Download The proteomics of plant cell membranes

Journal of Experimental Botany, Vol. 58, No. 1, pp. 103–112, 2007
Intracellular Compartmentation: Biogenesis and Function Special Issue
doi:10.1093/jxb/erj209 Advance Access publication 27 June, 2006
SPECIAL ISSUE PAPER
The proteomics of plant cell membranes
Setsuko Komatsu*, Hirosato Konishi and Makoto Hashimoto
National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan
Received 11 December 2005; Accepted 21 March 2006
Abstract
Membrane proteins are involved in many different
functions depending on their location in the cell.
Characterization of the membrane proteome can
bring new insights to the function of different plant
membrane systems and the subcellular compartments
where the proteins are found. Plant membrane proteomics can also provide valuable information about
plant-specific biological processes. Despite recent advances in the separation and techniques for the analysis of plant membrane proteins, characterization of
these proteins, especially the hydrophobic ones, is still
challenging. In this review, plant membrane proteomics data, compiled from the literature on Arabidopsis thaliana, are described. In addition, initial attempts
towards determining the physiological significance
of some proteins identified from membrane proteomics
in rice are also described.
Key words: Arabidopsis, cell membrane, plant, proteomics, rice.
Introduction
Plant cells contain many membrane systems specialized
to particular functions. Their lipid component provides a
barrier to solute movement, whilst membrane-associated
proteins undertake their unique biological roles. For example, the plasma membrane is an organized system that
serves both a structural role and acts as a communication
interface with the extracellular environment for the exchange of information and substances. Environmental
stresses cause significant intracellular restructuring in
plants (Buchanan et al., 2000), and the processing of
signals involved in responses to biotic and abiotic stresses
occurs in the plasma membrane. Therefore, a better knowledge of the plasma membrane proteome would help in
developing strategies to increase plants’ natural defences.
In plant cells, as well as in animal cells, the plasma membrane controls many primary cellular functions, such as
metabolite and ion transport, endocytosis, and cell differentiation and proliferation. All these processes involve
a large number of proteins with highly diverse structures
and functions.
The degree of association of proteins with a membrane
varies. Some proteins are embedded in the membrane lipid
core, while others are more peripheral, and associated by
reversible interactions with either lipids or other membrane
proteins (Marmagne et al., 2004). Plant membrane proteomics can give valuable information on plant-specific processes; however, the challenge for proteomics is to find
ways of extracting and identifying the entire set of mostly
hydrophobic plasma membrane proteins.
Proteome analyses at the level of subcellular structures
represent an analytical strategy that combines classical
biochemical fractionation methods to enrich for particular
compartments and tools for the comprehensive identification of proteins. One of the key potentials of this strategy is
the capability to enhance the understanding of the biochemical machinery of purified organelles for subsequent
functional studies (Ephritikhine et al., 2004). Proteomic
analysis of membrane proteins remains a major challenge.
Membrane proteins are more difficult to analyse than soluble proteins and they are generally under-represented
in datasets for several reasons that were summarized by
Ephritikhine et al. (2004). (i) Due to physico-chemical
heterogeneity, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation is not appropriate for
a comprehensive mapping of membrane proteins, (ii) many
hydrophobic proteins are not solubilized in the isoelectric
focusing sample buffer and precipitate at their isoelectric
point, and (iii) low abundance proteins, including rare
membrane proteins, are out of the detection limits of standard proteomic techniques. To address these difficulties,
Marmagne et al. (2004) used complementary methods for
the extraction of hydrophobic proteins and analysis by mass
* To whom correspondence should be addressed. E-mail: [email protected]
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104 Komatsu et al.
spectrometry on mono-dimensional gels, allowing the
identification of about 100 proteins, 95% of which had
never been found in previous proteomic studies. Alexandersson et al. (2004) used nano-flow reversed-phase
chromatography coupled ‘on-line’ to an electrospray
ionization mass spectrometer, and identified more than
200 putative plasma membrane proteins in Arabidopsis
thaliana green leaves. There are, however, only a limited
number of reports on plasma membrane proteins of rice
(Tanaka et al., 2004), because the plasma membrane of
monocot plants is difficult to isolate. One of the reasons
for this difficulty might be due to differences in cell-wall
polysaccharide composition between dicot and monocot
plants (White and Broadley, 2003).
Analyses of multiple complete genome sequences have
revealed that about 20–30% of all genes encode transmembrane proteins in both prokaryotes and eukaryotes.
The completely sequenced Arabidopsis thaliana (Arabidopsis Genome Initiative, 2000) and Oryza sativa (International Rice Genome Sequencing Project, 2005) genomes
enabled, for the first time, the systematic prediction of all
putative plant membrane proteins in these plant species.
The annotation of the Arabidopsis thaliana and rice
genomes has progressed at a rapid pace during the past
few years and by now, most of the predicted genes are
supported by full-length cDNAs (Kikuchi et al., 2003;
Yamada et al., 2003; Seki et al., 2004). Although rice is an
important crop, there are not many reports on proteomic
analyses of subcellular compartments of this plant. This
review is focused on our current knowledge of plant
membrane proteomics in Arabidopsis thaliana, but also
includes preliminary results from our recent proteomics
studies in rice.
Proteomics of Arabidopsis thaliana membranes
Proteomics of subcellular compartments in
Arabidopsis thaliana
The Institute for Genomic Research has annotated more
than 27 000 Arabidopsis proteins (Wortman et al., 2003)
and 25% of these are predicted to be integral membrane
proteins (Schwacke et al., 2003). The plasma membrane is
probably the most complex membrane system of the cell,
with a protein composition that varies with cell type,
developmental stage, and environmental exposure. It is
likely to harbour thousands of proteins. For instance,
receptor-like protein kinases alone, most of which probably
reside in the plasma membrane, are represented by more
than 600 genes in the Arabidopsis genome (Shiu and
Bleecker, 2001). Since the Arabidopsis genome was
completed in 2000 (Arabidopsis Genome Initiative, 2000),
efforts at characterizing the Arabidopsis chloroplast
(Peltier et al., 2000; Schubert et al., 2002; Ferro et al.,
2003; Froehlich et al., 2003), chloroplast membrane
(Eichacker et al., 2004; Huber et al., 2004; Peltier et al.,
2004), mitochondria (Kruft et al., 2001; Millar et al., 2001;
Millar and Heazlewood, 2003; Heazlewood et al., 2004),
mitochondrial membrane (Brugiere et al., 2004), peroxisome (Fukao et al., 2002; Carter et al., 2004), and tonoplast
proteome (Shimaoka et al., 2004), as well as the Arabidopsis cell wall proteome (Chivasa et al., 2002), have been
made. The Arabidopsis plasma membrane has been studied
using proteomic approaches also (Prime et al., 2000;
Santoni et al., 2000; Kawamura and Uemura, 2003;
Alexandersson et al., 2004; Marmagne et al., 2004).
Plasma membrane proteomics of Arabidopsis thaliana
Many plasma membrane proteomics studies have been
conducted (Prime et al., 2000; Santoni et al., 2000;
Kawamura and Uemura, 2003; Alexandersson et al.,
2004; Marmagne et al., 2004). Marmagne et al. (2004)
reported that about 100 putative plasma membrane proteins
were identified in plasma membrane fractions, obtained
from an Arabidopsis cell suspension culture, enriched
in hydrophobic proteins. Ninety-five per cent of these proteins represent newly identified plasma membrane proteins,
and 50 of these proteins had predicted transmembrane domains. Marmagne et al. (2004) used one-dimensional gels.
To the authors’ knowledge, no non-ionic or zwitterionic
detergent has been found to resolve plasma membrane
proteins quantitatively from eukaryotic cells in IEF for
the first dimension of 2D-PAGE. Thus, so far, it has not
been possible to use 2D-PAGE to resolve integral proteins
of eukaryotic plasma membranes.
Alexandersson et al. (2004) analysed highly purified
Arabidopsis plasma membranes from leaves and petioles
by mass spectrometry to identify integral and peripheral
proteins associated with the plasma membrane. In total,
238 putative plasma membrane proteins were identified,
of which 114 are predicted to have trans-membrane domains or to be glycosyl phosphatidylinositol anchored.
About two-thirds of the integral proteins identified have
not previously been shown to be plasma membrane proteins. Of the 238 proteins identified, 76% could be classified according to their function. Major classes represented
are transport (17%), signal transduction (16%), membrane
trafficking (9%), and stress responses (9%). Almost a
quarter of the proteins identified in this study have no
known function and more than half of these are predicted to
be integral membrane proteins (Alexandersson et al.,
2004). Functional characterization of these unknown proteins should provide clues that may lead to the identification of novel functions for plant plasma membranes.
Proteomics of other membrane systems of
Arabidopsis thaliana
The interest of combining several complementary extraction procedures to take into account specific features of
Proteomics of plant cell membranes
membrane proteins will be discussed in the light of recent
proteomics data, notably for the chloroplast envelope,
mitochondrial membranes and the plasma membrane from
Arabidopsis. Chloroplasts perform vital biosynthetic functions, and many processes are located exclusively within
these unique organelles including the light and dark reactions of photosynthesis, and the biosynthetic pathways for
fatty acids, vitamins, and amino acids. The envelope, a
two-membrane system surrounding all plastid types, is
involved in the synthesis of very specific components like
glycerolipids, pigments, and prenylquinones (Joyard et al.,
1998). In recent years, a number of comprehensive proteomic studies have focused on the chloroplast envelope
(Froehlich et al., 2003; Rolland et al., 2003; Eichacker
et al., 2004) and the thylakoid membrane and lumen
(Peltier et al., 2002, 2004; Schubert et al., 2002; Whitelegge, 2003; Friso et al., 2004). Peltier et al. (2004)
reported a simple, fast, and scalable off-line procedure
based on three-phase partitioning with butanol to fractionate membrane proteomes in combination with both ingel and in-solution digestions and mass spectrometry.
They analysed the salt-stripped thylakoid membrane of
chloroplasts of Arabidopsis, identifying 242 proteins of
which at least 40% were integral membrane proteins. The
functions of 86 proteins remain unknown. This analysis
showed strong differentiation in cellular functions between the two membrane systems and elucidated the
suborganellar localization of many chloroplast membrane
proteins (Peltier et al., 2004).
Mitochondria play a central role in eukaryotic cells by
providing ATP from oxidative phosphorylation. Mitochondria are also involved in many other cellular functions
including numerous catabolic or anabolic reactions and
apoptotic cell death (Balk et al., 2003; Newmeyer and
Ferguson-Miller, 2003). Recent developments in proteomics also open the path toward a deeper exploration of
mitochondrial function using 2D-PAGE, SDS–PAGE, or
blue native PAGE (Millar et al., 2001; Giege et al., 2003;
Millar and Heazlewood, 2003; Brugiere et al., 2004;
Heazlewood et al., 2004). Thus far, the Arabidopsis
mitochondrial membrane proteome is one of the best
characterized in plants. Brugiere et al. (2004) reported
that highly purified mitochondrial membrane proteins,
prepared from Arabidopsis cultured cells, provided the
most exhaustive view of the protein repertoire of these
membranes. Various extraction procedures were applied,
and LC-MS/MS analyses were then performed on each
membrane subfraction, leading to the identification of 114
proteins. About 40% of these proteins had not been
identified during previous proteomic studies performed on
mitochondria (Brugiere et al., 2004). Despite the large
number of previous proteomic studies of plant mitochondria, the strategy that was shown to be most efficient for
identifying new membrane proteins from chloroplast
envelope membranes was also efficient for mitochondrial
105
membranes. Comparative proteomics of mitochondrial
and chloroplast envelope/thylakoid membranes provides a
framework to which additional membrane proteins can be
added as their functions are experimentally determined.
Proteomics of rice membranes
Materials and methods for rice membrane proteomics
Isolation of vacuolar membranes: Rice seedling leaf sheaths
and roots were utilized for isolation of vacuolar membranes. All procedures were carried out at 4 8C. Fresh
tissues of rice seedlings were chopped and ground in
a homogenization medium consisting of 0.25 M sorbitol,
50 mM TRIS-acetate (pH 7.5), 1 mM EGTA, 1% polyvinylpyrrolidone (PVP), 10 lM phenylmethylsulphonyl fluoride, and 2 mM dithiothreitol (DTT) using a mortar and
pestle. The homogenate was filtered through a layer of
Miracloth (Calbiochem, La Jolla, CA, USA). The extract
was centrifuged at 3600 g for 10 min. The supernatant was
collected and centrifuged at 120 000 g for 25 min. The
precipitate was suspended in TRIS-sucrose buffer consisting of 0.5 M sucrose, 20 mM TRIS-acetate (pH 7.5), 1 mM
EGTA, 2 mM MgCl2, and 2 mM DTT, and the suspension
was overlaid with an equal volume of TRIS-sorbitol buffer
consisting of 0.25 M sorbitol, 20 mM TRIS-acetate (pH
7.5), 1 mM EGTA, 2 mM MgCl2, and 2 mM DTT. After
centrifugation at 120 000 g for 45 min, vacuolar membranes that formed a band at the interface between the two
solutions were collected, diluted with the TRIS-sorbitol
buffer, and centrifuged at 130 000 g for 25 min. The
resulting pellet was suspended in the TRIS-sorbitol buffer
and used as the vacuolar membrane fraction.
Isolation of plasma membranes: Rice seedling plasma
membranes were extracted from leaf blades, leaf sheaths,
and roots. A portion (70 mg) of fresh tissues was chopped
and ground in 210 ml of extraction buffer containing 0.4 M
sucrose, 75 mM MOPS/KOH pH 7.6, 5 mM EDTA/KOH
pH 7.5, 5 mM EGTA/KOH pH 8.2, and 10 mM KF
containing 1 mM DTT with 2% PVP using a mortar and
pestle on ice. These homogenates were filtered through four
layers of Miracloth. The filtrates were centrifuged at 9000 g
for 15 min and 42 000 g for 30 min at 4 8C, plasma
membrane-enriched fractions were obtained using a twophase partitioning method (Kawamura and Uemura, 2003).
Two-phase partitioning was repeated three times for the
root samples, and four times for leaf blade and leaf sheath
samples for increased purity.
Isolation of Golgi membrane: Nucleoside diphosphate and
(NDPase)-associated Golgi membrane were prepared from
cultured suspension cells as described by Mikami et al.
(2001). The NDPase-associated Golgi membranes were
further purified by a second centrifugation in a discontinuous density gradient consisting of 28%, 30%, and 34%
106 Komatsu et al.
sucrose in 50 mM glycylglycine–NaOH (pH 7.0) and 5 mM
MgCl2 at 100 000 g for 2 h. The Golgi membranes were
trapped on the 34% sucrose cushion. Enzymes characteristic of the endoplasmic reticulum, mitochondrion,
microbody, vacuolar membrane, and plasma membrane
were assayed as described previously (Mitsui et al.,
1990; Mikami et al., 2001).
Purity of membrane fractions: KNO3, Na3VO4, and NaN3
are specific inhibitors of V-, P-, and F-type H+-ATPases
that are specifically associated with the vacuolar membrane,
the plasma membrane, and the mitochondrial membrane,
respectively (Sze, 1985). The quality of isolated membranes was determined by assaying these specific ATPase
activities. ATPase activity was assayed in a reaction
solution containing 30 mM MES-TRIS (pH 6.5), 50 mM
KCl, 10 mM MgSO4, and 10 mM ATP with or without
inhibitors of ATPase, 100 mM Na3VO4 and 50 mM KNO3
and 2 mM NaN3. The solutions were incubated at 30 8C for
30 min. The reactions were stopped by the addition of
a solution containing 0.5% ammonium molybdate, 1%
SDS, and 1.96% H2SO4, to which ascorbate was added to
a final concentration of 10%. The reaction solution was
incubated at room temperature for 30 min, and the
absorbance at 750 nm was measured. Products of ATPase
activity were calculated from a standard curve generated
with K2HPO4.
Gel electrophoresis: Proteins (50 lg, 100 ll) of vacuolar
membranes solubilized with lysis buffer (O’Farrell, 1975)
were separated in the first dimension by isoelectric focusing
(IEF) or linear immobilized pH gradient (IPG) tube gels
(Daiichi Pure Chemicals, Tokyo, Japan) and in the second
dimension by SDS-PAGE. The IEF tube gel solution
consisted of 8 M urea, 3.5% acrylamide, 2% NP-40, 2%
ampholytes (pH 3.5–10 and pH 5.0–8.0, Amersham
Biosciences, Piscataway, NJ, USA), ammonium persulphate,
and TEMED. Electrophoresis was carried out at 200 V
for 30 min, followed by 400 V for 16 h and 600 V for 1 h.
For IPG electrophoresis, samples were applied to the acidic
side of gels and electrophoresis using IPG tube gels (pH
6.0–10) was carried out at 400 V for 1 h, followed by 1000
V for 16 h and 2000 V for 1 h. After IEF or IPG, SDSPAGE in the second dimension was performed using 15%
polyacrylamide gels. Proteins (20 lg) from plasma membranes were extracted by SDS-sample buffer, and subjected
to SDS-PAGE. The gels were stained with silver or
Coomassie brilliant blue (CBB), and image analysis was
performed. Images of two 2D-PAGE gels; one using IEF in
the first dimension and the other using IPG, were synthesized and the positions of individual proteins on the gels
were evaluated automatically using ImageMaster 2D Elite
software (Amersham Biosciences). The pI and Mr of each
protein were determined using 2-D markers (Bio-Rad,
Hercules, CA, USA).
Mass spectrometry analysis: The stained protein spots were
excised from gels, washed with 25% methanol and 7%
acetic acid for 12 h, and destained with 50 mM NH4HCO3
in 50% methanol for 1 h at 40 8C. Proteins were reduced
with 10 mM DTT in 100 mM NH4HCO3 for 1 h at 60 8C
and incubated with 40 mM iodoacetamide in 100 mM
NH4HCO3 for 30 min. The gel pieces were minced and
allowed to dry and then rehydrated in 100 mM NH4HCO3
with 1 pmol trypsin (Sigma, St Louis, MO, USA) at 37 8C
overnight. The digested peptides were extracted from the
gel slices with 0.1% TFA in 50% acetonitrile three times.
The peptide solution thus obtained was dried and reconstituted with 30 ll of 0.1% TFA in 5% acetonitrile and
then desalted with ZipTip C18 pipette tips (Millipore,
Bedford, MA, USA). The above peptide solution was
mixed with a-cyano-4-hydroxycinnamic acid and analysed
using MALDI-TOF MS (Voyager, Applied Biosystems,
Framingham, MA, USA). In another experiment, each
lane of the SDS-gel was cut out and sectioned into 20
pieces. Each section was further cut into smaller pieces and
digested as described above. These peptide solutions were
concentrated down to 10 ll by vacuum centrifugation and
reconstituted with 10 ll of 0.1% formic acid in water and
analysed using ESI-MS/MS (Nano-Frontier L, Hitachi
High-Technologies Co., Tokyo, Japan and/or Q-TOF
micro, Micromass Co., Manchester, UK). The mass spectra
were subjected to sequence database searches using Mascot
software (Matrix Science Ltd, London, UK).
For MALDI-TOF analysis, three criteria were used to
assign a positive match with a known protein. (i) The
deviation between the experimental and theoretical peptide
masses had to be less than 50 ppm. (ii) At least four different predicted peptide masses needed to match the observed masses for an identification to be considered valid.
(iii) The coverage of protein sequences by the matching
peptides had to reach a minimum of 10%. Furthermore, the
score that was obtained from the analysis with Mascot
software had to indicate the probability of a true positive
identification and the value had to be at least 50.
Plasma, vacuolar and Golgi membrane proteomics
of rice
Rice is not only a very important agricultural resource; it is
also a model plant for biological research because its
genome is smaller than those of other cereals (Devos and
Gale, 2000). Publication of the draft genome sequences for
Oryza sativa L. ssp. indica (Yu et al., 2002) and for Oryza
sativa L. ssp. japonica (Goff et al., 2002), and a complete
map-based sequence of chromosome 1 (Sasaki et al., 2002)
and chromosome 4 (Feng et al., 2002) for Oryza sativa L.
cv. Nipponbare provide rich resources for understanding
the biological processes in rice. Recently, the International
Rice Genome Sequencing Project (2005) presented a
map-based, finished-quality sequence that covers 95% of
Proteomics of plant cell membranes
the 389 Mb genome of rice, including virtually all of the
euchromatin and two complete centromeres. Once the rice
genome is completely sequenced, the challenge ahead for
the plant research community will be to identify the function, regulation, and type of post-translational modification
of each encoded protein. Also, whereas the genome is static;
the proteome is highly dynamic in its response to external
and internal cellular events. The responses of the proteome
can include changes not only to the relative abundance but
also to the post-translational modification of each protein.
In addition to tissue-specific analyses (Komatsu et al.,
2003; Komatsu and Tanaka, 2004; Komatsu, 2005), the
Rice Proteomics Project has analysed biological samples
that are specific to a subcellular compartment such as the
cell wall, plasma membrane, vacuole membrane, Golgi
membrane, mitochondrion, chloroplast, nucleus, and cytosol. Some of these results are described below. The plasma,
vacuolar and Golgi membranes, isolated from rice seedlings and suspension-cultured cells, were solubilized in
lysis buffer (O’Farrell, 1975), and the proteins were
separated by 2D-PAGE and analysed with Image-Master
2D Elite software. The 2D maps of the various subcellular
compartments resolved 464 proteins in the plasma membrane, 141 in the vacuolar membrane, and 361 in the Golgi
membrane (Tanaka et al., 2004). The most abundant
proteins on 2D-PAGE were analysed by Edman sequencing
and mass spectrometry. Edman sequencing showed that the
number of N-terminally blocked proteins varied widely
among the subcellular compartments. In mitochondria and
chloroplasts, respectively, 73% and 60% of the proteins
were N-terminally blocked (Tanaka et al., 2004). A much
larger proportion of the proteins were N-terminally blocked
in the plasma membrane (96%), vacuolar membrane (89%),
and Golgi membrane (98%). The SOSUI system software,
developed for the classification and secondary structure
prediction of membrane proteins (http://sosui.proteome.
bio.tuat.ca.jp/), was used to analyse the proteins identified
in the three membrane samples. This software predicted
transmembrane helices for 14 of the 58 plasma membrane proteins, 6 of the 43 vacuolar membrane proteins,
and 7 of the 46 Golgi membrane proteins sequenced in
this study.
To narrow down the possible role of the more abundant
proteins in subcellular membrane fractions of rice, the
identified proteins were categorized by criteria used by
Bevan et al. (1998). In the plasma membrane of rice, proteins with functions associated with metabolism, energy,
signal transduction, and defence categories were abundant.
By contrast, no proteins in the signal transduction and
defence categories were identified in the plasma membrane
proteome of A. thaliana, but proteins involved in metabolism and energy were present (Santoni et al., 1998).
The vacuolar membrane proteome is still poorly understood, but the vacuolar membrane of plant cells is known
to contain two electrogenic proton pumps, H+-ATPase
107
+
and H -translocating inorganic pyrophosphatase (PPase)
(Hedrich and Schroeder, 1989). In the Rice Proteomics
Project, the b subunit of ATPase was identified in the
vacuolar membrane proteome, but not the PPase. The a2
subunit of the 20S proteasome was detected in the vacuolar
membrane fraction, providing evidence that vacuoles might
participate in the degradation of denatured proteins. The
vacuolar membrane proteome also included a water channel
protein, a member of a family of vacuolar and plasma
membrane proteins that transport water molecules with
high efficiency and selectivity (Maurel, 1997). Signal
transduction proteins were abundant in the vacuolar
membrane and included a calmodulin-like Ca2+-binding
protein. Ca2+ pumps are widely distributed across plant
membranes, including the vacuolar membrane (Sze et al.,
2000).
The Golgi complex is a multifunctional organelle responsible for the biosynthesis of complex cell-surface
polysaccharides, the processing and modification of glycoproteins, and the sorting of polysaccharides and proteins
destined for various locations (Staehelin and Moore, 1995).
To understand better how these functions are carried out,
it will be necessary to survey the proteome of Golgi. For
proteins from the Golgi membrane fraction of rice, the
functional categories of metabolism, energy, and defence
were represented in abundance (Tanaka et al., 2004). A
reversibly glycosylated polypeptide, previously identified
as a protein localized to the Golgi membrane and involved
in the synthesis of xyloglucan and possibly other hemicelluloses (Dhugga et al., 1997), was detected in the Golgi
membrane proteome.
Data on the proteomics of rice membranes will be
valuable for resolving questions in functional genomics
as well as for genome-wide exploration of plant cell
function (Fig. 1).
Vacuolar membrane proteomics changes in
rice treated with gibberellin
Plant cells with defects in vacuole expansion cannot expand
(Schumacher et al., 1999). In these defective cells, the
vacuolar membrane cannot regulate the rapid uptake of
water by expanding vacuoles for a rapid osmoregulation
between the cytosol and the vacuole (Chaumont et al.,
1998). To analyse proteins that affect vacuolar functions,
the vacuolar membrane fraction was isolated using a discontinuous sucrose/sorbitol system. The purity of the
fraction was examined by assaying for H+-ATPase activity.
The sensitivity of H+-ATPase activity to nitrate, vanadate,
and azide was used to distinguish between vacuolar
membrane, plasma membrane, and mitochondrial enzymes,
respectively (Sze, 1985). The proportions of the total
activity that were sensitive to nitrate, vanadate, and azide
amounted to 60%, 10%, and 2%, respectively. The nitratesensitive fraction was thus enriched in the vacuolar
108 Komatsu et al.
Fig. 1. Numbers and percentages of proteins from each functional
category found in the plasma membrane, vacuolar, and Golgi membranes of rice. Proteins were categorized using the criteria of Bevan
et al. (1998).
membrane fraction, but it is clear that the preparation also
contained traces of plasma membrane and mitochondrial
contaminants.
Proteins were extracted from vacuolar membrane fractions of rice leaf sheath from plants treated with 5 lM GA3
for 48 h and from untreated control plants. Proteins were
separated in the first dimension by IEF or IPG and in the
second dimension by SDS-PAGE (Fig. 2; S Komatsu,
unpublished data). The abundance of 20 proteins increased
with GA3 treatment. These proteins were identified by
MALDI-TOF MS analysis. A number of them could be
identified by their sequence similarity with known plant
proteins such as ribosomal protein L (AF526214), glutathione S-transferase (AF402795), hypothetical protein
T20K18.50 (T06628), glyceraldehyde-3-phosphate dehydrogenase (AB106691 and X78307), Arabidopsis thaliana
36716 (AY087569), inorganic pyrophosphatase (AY153213),
RuBisCO SSU (X07515), 3-methylcrotonyl CoA carboxylase (AF251074), connectin/titin N2A-PEVK (AB100271),
clathrin assembly protein AP17-like protein (AF443601),
and dihydrolipoamide acetyltransferase (D21086). These
proteins contribute to vacuolar function in rice leaf
sheaths.
To examine changes in vacuolar membrane proteins in
response to GA3 in the root, proteins were extracted from
vacuolar membrane fractions of rice roots treated or not
with 0.1 lM GA3 for 48 h, and separated by 2D-PAGE. The
abundance of 10 proteins increased in the root vacuolar
membranes by GA3 treatment (Konishi et al., 2005). These
proteins were identified by MALDI-TOF MS analysis.
Some of them were similar to known plant proteins, such as
V-ATPase subunit B (AF375052), ubiquitin RiP-20
(AF216530), V-ATPase subunit A (P31450), ferredoxin
(AF010320), proteinase 2 precursor (S53952), and fructose-1,6-bisphosphate aldolase C-1 (D50301). Aldolase
C-1 increased in GA3-treated roots at low levels in roots
of Tan-ginbozu, a mutant of GA biosynthesis, and this
increase is reduced in roots treated with uniconazole-P or
ABA as compared with the control (Konishi et al., 2005).
These results suggest that aldolase C-1 may act as
a mediator between GA signalling and root growth. In
rice, three cytoplasmic aldolases (aldolase C-1, C-2, and Ca) and one chloroplastic aldolase (aldolase P) have been
reported (Hidaka et al., 1990; Tsutsumi et al., 1994;
Nakamura et al., 1996). Internal amino acid sequences of
protein 09 (Fig. 2) of the root vacuolar membrane (aldolase
C-1) were determined by sequence analysis of peptides
obtained using the Cleveland peptide mapping method
(Cleveland et al., 1977). Aldolase C-1 specific amino
acid residues N (115), S (191), E (195), S (222), E (234),
S (235), Q (241), and L (242) were detected, clearly
indicating that protein 09 identified was neither aldolase
C-2, aldolase C-a, nor aldolase P. Thus, aldolase C-1 is
likely to influence specifically vacuolar function in rice
roots.
Since Aldolase C-1 is involved in glycolysis, these
results suggest that GA3 enhances the metabolic rate of
glycolysis in rice roots. Based on immunoprecipitation
experiments (Konishi et al., 2005), aldolase C-1 activates
V-ATPase through physiological interactions. As a result,
the rate of cell growth of seedling roots may be efficiently
enhanced.
Proteomics of plant cell membranes
109
Fig. 2. Changes in the protein patterns of vacuolar membranes of rice leaf sheaths or roots treated with GA3. Proteins were extracted from the vacuolar
membranes of leaf sheaths treated or not with 5 lM GA3 and roots treated or not with 0.1 lM GA3 for 48 h, separated by 2D-PAGE with IEF and IPG in
the first dimension and SDS-PAGE in the second dimension, and detected by silver staining. The isoelectric point and relative molecular mass of each
protein were determined using 2-D markers (Bio-Rad). Circles show the positions of 20 and 10 proteins whose abundance increased in the vacuolar
membrane of leaf sheaths and roots, respectively, treated with GA3 as compared with control.
Plasma membrane proteomics changes in cold-treated
rice plants
Cold stress is one of the most severe environmental stresses
affecting plant growth and development. Plants that are
subjected to low temperature induce specific physiological
responses. Low-temperature-responsive proteins have been
identified using proteomic approaches. Such cold-response
proteins were participants in various metabolic pathways
such as protein biosynthesis, folding, and degradation, biosynthesis of cell wall components, and the energy pathway
in rice leaf tissue (Cui et al., 2005). Freezing-stress
provokes injury to the plasma membrane and induces the
expression of stress-related proteins. Kawamura and
Uemura et al. (2003) reported that the acidic protein
dehyrin, which increased during cold-acclimation, associates with the plasma membrane resulting in increased
freezing-tolerance. Lipoprotein-like proteins are also associated with acquired resistance to osmotic stress caused
by freezing in Arabidopsis leaves.
Plasma membrane fractions were prepared by an
aqueous-polymer two-phase partitioning method. The
purity of these fractions were determined by assaying for
H+-ATPase activity. The sensitivity of H+-ATPase activity
to vanadate and nitrate was used to distinguish between
plasma membrane and vacuolar enzymes, respectively
(Sze, 1985). The proportion of the total activity that was
sensitive to vanadate and nitrate amounted to 93% and
44%, respectively. The vanadate-sensitive fraction was thus
enriched in the plasma membrane fraction; however, the
preparation contained traces of vacuolar membrane contaminants. A number of plasma membrane proteomics
studies have been carried out in plants, but there are few
plasma membrane proteomic analyses in rice, except for
leaf blade plasma membranes, and only a very few of them
are related to cold stress. In this study, plasma membranes,
not only of leaf blades but also of leaf sheaths and roots,
were purified from cold-stressed rice seedlings (Fig. 3).
Plasma membrane proteins from rice roots were separated and many different proteins were identified. However,
many of these proteins were aquaporins. Proteins that
changed in abundance after cold stress were also detected.
In leaf blades of rice seedlings, more than 100 proteins were
determined as plasma membrane proteins by MALDI-TOF
MS and ESI-MS/MS. Chlorophyll a/b binding protein,
the large and small subunits of ribulose-bisphosphate
carboxylase/oxygenase (RuBisCO), putative photosystem I
reaction centre subunit II, glyceraldehyde-3-phosphate
dehydrogenase, and several unknown proteins were determined in the same fractions. Whether these are contaminants of the plasma membrane preparation or not cannot
be definitely determined before tagged fusion proteins
have been made or immunogold localizations have been
done. Only a few obvious contaminants, such as highly
110 Komatsu et al.
Conclusions
Fig. 3. Changes in the protein patterns of the plasma membrane from
rice leaf blades, leaf sheaths or roots upon exposure to cold. After cold
treatment at 5 8C, leaf blades, leaf sheaths and roots were collected, and
plasma membranes were purified. Plasma membrane proteins were
separated by SDS-PAGE, and stained with CBB. The protein bands that
change with cold treatment are marked on the figure with closed circles.
abundant soluble proteins like the RuBisCO large subunit
and chlorophyll a/b binding protein which are chloroplast
proteins, were found. It might be difficult to detect leaf
blade plasma membrane proteins in the absence of
chloroplast membrane contamination. On the other hand,
glyceraldehyde-3-phosphate dehydrogenase has been immunolocalized outside fungal cells and may be similarly
localized in plants (Gozalbo et al., 1998).
Among the proteins found differentially regulated in rice
leaf blades subjected to cold stress, several functionally
unknown proteins and the RuBisCO small subunit were
detected. The RuBisCO small subunit is generally translated as a precursor from a nuclear gene including a signal
peptide for chloroplast targeting (Dean et al., 1985);
however, the TargetP program (http://www.cbs.dtu.dk/
services/TargetP) predicted that the product of this
cDNA, for example, the mature RuBisCO small subunit
identified in this study, clearly has no signal peptide
(Kawamura and Uemura, 2003), suggesting that this protein may remain in the cytoplasm and become associated
with the plasma membrane during cold stress.
Due to the wide variety of physiological and biochemical
reactions carried out in different membrane systems, the
proteomes of plant subcellular compartments should be
fully described. This requires the ability to separate and
purify membranes successfully. However, no methods are
currently available to purify particular membranes from
plant cells to homogeneity, and cross-contamination with
other membranes and/or cytosolic constituents must be
expected. One way to circumvent this problem is to correlate the presence of particular proteins with the abundance
of different cell membranes and/or to confirm the location
of particular proteins using complementary techniques such
as fluorescence-tagging or immunochemical approaches.
Numerous membrane-associated proteins in plants are
still to be discovered and characterized (Ephritikhine et al.,
2004). To facilitate proteomic analyses, more powerful
bioinformatics tools capable of analysing large sets of
proteins have been developed, including tools devoted to
the systematic analysis of membrane protein structures
(Schwacke et al., 2004). In other respects, the functional
characterization of proteomes still lacks completely accurate and reliable programmes for functional annotation,
especially in the case of rice membrane proteins. Progress
should come from a combination of proteomic strategies
applied to membrane sub-proteomes. Taking into account
the dynamic nature of membrane proteomes arising from
the diversity between organs and organelles, and the
functional specificity of membrane systems, it is predicted
that post-translational modifications in response to stresses
should bring the first clues about protein functions.
Analysis of membrane proteins remains a major challenge for proteomics techniques based on 2D-PAGE. For
this reason, alternative methods based either on the use of
SDS–PAGE (Bell et al., 2001), or on the separation of
digested peptides have been described (von Haller et al.,
2001; Wolters et al., 2001). Although these methods have
been successful for the identification of membrane proteins,
they do not achieve the combination of quantitative analysis
and degree of separation of protein variants available
through the use of 2D-PAGE. The solubilizing power of
various nonionic and zwitterionic detergents as membrane
protein solubilizers for 2D-PAGE has been reported (Luche
et al., 2003). These results suggest that additional strategies
must be used in order to gain insight into the characterization of membrane proteins. Furthermore, many plasma
membrane proteins are probably only expressed in certain
cell types, at discrete development stages, or in response to
a particular stress. This dynamic nature means that the
large majority of plasma membrane proteins remain to be
identified. In addition, the functions of many of these proteins are presently unknown, many proteins identified in the
present study are functionally unclassified, and more than
half of these are predicted to be integral membrane proteins.
Proteomics of plant cell membranes
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