for the separation of peripheral and integral proteins in this study is especially effective to
the separation of V1 sector of V-type H+-ATPase from V0 sector, but other peripheral proteins were not completely released
from the tonoplast. The detection of the proteins without transmembrane domains in the integral fraction was not avoidable.
Proteins classified to this category contained twelve ribosomal proteins, which suggests that ribosomes might bind to the
outside of the vacuole (Table 5). Twenty-one proteins considered to be involved in the degradation of proteins, were identified (Table 5), consistent with the known function of vacuoles
in protein breakdown. It would seem likely that almost all of
these proteins would be localized in the vacuolar sap but bind
to the inside of the tonoplast.
Most of the other annotated proteins, which had one or no
transmembrane domain, have not previously been shown to be
associated with vacuoles (Table 5). The observation of the
small vesicle in the purified vacuole indicates that some of the
proteins without transmembrane domains might be from such
vesicular compartments. On the other hand, some of them have
a suggested vacuolar role, for example the FAD-linked oxidoreductase family protein (At4g20830) was similar to berberinebridge-forming enzyme (BBE) (Table 5). Because BBE has
been reported to occur in vacuoles (Bock et al. 2002), the identified FAD-binding protein could be localized to the vacuoles
and might bind to the inside of the tonoplast. Dynamin-like
protein was also detected (Table 5), although its encoding gene
was not defined because of the low sequence coverage in LCMS/MS analysis and because of the high homology between
dynamin-like protein 3 (ADL3) and ADL6 (Jin et al. 2001,
Mikami et al. 2000). ADL6 has been proposed to be involved
in the trafficking of cargo proteins from the trans-Golgi network to the lytic vacuoles (Jin et al. 2001).
Unannotated proteins
Thirty-four of the identified proteins were not annotated
(Table 6). Seventeen of them were predicted to have at least
two transmembrane domains. Almost all of the unannotated
proteins, which had more than two transmembrane domains,
could not be assigned to specific subcellular locations by the
TargetP program and may therefore be novel tonoplast proteins.
Conclusion
By comprehensive proteomic analysis of purified tonoplast, 163 proteins were identified. They included almost all
subunits of the major tonoplast proteins, the V-type H+-ATPase
and the V-type H+-PPase, as well as a broad range of other
transporters. There were also numerous proteins with one or no
transmembrane domains whose function could be predicted
from existing databases. Few proteins from other organelles
681
were detected, which represents a major advance over previous
attempts to analyze tonoplast proteins by proteomic methods.
Materials and Methods
Plant material and culture
A. thaliana suspension-cultured cells [Arabidopsis Col-0 cell
suspension supplied by courtesy of Dr. Umeda (University of Tokyo)]
(Mathur et al. 1998) were cultured in modified Murashige and Skoog
medium supplemented with 4.5 µM 2,4-D and 3% sucrose. Two to
3 ml of cell suspension was transferred to 20 ml of fresh medium every
7 d. Cells were cultured with shaking at around 23°C in the dark.
Protoplast and vacuole isolation
Protoplasts and vacuoles were isolated by modifying a method
described in Massonneau et al. (2000). Cells were sedimented at
200×g for 10 min and subsequently resuspended in medium A
[500 mM sorbitol, 10 mM MES adjusted to pH 6.0 with Tris, 1 mM
CaCl2, 1.0% Cellulase Y-C (Seishin Pharmaceutical Co., Ltd., Tokyo,
Japan), 0.1% Pectolyase Y-23 (Seishin Pharmaceutical Co., Ltd.,
Tokyo, Japan)]. The cells were incubated for 120–180 min at 31°C
with shaking at 120 rpm and the released protoplasts were collected by
centrifugation at 200×g for 10 min. In order to reduce disruption of
protoplasts, the medium containing them was underlayered with
medium B (30 mM HEPES adjusted to pH 7.2 with Tris, 30 mM Kgluconate, 2 mM MgCl2, 2 mM EGTA) supplemented with 400 mM
sucrose and 50% Percoll (medium I). The sedimented protoplasts were
mixed with an appropriate volume of medium I and a gradient was
formed by overlaying medium II (medium B containing 400 mM
sucrose and 7.5% Percoll) and medium III (medium B containing
400 mM sorbitol). After centrifugation (800×g, 10 min), purified protoplasts were obtained in the interfaces between medium I and medium
II or between medium II and medium III. The latter protoplasts were
discarded to avoid contamination of the vacuolar fractions with protoplasts. One volume of the remaining purified protoplasts was mixed
with one volume of medium B and incubated on ice for 5 min. A mixture of protoplasts, vacuoles and lysate was mixed with an appropriate
volume of medium IV (medium B containing 200 mM sorbitol and
25% Percoll) and a gradient was formed by consecutive overlaying
with medium V (medium B containing 200 mM sucrose and 7.5% Percoll), medium VI (medium B containing 200 mM sucrose and 5% Percoll), medium VII (medium B containing 200 mM sucrose and 2.5%
Percoll) and medium VIII (medium B containing 200 mM sucrose).
After centrifugation (800×g, 10 min), purified vacuoles were recovered
from the interface between medium VII and medium VIII.
Purification of tonoplast
Isolated vacuoles were treated with 50 mM KCl and 0.05% deoxycholate to remove surface proteins from the tonoplast, and then frozen at –80°C to rupture the vacuoles. The thawed sample was then
centrifuged at 120,000×g at 4°C for 75 min to yield a pellet that contained the purified tonoplast. The supernatant was used as the vacuolar
sap fraction. About 10 µg of tonoplast proteins were obtained from
10 g FW suspension-cultured cells.
In some experiments, the tonoplast sample was further fractionated to integral proteins and tightly bound peripheral proteins. The
purified tonoplast sample was stirred in 100 mM Na2CO3 and 500 mM
KSCN at 4°C for 30 min and again centrifuged at 120,000×g at 4°C
for 75 min. The membrane spanning proteins were collected in the pellet, and the tightly bound peripheral proteins were collected in the
supernatant.
682
Proteome of tonoplast
Measurement of enzyme activities
V-type H+-ATPase was used as a marker for the tonoplast and its
activity was measured as Bafilomycin-sensitive ATP hydrolysis
according to Dietz et al. (1998). Two µg protein of each sample was
used for activity measurement. The marker enzyme for the plasma
membrane was P-type H+-ATPase whose activity was measured as
vanadate-sensitive ATP hydrolysis. The composition of measuring
buffer was 25 mM MES, 50 mM KCl, 2 mM NaMoO4, 2 mM MgSO4,
1 mM NaN3, 0.05% Brij58 and 1 µM Bafilomycin A1. Its pH was
adjusted to 6.5 with Tris. For Golgi, latent IDPase was measured. The
composition of measuring buffer was 25 mM Tricine, 50 mM KCl,
2 mM NaMoO4, 3 mM MgSO4, 1 mM NaN3 and 0.05% Brij58. Its pH
was adjusted to 7.5 with Tris. Since enzyme activities were not prominent in protoplast sample, 10 µg protein was used for measurements of
P-type H+-ATPase and latent IDPase of protoplasts. In all measurements, inorganic phosphates liberated from substrates were measured
with Bencini method (Bencini et al. 1983).
SDS-PAGE and Western blotting
Conventional SDS-PAGE of the tonoplast fraction was performed
with 7.5% acrylamide gel. Western blot analysis was carried out using
antibodies for V-type H+-ATPase a subunit (Matsuura-Endo et al.
1992), H+-PPase (Takasu et al. 1997), P-type H+-ATPase (Kinoshita
and Shimazaki 1999) and binding protein (BiP; a member of the heatshock 70 family) (Hatano et al. 1997).
Mass spectrometry
After SDS-PAGE, the stained bands were excised from the
SYPRO Ruby-stained gel. The smearing part was cut off at intervals of
1 mm. The excised gel pieces were washed, reduced, alkylated and
digested with modified trypsin (Promega) according to Shevchenko et
al. (1996). The peptides were then extracted with 5% (v/v) formic acid
solution and acetonitrile. After concentrating to about 10 µl, the tryptic peptides were made up to 20 µl with 0.1% (v/v) aqueous formic
acid. The samples were injected into an Agilent 1100 capillary HPLC
system (Agilent Technologies, CA, U.S.A.). The peptides were
separated on a 75 µm i.d. × 50 mm HiQ sil C18V column (KYA
Technologies Co., Tokyo, Japan) using a gradient from solution A
(10% acetonitrile: 90% water: 0.1% formic acid) to solution B (90%
acetonitrile: 10% water: 0.1% formic acid) over 70 min at a flow rate
of 200 nl min–1.
The LC system was directly coupled to a Q-TOF Ultima mass
spectrometer (Waters Co., Milford, MA, U.S.A.) through a PicoTip
(New Objectives, MA, U.S.A.). MS and MS/MS data were acquired
and processed automatically using MassLynx 4.0 software (Waters
Co., Milford, MA, U.S.A.). The spectra were calculated externally
using fragments from (Glu1)-fibrinopeptide B (Sigma-aldrich, Saint
Louis, MO, U.S.A.). Database searching was carried out using MASCOT (Matrix Science, U.K.). The Arabidopsis protein database from
TIGR 4.0 release and mitochondrial and chloroplast genomes were
used for the database searching. For identification of proteins, the peptide sequence was checked manually.
Acknowledgments
We greatly appreciate Dr. Rob Reid (University of Adelaide,
Adelaide, Australia) for his kind discussion and correction of this manuscript. We also thank to Dr. Csaba Koncz (Max-Planck-Institut für
Züchtungsforschung) and to Dr. Masaaki Umeda (The University of
Tokyo) for their kind supply of Arabidopsis suspension culture cells.
This work was supported by CREST of JST (Japan Science and Technology Corporation) and a Grand-in-Aid for Scientific Research on
Priority Areas (B) (10219202) by the Japanese Ministry of Education,
Culture, Sports, Science and Technology, Japan Society for the Promotion of Science. This work was also supported in part by the New
Energy and Industrial Technology Development Organization subsidized by the Ministry of Economy, Trade and Industry of Japan.
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(Received March 19, 2004; Accepted April 17, 2004)