Plant Physiology and Biochemistry 42 (2004) 943–962

www.elsevier.com/locate/plaphy

Review
Plant membrane proteomics
Geneviève Ephritikhine a,1,*, Myriam Ferro b,1, Norbert Rolland c,1
a
Institut des Sciences du Végétal, CNRS (UPR 2355), Bâtiment 22, avenue de la Terrasse, 91198 Gif sur Yvette cedex, France
b Laboratoire de Chimie des Protéines ERM-0201 (Inserm/CEA), Département Réponse et Dynamique Cellulaires, CEA Grenoble, 17, rue des Martyrs,
38054 Grenoble cedex 9, France
c Laboratoire de Physiologie Cellulaire Végétale UMR 5168 (CEA/CNRS/Inra/Université Joseph Fourier), Département Réponse et Dynamique
Cellulaires, CEA Grenoble, 17, rue des Martyrs, 38054 Grenoble cedex 9, France
Received 16 September 2004; accepted 9 November 2004
Available online 22 December 2004

Abstract

Plant membrane proteins are involved in many different functions according to their location in the cell. For instance, the chloroplast has
two membrane systems, thylakoids and envelope, with specialized membrane proteins for photosynthesis and metabolite and ion transporters,
respectively. Although recent advances in sample preparation and analytical techniques have been achieved for the study of membrane pro-
teins, the characterization of these proteins, especially the hydrophobic ones, is still challenging. The present review highlights recent advances
in methodologies for identification of plant membrane proteins from purified subcellular structures. The interest of combining several comple-
mentary extraction procedures to take into account specific features of membrane proteins is discussed in the light of recent proteomics data,
notably for chloroplast envelope, mitochondrial membranes and plasma membrane from Arabidopsis. These examples also illustrate how, on
one hand, proteomics can feed bioinformatics for a better definition of prediction tools and, on the other hand, although prediction tools are not
100% reliable, they can give valuable information for biological investigations. In particular, membrane proteomics brings new insights over
plant membrane systems, on both the membrane compartment where proteins are working and their putative cellular function.
© 2005 Elsevier SAS. All rights reserved.

Keywords: Arabidopsis; Membrane proteins; Proteomics; Plant; Subcellular localization

1. Introduction The first major difference between genome and proteome

With the recent advance of the genome-sequencing
projects, thousands of known and hypothetical proteins have
been identified in plant genome sequences. Proteome analy-
ses of plant cells has become a technology to provide infor-
mation on functional genomics, including identification or
validation of hypothetical open reading frames (ORFs) from
genome sequences, determining the subcellular localization
of proteins, identifying components of a multifunctional pro-
tein complex, and identifying novel components involved in
plant cell metabolism.
* Corresponding author. Tel.: +33 1 69 82 37 93; fax: +33 1 69 82 37 68.
E-mail address: genevieve.ephritikhine@isv.cnrs-gif.fr
(G. Ephritikhine).
1
Equally contributing authors.
0981-9428/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.plaphy.2004.11.004
is that the genome is static, while the proteome of living cells
is dynamic, altering in response to the individual cell’s meta-
bolic state and reception of intracellular and extracellular sig-
nal molecules. The analysis of proteins is thus the most direct
approach to the dynamic of plant cell metabolism [57].
In addition, plant cells are compartmentalized and this
compartmentalization is supported by subsets of gene prod-
ucts that are specifically targeted to particular subcellular
organelles. This subcellular compartmentalization of plant
cells provides distinct and suitable environments for various
biochemical processes. Thus, protein localization is linked to
cellular function. However, to date, the subcellular localiza-
tion and in planta functions of most of the known and puta-
tive gene products identified in the sequenced plant genomes
remain to be determined. There is thus a need for proteome
944 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962
strategies, as the analytical complement of genome-
sequencing, to address the organization levels of plant cells
[17].
Proteome analyses at the level of subcellular structures rep-
resent an analytical strategy that combines classical biochemi-
cal fractionation methods (when available), to enrich for par-
ticular compartments, and tools for the comprehensive
identification of proteins. Among the key potentials of this
strategy is the capability to enhance the understanding of the
biochemical machinery of the purified organelle for subse-
quent functional studies.
Membranes play a critical role in eukaryotic cells by pro-
viding a physical barrier between the cell and its environ-
ment and these various subcellular compartments. As the
result of unique and compartment-specific compositions (lip-
ids, proteins, pigments...), these membranes have character-
istic functional properties.
In this review, we focus on plant membrane proteomics
and on recent advances in methodologies for identifying these
membrane proteins from subcellular structures.
1.1. Limits of classical methodologies for the identification
of membrane proteins
A membrane proteome can be defined as the entire comple-
ment of membrane proteins present in a cell under a specific
condition, and at a specific time [90]. Proteomic analysis of
membrane proteins remains a major challenge. Although they
represent almost one-third of eukaryotic genomes, mem-
brane proteins are more difficult to analyze than soluble pro-
teins and their representation in datasets is generally low. This
is especially true with integral membrane proteins for the fol-
lowing reasons: (a) due to physico-chemical heterogeneity of
these proteins, 2-D gel separation is not appropriate for a com-
prehensive mapping of membrane proteins, (b) many hydro-
phobic proteins are not solubilized in the non-detergent iso-
Fig. 1. Outline of the different steps involved in the proteomic study of plant membrane proteins from their purification to their functional characterization.
Subcellular fractionation, extraction procedures, analytical techniques, in silico approaches and functional studies will be developed in the different sections of
the present paper.
electric focusing sample buffer and precipitate at their
isoelectric point and (c) low abundance proteins, including
rare membrane proteins, are out of the scope of standard pro-
teomic techniques (for reviews, see [64,68,89,90]).
It is now clear that subcellular fractionation and biochemi-
cal enrichment of these proteins are required, as a first step,
to overcome these abundance issues. More recently, various
procedures were developed to separate proteins as a second
step prior to MS-based protein identification. First, solubili-
zation of membrane proteins was improved using the power
of various non-ionic and zwitterionic detergents as plant
plasma membrane protein solubilizers for 2-D electrophore-
sis [45]. Second, alternatives to the first dimension of classi-
cal 2-D gels were developed, e.g. blue-native-PAGE separa-
tions of plant mitochondrial membrane complexes [41],
sequential extractions based on the physico-chemical prop-
erties of the membrane proteins of various plant membrane
systems [8,20,23,48,62] or anion exchange chromatography
of ASB-14-solubilized membrane proteins [72].
1.2. Proteomics of subcellular structures and their
membrane systems: a rapid survey
Subcellular fractionation represents the first step of sub-
cellular proteome analyses (Fig. 1) and many procedures
established in the past decades can be adapted to determine
the protein content of these structures. To date, while plastids
and mitochondria represent the major investments for the
analysis of the subcellular proteomes of plant cell organelles
and membranes, studies concerning other cell structures were
reported (for recent reviews, see [12,17,50,57]). Over the last
years several subcellular proteome studies have been pub-
lished on various plant cells compartments, extracted from
leaves and/or cell cultures deriving from various plant or algal
species (Arabidopsis, Chlamydomonas, pea, rice, tomato,
tobacco, spinach...).
 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962
1.2.1. Plastids and their membrane systems (inner
and outer envelope and thylakoids)
Plastids, and especially chloroplasts, conduct vital biosyn-
thetic functions, and many reactions are located exclusively
within these unique organelles (photosynthesis, amino acids
biosynthesis, fatty acids and vitamins biosynthesis...). A two
membrane system, the envelope, surrounds all plastid types.
This plastid envelope is particularly involved in the synthesis
of very specific components like glycerolipids (MGDG,
DGDG, SL and PG), pigments (chlorophylls, carotenoids),
prenylquinones (plastoquinone-9, a-tocopherol...) [37]. In
recent years, a number of comprehensive proteomics studies
and review articles have focused on the chloroplast envelope
[20–22,24,66,75] and the thylakoid membrane and lumen
[23,35,61,73,88]. Most of these studies were reporting analy-
ses performed on thylakoid or envelope membrane proteins
thus leading to identification and subcellular localization of
more than 700 plastid membrane (envelope and thylakoid)
proteins. Information concerning most of these identified pro-
teins can be retrieved from the plastid proteomics database
(PPDB; http://ppdb.tc.cornell.edu/ [62]).
1.2.2. Mitochondria and their outer and inner membranes
Mitochondria play a central role in eukaryotic cells by pro-
viding ATP by the process of oxidative phosphorylation. Mito-
chondria are also involved in many other cellular functions
including numerous catabolic or anabolic reactions and apo-
ptotic cell death [3,56]. The recent development of proteom-
ics also opens the path toward a deeper exploration of the
mitochondrial functions and recent works dealing with plant
mitochondria proteome analysis from Arabidopsis [8,29,32,
41,51,52], pea [5], Chlamydomonas reinhardtii [83] and rice
[31,40] has proven the usefulness of the approach. It is impor-
tant to note that, when membrane proteins were considered
in some of the above cited investigations, several alternatives
to 2-D electrophoresis (1-D or 2-D SDS-PAGE, BN-PAGE
separations...) were used. As a consequence, and to date, the
plant mitochondrial membrane proteome is one of the best
characterized and a dozen of mitochondrial proteome analy-
ses were already collected in a mitochondrial proteome data-
base (http://www.mitoz.bcs.uwa.edu.au/; see [32]).
1.2.3. Vacuole and tonoplast
The vacuole contributes to cell growth by occupying most
of the volume of the cell, stores secondary metabolites and
excess nutrients, and acts as a repository for substances that
might otherwise interfere with cytoplasmic homeostasis (for
review, see [46]). A large number of proteins in the tonoplast,
including pumps, carriers and ion channels support the vari-
ous functions of the plant vacuole. Until recently, few pro-
teins involved in these activities had been identified at the
molecular level. Recent proteomic studies were performed
on the total vacuole or on the tonoplast and allowed identifi-
cation of many new Arabidopsis tonoplast membrane pro-
teins [70,77,81].
945
1.2.4. Plasma membrane
The main function of plasma membrane is to regulate the
exchanges of information, ions and metabolites between the
cell and its surrounding environment. The plant plasma mem-
brane carries additional functions, such as cell wall biosyn-
thesis and responses to biotic and abiotic factors. Due to the
lack of specific targeting sequences to identify plasma mem-
brane proteins from genomic sequencing information, very
few proteins were identified in this membrane system. A pio-
neering effort was developed which resulted in the first cata-
logue of protein present in the Arabidopsis plasma mem-
brane [67,69]. However, while based on the extraction and
solubilization of proteins using various detergents and the use
of 2-D electrophoresis, the majority of proteins identified in
these studies corresponded to peripheral proteins. Based on
the progress achieved in the Arabidopsis genome-sequencing
project, coupled to sensitive and rapid identification of pro-
teins by mass spectrometry and to the use of various extrac-
tion and separation procedures, more recent proteomics
approaches have been developed to analyze the complex func-
tions of this membrane system [48,58,59].
1.2.5. Peroxisomes/glyoxisomes and their membrane
In the cells of higher plants, peroxisomes differentiate into
at least three different classes: glyoxysomes, leaf peroxi-
somes, and unspecialized peroxisomes. These organelles con-
tain enzymes of the fatty-acid b-oxidation cycle, the glyoxy-
late cycle, the photorespiration pathway and the H2O2-
scavenging pathway (for review, see [30]). To better
understand the functions of such organelles, proteomic analy-
ses were also recently performed on peroxisomes and gly-
oxysomes from Arabidopsis cotyledons [25,26]. Most of the
proteins identified during these studies derived from the
soluble phase of these organelles and further investigations
are required to examine their membrane composition.
1.2.6. Nuclei and the nuclear envelope
The nucleus regulates various biological activities, includ-
ing gene expression. It stores genes on chromosomes, pro-
duces messengers that code for protein, transports regulatory
factors and also participates to many signaling responses. The
nucleus is separated from other cell compartments by a
double-membrane system (the nuclear envelope) that is punc-
tuated by pores formed by the aqueous central channel of
supramolecular proteinaceous structures called Nuclear Pores
Complex (for review, see [34]). Recent reports on the nucleus
of Arabidopsis have been published and highlighted their
molecular functions [2,11,38]. Most of the proteins identi-
fied during these studies derived from the nuclear matrix and
the identification of very few membrane proteins was reported.
The development of purification strategies to isolate pure
nuclei in detergent-free media [10,91] will probably lead to a
better coverage of the nuclear envelope proteome.
1.2.7. The endoplasmic reticulum and the Golgi apparatus
The endoplasmic reticulum (ER) is central to the synthe-
sis, sorting and storage of protein and lipid reserves. A first
946 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962
study was published which described the proteomics analy-
sis of organelles from Arabidopsis, including ER and the
Golgi apparatus [63]. More recently, ER has been prepared
and analyzed from germinating and developing castor bean
endosperm [47]. Sample complexity has first been reduced
by separation of ER proteins into lumenal, peripheral mem-
brane and integral membrane sub-fractions. A combination
of 1-D and 2-D gel electrophoresis was used to study the com-
plexity of the sample thus leading to identification of both
soluble and membrane proteins. More extensive analysis will
have to be performed but the basic separation technologies
for a plant ER preparation have been established.
1.2.8. Other cell structures
Finally, some non-containing membrane structures were
analyzed that are thus out of the scope of this paper. It is
however important to cite a pioneering effort developed which
resulted in the first catalogue of protein present in the pri-
mary cell walls of suspension cultured cells of five plant spe-
cies, Arabidopsis, carrot, french bean, tomato, and tobacco
[65] and the more recent proteomics approach which have
been developed to further analyze the complex functions of
this plant structure [7].
2. Extraction of membrane proteins from specific
membrane systems
2.1. Diversity of plant membrane systems: biochemical
models versus genetic models
It is important to note that the biochemical models used to
develop subcellular fractionation technologies and to ana-
lyze the proteome content of a plant cell membrane system
does rarely correspond to species for which an extensive (if
not complete) genetic information was available. In the past
decades, biochemists trying to purify a rare enzyme using
classical biochemical approaches almost never used Arabi-
dopsis as a model system. The strategy was to choose an eas-
ily available plant system and to test the specific activity of
their target enzyme. When poorly active or stable in these
species, other plant species where tested for the specific activ-
ity of the target enzyme and the selected plant model was
used for further subcellular fractionation and/or purification
studies. As a consequence, many protocols designed to iso-
late pure organelles and their membrane systems are not
immediately adaptable to subcellular proteomic studies for
several reasons: (a) purity of the protein sample is not vali-
dated, or the enriched fraction contains too many contami-
nants (b) the amount of genetic information is too low in the
corresponding plant system and (c) the yield of protein is too
low in the purified membrane fraction.
To only cite examples about organelle purifications, extrac-
tion of pure mitochondria from Arabidopsis leaves or any
other Arabidopsis tissue was poorly referenced and these
mitochondria are generally contaminated (and inactive) when
extracted from tissues. Pure and active mitochondria can eas-
ily be extracted from potato tubers or pea tissues (roots, leaves,
seeds...). When purified, the mitochondria are easy to brake
(using osmotic shock) and the expected yield of membrane
proteins is close to 50%. Chloroplasts can be extracted from
Arabidopsis but also from many other plant species (tobacco,
pea, spinach, rice, maize...). One major limit comes from the
possibility to sub-fractionate these organelles. Pure and active
spinach chloroplasts can be easily broken by a simple osmotic
shock. As a consequence, sub-plastidial fractions are gener-
ally highly pure and poorly cross contaminated. On the con-
trary, some other chloroplasts (Arabidopsis, pea...) require
mechanical processes to liberate their soluble and membrane
sub-components (envelope, stroma, thylakoids). A conse-
quence of these treatments is the induced cross-contamination
of the envelope and thylakoid membranes (e.g. using mechani-
cal process or freeze/thaw cycles) resulting from creation of
mixed membrane vesicles.
2.2. Subcellular fractionations: methods
The best methods to obtain pure plant organelles still rely
on (a) tissue and cell breakage using mechanical processes
(Waring blendor...) and recovery of cell contents and debris
in buffered medium (to limit proteolysis attack due to acidity
of the vacuolar content) and an osmoticum (to maintain the
integrity of the organelles), (b) a series of differential cen-
trifugations, to enrich the targeted intact organelle and to
eliminate soluble proteins (deriving from the cytosol, vacu-
ole and broken organelles) and cell debris, (c) Percoll-based
gradients to purify the targeted organelle (e.g. plastids or mito-
chondria) or two-phase partitioning in aqueous polymers (e.g.
plasma membrane) or sucrose gradients (e.g. ER or tono-
plast). Percoll-purified plastids or mitochondria contain very
few protein contaminants from the cytosol [8,20]. It is sus-
pected that the abrasive nature of the Percoll beads elimi-
nates all protein contaminants from the outer surface of the
so-purified organelles. Furthermore, Percoll is osmotically
inactive and can be combined with a constant osmoticum con-
centration in the whole gradient. It is thus surprising to find
that some recent subcellular proteomics approaches still rely
on plant organelles purified on sucrose gradients knowing that
the lack of correspondence of iso-osmolarity and iso-density
in these sucrose gradients is often poorly compatible with
organelle integrity. Then, sub-organellar fractionation relies
on osmotic or mechanical breakage of the organelles and fur-
ther fractionation using differential centrifugation (e.g. crude
mitochondrial membrane pellet versus soluble content of the
matrix in the supernatant) or sucrose gradients (when several
sub-organellar fractions needs to be purified; e.g. envelope
and thylakoid membranes from broken chloroplasts, photo-
systems and or LHC protein complexes from thylakoid mem-
branes...).
The first two steps described above for preparing organelle
suspensions, i.e. tissue homogenization and differential cen-
trifugation, are also prerequisite for the purification of other
 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962
cell membrane systems constituting the microsomal fraction.
Then, the purification step depends on the final objective.
When focusing on plasma membrane, the two-phase parti-
tioning in aqueous polymers (a mixture of dextran and poly-
ethyleneglycol) is the most suitable technique, as the PEG
upper phase is highly enriched in plasma membrane while all
the endomembranes are concentrated in the lower phase. Puri-
fication on sucrose gradients remains the most commonly used
procedure to split microsomes in their different membrane
components (reticulum endoplasmic, Golgi membranes, tono-
plast...) on the basis of their density. An alternative is the sepa-
ration of the different membrane systems from microsomes
using the free flow electrophoresis technique.
2.3. Sample purity
The aim of organelle proteomics is first to establish the
protein repertoire of various subcellular compartments. Pro-
teins identified in subcellular membrane proteomic studies
can only be assigned to an organelle or a membrane if there is
no contaminant present in the sample preparation. As a result,
the majority of plant organelle proteomic studies have focused
on the chloroplast and mitochondria, which can be isolated
rather easily. However, the isolation of components of the
endomembrane system is far more difficult due to their simi-
lar sizes and densities.
Purity should be the first priority before yield. To give one
example, the yield of highly purified chloroplasts from spin-
ach or Arabidopsis leaves is close to 2% of the original leaf
contents [20]. As a consequence, and especially when
organelle purification is a first step before sub-fractionation,
it is expected to start from large amounts of tissues (which
might be limiting for small plants like Arabidopsis).
During subcellular fractionation, enzymatic marker activi-
ties should be tested to estimate the contamination deriving
from other subcellular compartments (Table 1). Alterna-
tively, enrichment of immunoreactivity for known marker pro-
teins of the target subcellular compartments, together with a
decrease of the immunoreactivity for markers deriving from
other cell structures are monitored (Table 1). These ap-
proaches are often combined to other alternatives, relying on
compartment-specific methods (i.e. pigment composition of
a membrane, microscopy, detection of major component...)
which help to validate integrity or purity of the target
organelles or subcompartments (Table 1). It appears from
Table 1 that most of the samples analyzed were not tested for
contamination with specific markers deriving from all other
subcellular compartments or membrane systems. However,
it also appears that, most of the times, only specific structures
contaminate the analyzed membrane systems. To cite some
examples, envelope preparations were mostly contaminated
with stroma- and thylakoid-deriving proteins while plasma
membrane contaminants were mainly deriving from the cyto-
sol (Fig. 2). Anyway, whatever the level of analyses origi-
nally performed to detect contaminating proteins in the puri-
fied membrane fraction, complementary approaches are
947
required to validate subcellular proteomics data (see below,
Section 4).
2.4. Membrane fractionation: biochemical methods for
the recovery of membrane proteins
The real challenge of membrane proteomics, is to find the
way of extracting and identifying the whole set of membrane
proteins, including the integral proteins (Fig. 1). Firstly, one
should start from highly purified membrane systems to ana-
lyze rare and hydrophobic membrane proteins present in these
structures. Complexity of the membrane fractions to be ana-
lyzed can be reduced using different strategies and several
methods have been developed to analyze membrane proteins
from the less to the more hydrophobic ones [8,20,23,48,62].
The development of such technologies allowed revealing the
hydrophobic protein composition of a particular membrane
system in a given tissue, thus improving our understanding
of the plant membrane systems.
2.4.1. Sonication of membrane vesicles
Considering the purity of membrane fractions deriving
from organelles, not every proteins present in these prepara-
tions can be considered as a genuine membrane protein. Some
proteins deriving from the soluble compartments are always
trapped in the purified membrane vesicles deriving from
organelles. Further treatments of these vesicles are thus
required to eliminate these contaminating soluble proteins.
Too aggressive treatments of these vesicles will disrupt the
membrane structure and eliminate some genuine membrane
interacting proteins. An easiest way to eliminate these con-
taminating proteins thus remains sonication of the mem-
brane vesicles. Since the vesicles are broken and reformed
during sonication, soluble proteins are released and diluted
in the soluble fraction and can be eliminated after re-
centrifugation of the membrane proteins.
Depending on the analyzed membrane system, such a treat-
ment can eliminate up to 50% of the original content of the
purified membrane protein fraction (e.g. crude envelope mem-
brane fraction contaminated with major stroma proteins). Such
a treatment was demonstrated to remove the major part of the
contaminating RuBisCO from purified envelope membrane
fractions while not affecting the interaction of the peripheral
membrane proteins ceQORH [54]. To cite few examples, this
treatment was applied for studies on membrane vesicles deriv-
ing from plastid envelope membranes [20], mitochondrial
membranes [8] and plasma membrane [48].
2.4.2. Salt treatments
Salt (NaCl, KBr, KNO3...) treatments were demonstrated
to abolish electrostatic interactions of peripheral membrane
proteins with integral membrane proteins or the polar head of
lipids. As a consequence, many peripheral proteins are elimi-
nated after these types of treatments when most of the inte-
gral membrane proteins are still embedded in the vesicle bilay-
ers. As stated above, sonication of the membrane vesicles in
948 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962

Table 1
Markers and methods used for characterization of the purity of subcellular fractions in some recent studies related to plant proteome investigations. “–”
Indicates the lack of data for such markers or methods in referenced studies
Compartments Sub compartments Immunological markers Enzymatic markers Other methods used
Mitochondria Inner membrane Nad-9 [48] Cytochrome-c oxidase Oxygen consumption by isola-
[12,25,26,52] ted mitochondria [52], subunits
of the glycine decarboxylase
complex in green tissues as
major component in 2D gels [5]
Outer membrane Tom-40 [48]
Matrix T-subunit of the glycine decar- Malate dehydrogenase [40]
boxylase complex [8], Cpn10
[25]
Plastids Inner envelope membrane E37 [48], ceQORH, IE18 – Pi/triose-P as major component
[20,54] in 1-D gels [20]
Outer envelope membrane OEP24 [54] OEP24 as major component in
1-D gels [54]
Thylakoid membrane LHCP [8,20,35], OEE2 [2] – Chlorophyll content of thy-
lakoids [20,21]; BN-PAGE
detection of photosynthetic
complexes [41], carotenoids
content of plastid membranes
[52], LHCP as major compo-
nent in 1-D gels of thylakoids
[20,21]
Stroma Cpn20 [25] Alkaline pyrophosphatase [52] RbcL protein as major compo-
nent in 1-D gels [20,21]
Plasma membrane – P-type H+-ATPase [48,38] P-type H+-ATPase [48,38] –
Vacuole Tonoplast c-TIP [48], V-type H+-ATPase V-type H+-ATPase [77] –
( [38]
Soluble proteins RD21A [25] – –
Nuclei Envelope membranes – – Photomicrograph of nuclei-
enriched fractions [38]
Matrix Histone H1 [2,38], REP1 [2] –
Peroxysomes Membranes – – –
Soluble proteins Catalase [26], hydroxy- Catalase [25,26,52] –
pyruvate reductase [26]
Cytosol – Glutathione-S-transferase [2], Alcohol dehydrohenase [52] –
aconitase [25]
ER – BiP [25,77] – Protein disulfide isomerase or
calreticulin as major compo-
nents in 1-D gels [47]
Golgi – – Latent IDPase activity [77] –
Cell wall – – – Integrity of cellular compart-
ments by electron microscopy
[7]

the buffer used for the salt treatment helps to remove periph-
eral proteins from the inner surface of membrane vesicles
(50% of proteins to be removed). Non-sonicated chloroplast
membrane vesicles still store some large amounts of contami-
nating stroma proteins. Salt treatments were performed dur-
ing the analysis of plastid envelope membranes [20], mito-
chondrial membranes [8] and plasma membrane [67].
2.4.3. Alkaline treatments
Alkaline treatments of membrane vesicles are expected to
eliminate not only peripheral membrane proteins but also
some lipid anchored proteins and proteins that interact through
hydrophobic interactions with integral membrane proteins or
the polar head of lipids. As a consequence, many proteins are
lost after these types of treatments and essentially true inte-
gral membrane proteins remain attached to the vesicle bilay-
ers. As stated above, sonication of the membrane vesicles in
the buffer used for the alkaline treatment is essential to elimi-
nate the proteins from both surfaces of membrane vesicles.
The use of membrane washes with carbonate buffer or NaOH
was applied to proteome analysis of plastid envelope mem-
branes ( [20] and unpublished results), mitochondrial mem-
branes [8,51], plasma membrane [48,67] and thylakoid mem-
branes [23].
2.4.4. Organic solvent extractions
Treatments of membrane vesicles by organic solvents like
chloroform/methanol (C/M), n-butanol... are expected to
extract the most hydrophobic proteins of the analyzed mem-
brane system. It was originally demonstrated that most of the
 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962
Fig. 2. Number and nature of contaminating proteins identified during pro-
teomics analyzes performed on chloroplast envelope [20], mitochondrial
membranes [8] and plasma membrane [48]. Note that major contaminants of
analyzed membrane systems mainly derived from specific compartments of
the cell (e.g. other sub-plastidial compartments for the chloroplast envelope,
cytosolic proteins for the plasma membrane).
proteins that are extracted in organic solvents contain a high
ratio of residues number on transmembrane domains (res/TM
ratio) [75]. It is thus expected that not only being an integral
protein is required to be soluble in the organic phase, but this
protein must also contain very few and short hydrophilic
domains. This method is versatile and could be applied to
various membrane systems including plastid envelope mem-
branes [20,21,75], thylakoid membranes [22,23], mitochon-
drial membranes [8,51] and plasma membrane [48].
Three major limits were identified for such a treatment.
First, following the treatment, the hydrophobic proteins (few
percent of the original membrane content) are recovered in
an organic solution containing 100% of the original lipids
and pigments. Elimination (evaporation under nitrogen or
argon) of the organic solvent allows recovering a pellet con-
taining all these components. As a consequence, it is almost
impossible to re-solubilize these proteins in classical sample
buffers and to analyze them on a classical SDS-PAGE. One
949
option relies on the elimination of lipids and pigments using
washes of the proteins/lipids/pigments pellet with cold acetone
or isopropanol. This was demonstrated to be poorly efficient
and lowers the yield of hydrophobic membrane proteins puri-
fication by losing many proteins in the washes [75]. An alter-
native relies on the direct precipitation of hydrophobic pro-
teins in the organic phase using 4 vol. of cold acetone [20,21].
This allows removing lipids and pigments from the protein
sample and strongly limits the problems with re-solubilization
and migration of the hydrophobic proteins.
The second major limit concerns the original content of
the purified membrane fraction. The proteins that are not
soluble in the organic solvents are expected to precipitate. As
a consequence, if the original membrane fraction contains a
too high proportion of poorly hydrophobic membrane pro-
teins and many hydrophilic contaminants, this induces the
precipitation of all the proteins present in the original sample.
It is thus very important to limit the original protein sample
to genuine membrane proteins. An alternative relies on the
use of previous salt or alkaline treatments of the purified mem-
brane vesicles. This allows lowering the proportion of poorly
hydrophobic proteins in the sample and thus limits the loss of
hydrophobic proteins induced by precipitation of hydro-
philic proteins.
The last major limit concerns the yield of proteins extracted
from the purified membrane fraction. This treatment being
selective for highly hydrophobic proteins [75], most of the
original content of the membrane vesicles, including some
true integral membrane proteins, will be lost after this type of
extraction.
2.4.5. Differential extractions using detergents
These treatments are expected to solubilize proteins from
the most hydrophilic to the more hydrophobic ones. As
expected, the more the protein is embedded in the membrane
bilayer, the higher the concentration of detergent will be
required to solubilize it. The reproducibility of the efficiency
of such treatments depends on the lipid content in the target
membrane and the ratio between detergent and protein con-
centration in the sample. This type of treatment was used to
validate the suspected hydrophobicity of some plastid enve-
lope proteins [54,75], but also to lower the proportion of
poorly hydrophobic proteins in original membrane protein
samples before organic solvent extractions [20,48,67] or gel
separation [23].
3. Identification of plant membrane proteins: specific
features and bioinformatics approaches
3.1. Complementary methods: towards an exhaustive
catalogue of protein content
Various proteomic studies of plant membrane systems pro-
vide evidence for the interest in using a wide set of methods
to get the most exhaustive repertoire of a complex protein
950 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962

Table 2
Recent proteomics studies of plant membrane systems provide evidence for the interest in using a wide set of methods to get the most exhaustive repertoire of
a complex protein mixture. Extraction procedures used to identify membrane proteins in a (non-exhaustive) list of recent plant membrane proteomic studies
Membrane system
Plasma membrane
Plasma membrane
Plasma membrane
Mitochondrial membranes
Mitochondrial membranes
Mitochondrial membranes
Tonoplast
Tonoplast
Chloroplast envelope
Chloroplast envelope
Thylakoid membrane
Thylakoid membrane
Extraction procedure Electrophoretic and MS methods Specificity References
1-Na2CO3 treatment, 2-PI-PLC 1DE, LC–MS/MS Recovery of GPI-anchored- [18]
treatment + two-phase separation proteins
C/M extraction, NaOH treatment 1DE, MALDI, ESI-MS/MS, Differential extraction procedures [48]
LC–MS/MS
Cold Triton X-100 treatment 1-D and 2DE, LC–MS/MS Recovery of Triton-insoluble frac- [55]
tion (lipid rafts)
C/M extraction, NaOH treatment, 1DE, LC–MS/MS Differential extraction procedures [8]
NaCl treatment
Sub-fraction of the mitochondrial LC–MS/MS Identification of protein import [44]
membrane fraction proteins
Na2CO3 treatment, C/M extraction 1DE, ESI-MS/MS Identification of carrier proteins [51]
Na2CO3 and KSCN treatment 1DE, LC–MS/MS Membrane and peripheral proteins [77]
(pellet and soluble fractions)
n-Dodecyl b-D-maltoside treat- Gel filtration and ion exchange, Improved separation of proteins [81]
ment 1DE, MALDI-MS
C/M extraction, NaOH treatment, 1DE, LC–MS/MS Differential extraction procedures [20]
NaCl treatment
– 1DE + LC–MS/MS, or MudPitt Complementary MS-based [24]
approaches
Na2CO3 treatment, C/M fractiona- 1DE and 2DE, LC–MS/MS Differential extraction procedures [23]
tion
KNO3/KBr treatment, Three phase 1DE, LC–MS/MS Differential extraction procedures [62]
partitioning (n-butanol, ammo-
nium sulfate

mixture (Table 2). Using different extraction procedures and
analytical techniques allow proteins with different protein
dynamic range and hydrophobicity to be identified. For
instance, since the phosphate/triose-phosphate transporter rep-
resents about 20% of the chloroplast envelope protein con-
tent, it is important to use different fractionation methods to
get access to more minor proteins (see Section 2). As quoted
below, complementary approaches can be used at different
levels (protein purification and extraction, protein separa-
tion, mass spectrometry analysis) to get the most exhaustive
or the most relevant repertoire of a membrane system (Fig. 1).
3.1.1. Using different extraction procedures from enriched
membrane fractions
Referring to the proteomic analysis of plastid envelope,
mitochondrial membranes and plasma membrane [8,20,48],
it was shown that different proteins could be identified when
using complementary extraction methods, i.e. C/M extrac-
tion, alkaline and saline treatments. Indeed for the chloro-
plast envelope and mitochondrial membranes sets of data, less
than 10% of proteins were identified from the three extrac-
tion procedures altogether, while more than 60% of proteins
were identified from only one extraction procedure (Fig. 3).
Similarly, for the plasma membrane samples, a weak overlap
was observed between these three extraction procedures, even
though protein identification from the NaCl fraction was per-
formed in a more exhaustive way (Fig. 3). A difference in
protein dynamic range and in stringency, with respect to pro-
tein hydrophobicity, might explain the complementarities of
the extraction methods, regardless of the subsequent analyti-
cal techniques used for protein identification. For instance
C/M extraction allowed some minor and hydrophobic pro-
teins to be concentrated and further identified, whereas these
proteins were not identified from salt treatment experiments.
Complementary and differential extraction procedures,
using organic solvents (C/M or n-butanol), were also per-
formed for the in-depth analysis of the thylakoid membrane
proteome, altogether with complementary electrophoretic
techniques (1-DE and 2-DE) [23,62]. Different salt treat-
ments and/or organic solvent extraction were also used for
the analysis of the tonoplast [77] and the mitochondrial mem-
branes [51].
Targeted extraction procedures have been described to
recover and identify specific proteins. From a plasma mem-
brane fraction of Arabidopsis, Elortza et al. [18] described
the specific recovery of GPI-anchored proteins using a treat-
ment with phosphatidylinositol phospholipase C and a two-
phase partitioning. Other plant plasma membrane proteins
were specifically retrieved using a cold Triton X-100 treat-
ment. Using such a procedure, which is commonly used to
study lipid rafts, the data obtained indicated that tobacco
microdomains are able to recruit specific membrane proteins
and exclude others [55]. Other strategies have been recently
described for the specific recovery of plasma membrane pro-
teins. For instance biotinylation of cells allows the labeling
of cell surface proteins, only, to be performed. A subsequent
affinity purification step, using streptavidin, allowed labeled
plasma membrane proteins to be collected and to be further
analyzed by mass spectrometry [92].
 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962
Fig. 3. Number of proteins identified from different extraction procedures
for chloroplast envelope [20], mitochondrial membranes [8] and plasma mem-
brane [48]. C/M: C/M extraction from the membrane; NaOH: NaOH treat-
ment of the membrane; NaCl: NaCl treatment of the membrane. Proteins
recovered from the NaCl plasma membrane treatment underwent a more
exhaustive analytical strategy, involving multiple LC–MS/MS analyses, com-
pared to the two other treatments (G. Ephritikhine, unpublished results). Figu-
res into brackets indicate the number of proteins.
3.1.2. Analytical techniques for the identification of plant
membrane proteins
Besides the ability to use different strategies to collect
membrane proteins, complementary analytical techniques
(separation techniques, mass spectrometry) can be used for a
better overview of membrane protein content. One-
dimensional SDS-PAGE is widely used for the separation of
membrane proteins and can be completed by 2-D electro-
phoresis for a better coverage of the less hydrophobic mem-
brane proteins [23,55,67]. Additional separation techniques
can be used in order to make the protein mixture less com-
plex. For instance Szponarski et al. [81] used both gel filtra-
tion and ion exchange to separate proteins from tonoplast
preparations. Also complementary mass spectrometry-based
procedures, such as MudPit and LC–MS/MS for SDS-PAGE
separated bands, were carried out for the analysis of the chlo-
roplast envelope [24]. Almost 400 non-redundant proteins
were thus identified, from which 38% and 27.8% were exclu-
sively identified from MudPit and LC–MS/MS analyses,
respectively [24].
In most cases, membrane proteins separated by SDS-
PAGE, are in-gel digested by trypsin and tryptic fragments
951
are analyzed by LC–MS/MS analyses (Table 2). Alternative
gel-free methods can be used and imply the in-solution diges-
tion of membrane proteins. Digestions with trypsin in 60%
methanol [6], proteinase K in high pH buffer, or CNBr in
90% formic acid [90] have been described in the case of
membrane-enriched protein fractions. As those fractions were
highly complex, MudPit analyses were performed to get as
much identification as possible [43]. In the context of the study
of plant membrane systems such an approach has not been
applied.
3.2. Physico-chemical properties of plant membrane
proteins according to the analytical method used
We described above (see Section 2) the different methods
for the recovery of plant membrane proteins: subcellular frac-
tionation combined with different extraction and/or treat-
ment procedures. Based on experiments performed on three
different plant membrane systems, involving the same meth-
odologies [8,20,48], we investigated the physico-chemical
properties of the identified proteins according to the extrac-
tion procedure used. Fig. 4 shows the ratio Res/TMD (Res
= number of amino acids and TMD = predicted transmem-
brane domains) according to the protein molecular weight.
The lower Res/TMD is, the more hydrophobic a protein can
be considered (e.g. Res/TMD < 200). Besides, lines can be
drawn on these diagrams that correlate proteins with the same
number of TMDs. Organic solvent extraction appears to select
for low Mr and high hydrophobicity with a poor contamina-
tion with hydrophilic proteins for the chloroplast envelope
and the mitochondrial membranes. The situation is less clear-
cut for the plasma membrane, from which C/M extraction
allowed a relative higher percentage of hydrophilic proteins
to be identified, compared to the two other membrane sys-
tems. The ability of C/M extraction to recover hydrophobic
proteins was also demonstrated in the case of the thylakoid
membrane proteome [23]. On the contrary, saline (NaCl) treat-
ment appears to be less efficient to identify highly hydropho-
bic proteins (Fig. 4). Most of the proteins identified from
saline treatment are predicted to contain no or only one trans-
membrane domain. This difference in predicted hydropho-
bicity explains the low value of cross identification using C/M
extraction and NaCl treatment (Fig. 3). On the other hand,
alkaline (NaOH) treatment appears to be a good compro-
mise, especially for mitochondria and chloroplasts, to retrieve
a wide range of proteins, regarding their physico-chemical
properties. Indeed, this procedure clearly selects for more
intrinsic proteins when compared to saline treatment since
less hydrophilic contaminants are recovered. However, this
selection is less stringent than C/M extraction since proteins
are identified in a wide range of size and hydrophobicity.
Indeed, whatever the extraction procedure used, the distribu-
tion of the physico-chemical properties displayed by plasma
membrane proteins is spread over wider res/TM and Mr
ranges (Fig. 4).
When referring to the protein pI, it appears that the C/M
extraction seems to be rather selective for basic proteins. This
952 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962

Fig. 4. Molecular weight and Res/TMD ratios of some identified plant membrane proteins. Res/TMD: Res = number of the protein amino acids; TMD = number
of a-helical transmembrane domains predicted by HMMTOP. The grayed rectangle at the bottom left of each diagram indicates proteins with a Res/TMD < 200
(hydrophobic proteins) and a molecular mass < 40 kDa. The grayed rectangle, at the top of each diagram (Res/TM > 800), points out proteins with 0 TMD. On
NaCl diagrams two lines indicate proteins with one or two TMDs.

bias could however be due to the nature of the extracted pro-
teins. Indeed, whatever the extraction procedure used, most
of the proteins identified from the plastid envelope, and espe-
cially proteins localized in the inner membrane, were shown
to be basic [21,80] (see Section 3.3.).
3.3. In silico approaches: is it possible to identify plant
membrane proteins via an “in silico” approach?
Plant membrane proteins show general and specific fea-
tures with respect to their function, their localization, their
sequence and their physico-chemical properties. Many plant
membrane proteins are characterized by specific structural
features, like transmembrane a-helices or b-barrel, because
they are actually embedded in a membrane. Although b-barrel
prediction is a difficult task, the calculation of TMDs can be
achieved by different experienced tools (e.g.: HMMTOP,
TMHMM) with reasonable level of confidence. Such predic-
tion tools allowed plant membrane protein databases to be
set up, on the basis of A. thaliana and Oryza sativa protein
databases: http://aramemnon.botanik.uni-koeln.de/ [74] and
http://www.cbs.umn.edu/arabidopsis/.
In addition to TMDs or b-barrel structures, some plant
membrane proteins also contain a transit peptide which allows
them to be targeted to a specific membrane system such as
chloroplast or mitochondrial membranes. Other features such
as the pI, the protein molecular weight, the cystein content
can be specific of a membrane protein and its localization in
a plant cell [21,39,71,80]. Such specific features can be pre-
dicted or calculated by bioinformatic tools to mine protein
databases. For instance, in a previous study, we showed that a
combination of (a) the prediction of a chloroplastic transit
peptide, (b) a Res/TMD < 100 (Res = number of amino acids),
(c) TMD ≥ 4 and (iv) pI > 8.8 is characteristic of chloroplast
inner envelope transporters. Using these parameters, the Ara-
bidopsis protein database was mined and 136 proteins were
retrieved, that showed the features quoted above. Amongst
these proteins only four are known to belong to another sub-
cellular compartment (see [21] and Fig. 5) and 24 were iden-
tified in proteomic studies performed on Arabidopsis or spin-
ach envelope fractions [20,21]. Schleiff et al. [71] also
described the use of bioinformatic tools in order to predict
chloroplast outer membrane proteins. b-Barrel proteins were
selected using two different bioinformatic tools for which
parameters had to be optimized. b-Barrel proteins having a
pI > 7 and not predicted to be located in the chloroplast (most
of the chloroplast outer membrane proteins lack a chloro-
plast targeting signal) were further selected as candidates for
being chloroplast outer membrane proteins. After manual
inspection, 891 proteins were eventually considered as poten-
tial chloroplast outer membrane proteins. Predictions for both
the inner and the outer membranes of the chloroplast enve-
lope were based on the knowledge of these membrane sys-
tems and the proteomic analyses which feed and/or confirm
 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962
Fig. 5. Classification of chloroplast envelope retrieved by an in silico approach
[21]. Putative chloroplast innermembrane envelope transporters were retrie-
ved from the Arabidopsis protein database using a combination of bioinfor-
matic tools to predict (a) a plastid transit peptide, (b) a Res/TMD < 100 (Res
= number of amino acids), (c) TMD ≥ 4 and (d) pI > 8.8. Functions were
assigned according to protein database annotations and literature. Proteins
involved in lipid and pigment metabolism and proteins with other functions
are known to be located in the plastid envelope. Proteins that are similar to
Synechocystis counterparts have functions but, due to their similarity with
Synechocystis gene products, are very likely to be located in the plastid.
Transporters are known to be located in the plastid or have an unknown
localization.
in silico approaches. Sun et al. [80] described a detailed inves-
tigation of distinctive proteome properties in order to set up
rules for the prediction of different plastid subproteomes.
These subproteomes include the integral inner envelope and
the thylakoid proteome. Based on curated plastid subpro-
teomes, collected from published experimental data sets, Sun
et al. [80] underlined specific features for proteins of the chlo-
roplast inner envelope and the thylakoid membrane (Table 3):
protein size, Gravy index, pI, cystein content and number of
TMDs seem significantly different between these two mem-
brane systems [80].
Specific features of non-plastids plant membrane systems
were not investigated yet. In order to get an overview of some
parameters important for plant membrane proteins (e.g.
TMDs, subcellular location prediction, pI), as shown above,
we retrieved, from the SwissProt-Trembl protein database,
Arabidopsis and/or other plant (Viridiplantae) proteins known
to be located in various plant membrane systems and inves-
tigated the features of these proteins. Although such a set of
proteins may certainly be biased, due to the way to retrieve
Table 3
Specific features of membrane proteins from the chloroplast envelope and
the thylakoids (adapted from Sun et al. [80]). The protein size is given via
the number of amino acids (aa). For protein size, Gravy, pI and cystein content
unprocessed proteins were considered and the median values are given. (*)
For TMDs the values were assessed from a frequency distribution curve and
calculated from the sequence portion from which a predicted transit peptide
was withdrawn
Envelope Thylakoid
Protein size ~450 aa ~250 aa
Gravy ~0.1 ~–0.02
pI ~9 ~7
Cystein content 0.011 0.005
TMDs* 1–4 or >9 0–3
953
these proteins (Fig. 6), specific features can be confirmed or
pointed out for some membrane systems, as shown for the
inner and outer membranes of the chloroplast envelope. Fig. 6
summarizes the pI values and the number of putative TMDs
for different plant membrane proteins. Most of proteins from
outer membrane systems (chloroplast and mitochondria) show
less than two a-helical domains. This is consistent with the
fact that outer membrane proteins are more likely to contain
b-barrel structures [71]. Thylakoid membrane proteins that
are highly hydrophobic (TMDs ≥ 4) are mainly acidic (pI
< 7) whereas highly hydrophobic inner membrane chloro-
plast proteins are mainly basic (pI > 8). The later set of pro-
teins gathers a lot of transporters of the chloroplast envelope
[21]. Similarly about 70% of highly hydrophobic inner mem-
brane mitochondrial proteins are basic (pI > 8). These out-
lined different properties of thylakoid and chloroplast inner
envelope membrane proteins are in good agreement with the
observations of Sun et al. [80] (Table 3). For plasma mem-
brane, proteins containing more than six TMDs are mainly
acidic whereas a set of proteins containing six TMDs are basic.
These basic proteins with six TMDs are mainly aquaporins
whereas the acidic proteins containing more than six TMDs
are mainly ATPases. Similarly to the chloroplast inner enve-
lope proteins that are basic and highly hydrophobic (TMDs ≥
4), highly hydrophobic plasma membrane proteins are mainly
involved in transport functions (pI > 8) (aquaporins and other
transporters). Table 4 summarizes some of the general fea-
tures that could be assigned to plant membrane proteins
according to their subcellular localization. Some specific fea-
tures assigned to mitochondrial or chloroplast envelope mem-
brane proteins can be predicted by bioinformatic tools to allow
in silico targeted databases to be generated. Nevertheless the
features quoted in Table 4 are just indicative. Indeed, for
instance, Sun et al. [80] pointed out that the in silico analysis
of physico-chemical properties should be done after removal
of cleavage transit peptide, for thylakoid and chloroplast enve-
lope proteins. Other parameters, like the cystein content and
the protein molecular weight, were shown to be worth being
taken into account for chloroplastic membrane systems [80].
Table 4 indicates also the possibility to predict the subcel-
lular localization for chloroplastic and mitochondrial mem-
brane proteins. The issue of the in silico subcellular localiza-
tion has been addressed in the case of plant proteins, and
bioinformatic tools have been tested for their reliability. For
instance many prediction programs (TargetP, Predotar, Mito-
Prot II, SubLoc and iPSORT) were tested for their ability to
predict mitochondrial localization [32]. It was concluded that
those programs predicted mitochondrial targeting rather
loosely and that caution must be taken with respect to the
so-generated in silico data. Predictions must remain indica-
tive and not informative, as can be experimental data. Plas-
tidial and mitochondrial localization can be predicted based
on the occurrence of a specific transit peptide. No specific
prediction programs exist for the plasma membrane or the
tonoplast. Most outer mitochondrial and plastidial mem-
brane proteins do not contain any transit peptides. Most inner
954 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962

Fig. 6. Number of TMDs (y axis) and pI (x axis) of some plant membrane proteins collected from the SwissProt-Trembl database. Proteins were retrieved from
the SwissProt-Trembl database (version of April 2004) using the searching tool SRS (http://au.expasy.org/srs5bin/cgi-bin/wgetz). The following key words
were entered in order to retrieve the proteins: Viridiplantae, chloroplast, envelope, inner membrane, outer membrane for the chloroplast envelope; Arabidopsis,
mitochondria, membrane for the mitochondria membranes; Arabidopsis, thylakoid and membrane for the thylakoid membrane; Arabidopsis and plasma mem-
brane for the plasma membrane; Arabidopsis, vacuol*, membrane and tonoplast for the tonoplast (different combination of key words were used). SwissProt-
Trembl annotations were checked for consistency with the subcellular localization and redundant proteins were removed, especially for searches that used
Viridiplantae as a key word. Molecular weight and pI were calculated according to http://ca.expasy.org/tools/pi_tool.html and TMDs were calculated using
HMMTOP (http://www.enzim.hu/hmmtop/).

mitochondrial and plastidial membrane proteins contain a
transit peptide. Nevertheless, some of these inner membrane
proteins do not possess any transit peptide (see below and
[54]). A better knowledge of the protein import systems to
and via cell membranes would help in refining prediction tools
for subcellular localization. Also proteomic analyses could
help in feeding predictive tools with learning sets retrieved
from experimental data. For instance, in the context of sub-
cellular localization assessment, proteomic data provide infor-
mation like the sequence of peptides which contain the pro-
cessed N-terminus of some proteins. These N-terminal
peptides may indicate if a transit peptide exists and if so, where
is precisely located the cleavage site as shown for the chlo-
roplast envelope [20]. Ferro et al. [20] showed the identifica-
tion of N-acetylated peptides and/or non-tryptic peptides that
were located close to protein N-termini indicated the loca-
tion of a probable cleavage site. The investigation of the
N-termini of mature proteins necessitates a careful examina-
tion of proteomics data [20] or a targeted approach towards
processed protein N-termini [28]. To do so, Gevaert et al. [28]
used specific chemical derivatization of protein N-termini with
subsequent tandem mass spectrometry analysis to isolate and
get sequence information for N-terminal peptides.
On one hand, proteomics data can feed bioinformatics for
a better definition of prediction tools. On the other hand,
although not 100% reliable, prediction tools can give valu-
able information for biological investigations. For instance,
two phosphate transporters, (Pht2;1 and a putative Pi trans-
porter P56-4, were found via a targeted proteomic approach
to be located in the chloroplast envelope whereas P56-4 was
not predicted to be located in plastids by the prediction tool
ChloroP. Besides an additional putative phosphate trans-
porter (P56-2), which was not identified by proteomics, was
found to be located in plastids via an in silico approach. Using
GFP fusion proteins, those three proteins were found to be
actually located in chloroplasts (see Section 4) suggesting
that they may all participate in phosphate transport across the
chloroplast envelope [20].
4. Proteomics for new insights over plant membrane
systems
The major interest of sub-proteomics approaches dealing
with membrane systems is that they bring clues on both the
membrane compartment where proteins are working and their
putative cellular functions.
4.1. Subcellular location of membrane proteins
Membrane proteomics is a way to check or to assign the
subcellular compartmentation of a protein. Through the sur-
 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962 955

Table 4
Main specific features associated to plant membrane proteins deriving from various subcellular compartments. The arbitrary classification of different plant
membrane proteins, according to their localization and function, was achieved according to previously published observations [8,20,21,48,71,80] and by taking
into account data presented in Fig. 6
Subcellular Membrane system Sub-compartment Function pI > pI TM > TM ≥ TM cTP mTP b-Barrel Main known
compartment 8 <8 6 4 <4 function
Plasma Plasma membrane + – – + – – – +/– Aquaporins +
membrane (1) transporters
Plasma membrane – + + + – – – – ATPases + others
(2)
Chloroplast Chloroplast enve- Inner membrane Transporter + – +/– + – + – – Transporters
lope
Other function +/– +/– – – + + – +/– Lipid and vitamin
metabolism
Outer membrane + +/– – – + – – + Porins (pI > 7
[71])
Thylakoid mem- – + – – + + – +/– Some photosys-
brane (1) tems + other func-
tions
Thylakoid mem- – + +/– + + + – +/– Photosystems,
brane (2) cytochromes, ATP
synthases
Mitochondria Mitochondrial Inner membrane Transporter + – +/– + – – +/– – Transporters
membranes
Other function +/– + – – + – + +/– TIM proteins +
other functions
Outer membrane + +/– – – + – – + Porins
Vacuole Tonoplast +/– + – + + – – +/– Aquaporins,
ATPases, ATP
synthases

vey of hydrophobic proteomes dealing with different mem-
brane systems, numerous membrane proteins found a defini-
tive or at least a putative location. This is shown in Fig. 2 for
newly identified proteins from membranes of chloroplasts
[20], mitochondria [8] and from plasma membrane [48] and
in Table 2 for proteins putatively associated to thylakoids and
tonoplast. Nevertheless, in all these cases, compartment
assignments for previously unidentified proteins rely on the
purity of the membrane fraction they originated from and
therefore remain indicative. Indeed, the quality of the puri-
fied membrane fractions can be assessed by biochemical tests
and further confirmed by the MS-based identification of pro-
teins either well known as resident in a membrane compart-
ment, or conversely, as proteins associated to other compart-
ments. For example, the plasma membrane localization of
newly identified plasma membrane proteins was strength-
ened because of the presence of H+-ATPase and PIPs in the
plasma membrane fraction and also the lack of major pro-
teins from other organelles like RuBisCO from chloroplast
membranes or cytochrome-c reductase from mitochondrial
membranes [48]. Other proteins already described as associ-
ated to a given membrane system but in other plants, are newly
identified and localized in the Arabidopsis model, thus con-
firming their localization in that membrane compartment. This
is the case for several proteins previously identified in tobacco
chloroplast envelope like the proteins TPT, HP36 and OEP21
[20] or in plasma membrane fractions isolated from other
diverse species like tomato (LePT1) or tobacco (NtSYR1)
[48].
The discovery of proteins in a membrane sub-proteome,
when they were expected in other membrane system on the
basis of their putative biochemical or physiological func-
tions, is more surprising and lead to develop complementary
studies to further validate the new subcellular localization.
For instance, Ferro et al. [21] found a protein, namely the
IEP60 H+/Pi transporter, homologous to the Arabidopsis
Pht2;1 Pi transporter (At3g26570) in proteomic analyses of
spinach chloroplast envelope. The Pht2;1 Pi transporter was
previously suggested to be localized in the plasma mem-
brane and involved in the uptake and intercellular movement
of Pi in Arabidopsis shoots [16]. In support to proteomic
analyses, experiments based on transient expression of
Pht2;1::GFP fusions in Arabidopsis leaves and western blot
analyses demonstrated unambiguously the localization of this
protein in the inner membrane of chloroplast envelope, exclu-
sively [21,84]. Likewise, the identification of proteins from
the voltage dependent anion channel (VDAC) family in the
plasma membrane proteome was unexpected [48]. The plasma
membrane targeting of AtVDAC1 (At5g15090) was further
confirmed by transiently co-expressing in onion epidermal
cells the translational fusions AtVDAC1::DsRed and
Nramp3::GFP, a tonoplast marker (Fig. 7A–C) [48]. Stron-
ger evidence was provided by stably expressing
AtVDAC1::GFP fusions in the Arabidopsis mutant vdac1-1
background (Fig. 7D–F). In any case, the ultimate proof for
assigning a membrane compartment to a candidate protein
will arise from subcellular localization studies especially
devoted to that question. Up to now, no systematic subcellu-
956 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962

Fig. 7. Plasma membrane targeting of the AtVDAC1 and AtPT2 proteins. The channel protein AtVDAC1 (At5g15090) and the phosphate transporter AtPT2
(At2g38940) were identified in the hydrophobic plasma membrane proteome [48]. A, B and C: AtVDAC1-DsRed and NRamp-GFP fusion proteins were
transiently co-expressed into onion epidermal cells. A and B, the fluorescence signal associated with the expression of AtVDAC1-DsRed (A) with NRamp-GFP,
a tonoplast marker (B). C, overlay of the two pictures. D, E and F: AtVDAC1-GFP fusion proteins were stably expressed in the vdac1-1 mutant plant (Mar-
magne and Ephritikhine, unpublished data). D, fluorescence signal associated to root cells. E and F close view of root cells in transmission (E) and in fluores-
cence (F). G, H and I: AtPT2-GFP fusion proteins were transiently expressed into Arabidopsis protoplasts (Marmagne and Ephritikhine, unpublished data). G,
the transmission image of the protoplast; H and I, two magnifications of the fluorescence signal associated with expression of AtPT2- GFP fusions. Pictures
kindly provided by A. Marmagne. Their localization was visualized by confocal microscopy, 24 h post-transformation in transient expression experiments and
on T1 progeny of the vdac1-1 transformed plants.

lar localization study performed from a unique plant mem-
brane proteome was published. Marmagne et al. [48] reported
on plasma membrane proteome and brought direct evidence
for the plasma membrane assignment of several new pro-
teins, belonging to different physico-chemical and func-
tional classes. An additional example is shown in Fig. 7 (G–I),
with the plasma membrane targeting of a new gene product,
AtPT2 (At2g38940), a phosphate transporter identified in the
plasma membrane proteome. Beside MS-based proteomic
approaches, global localization experiments provide an alter-
native approach for subcellular localization determination.
The high-throughput analysis of around 60% of the epitope-
tagged proteome of yeast is a good illustrative example [42].
To a lesser extent, about 100 human proteins were analyzed
after expressing GFP fusion plasmids in mammalian cells
[79]. In plants, with regards to the rapid and increasing
progress in methodologies (epitope-tagging, high-throughput
cloning) and in functional genomics, there is no doubt that
extensive subcellular localization studies would emerge soon.
Recently, Escobar et al. [19] developed an innovative strat-
egy based on a high-throughput viral expression system in
Nicotiana benthamiana of cDNAs fused to the 5′ or 3′ end of
the gene for green fluorescent protein (GFP). Several hun-
dreds of cDNA-gfp fusions were screened daily. More than
half of the members of the library carrying cDNA fusions
that expressed fluorescence displayed discrete, non-cytosolic,
subcellular localizations. But still, the characteristics of the
membrane proteins will raise specific difficulties and will be
 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962
limiting. Mis-targeting or protein aggregation in the cytosol
or Golgi vesicles, in particular for plasma membrane pro-
teins, can lead to inconclusive results about their subcellular
localization (Ephritikhine, unpublished results). In addition,
to localize with accuracy proteins associated to sub-proteomes
like inner and outer membranes of the chloroplast envelope
or inner and outer mitochondrial membranes will require per-
forming western blot analyses on sub-fractions in addition to
confocal microscopy.
An increasing number of studies demonstrate that some
proteins are targeted simultaneously to more than one com-
partment [78]. The dual localization of soluble proteins tar-
geted to both mitochondria and plastids is supported by
increasing data based on in silico sequence analyses com-
bined with the molecular characterization of the targeting sig-
nals [15]. This is not the case for proteins associated to the
different cell membrane systems, mostly because clear tar-
geting signatures are not yet identified. Comparison between
different membrane proteomes may reveal new features on
the subcellular localization of proteins, already described as
associated to a given membrane compartment. Analysis of
the hydrophobic proteomes of plasma membrane [48] and
mitochondrial membranes [8] showed that the protein AtV-
DAC1 is present in the two proteomes. Likely related to these
results, VDAC proteins of Lotus japonicus seemed to local-
ize not only to mitochondria but also to undetermined vesicles
in root nodules [87]. Such a double localization of a VDAC
channel is not totally surprising as it is now well accepted,
for animal models, that the same protein VDAC can be tar-
geted to both plasma membrane and mitochondria, depend-
ing on an alternative splicing in the 5′ UTR region [9].
The identification of one or two target membrane compart-
ment(s) for new or already characterized proteins, raise the
crucial question of the pathways and mechanisms involved
in their final localization(s). For example, still considering
AtVDAC1 and the other members of the AtVDAC family, a
double question is now addressed. First the mechanisms con-
trolling the subcellular dual-targeting of the protein AtV-
DAC1 but also, the processes involved in the targeting of the
other VDAC channels likely to the outer mitochondrial mem-
brane. But the interest in searching for the mechanisms under-
lying the protein targeting to a membrane compartment is not
restricted to proteins displaying multiple localizations. The
protein ceQORH (At4g13010) was first identified in Arabi-
dopsis on the basis of homologies with peptides encoding a
quinone oxidoreductase identified from the spinach chloro-
plast envelope proteome [75]. The protein ceQORH likely
belongs to a new type of electron-transfer chain [36] and is
localized in the inner membrane of the chloroplast envelope,
though it does not possess any cleavable targeting sequence
[54]. This contrasts with other proteins from this membrane
compartment, suggesting the existence of an original plastid
import mechanism. Such examples illustrate unexpected ques-
tions generated by increasing proteome analyses and show
that proteomic approaches are the first step towards the
molecular and functional characterization of proteins.
957
4.2. Functions
In order to assign putative functions to proteins several
bioinformatics tools are available. Database searches can be
performed using generic databases (NCBI, http://www.ncbi.
nlm.nih.gov/ or SwissProt + TREMBL, ftp://us.expasy.org/
databases/) and A. thaliana databases (NCBI A. thaliana
specified or TAIR, ftp://ftp.arabidopsis.org/home/tair/). Func-
tional classification relies on the use of in silico tools allow-
ing sequence alignments, like BLAST for example
(http://www.ncbi.nlm.nih.gov/blast) and the search for con-
served functional domains, CDD for instance (NCBI con-
served domain database (CDD, http://www.ncbi.nlm.nih.gov/
Structure/cdd/cdd.shtml). CDD currently contains domains
derived from two commonly used collections, Smart and
Pfam, plus contributions from colleagues at NCBI, such as
COG. A recent bioinformatic tool, GO (Gene Ontology™
Consortium, http://www.genontology.org), was developed to
produce a controlled vocabulary that can be applied to all
organisms, including plant species, A. thaliana and O. sativa.
GO is based on three organizing principles, molecular func-
tion, biological process and cellular component.
Facing the avalanche of proteomic data, the major goal in
the very near future is to identify the functions of the all sets
of identified proteins, the majority of which being previously
proteins [4]. For instance, amongst the hundred of proteins
identified in the plasma membrane hydrophobic proteome
[48], 95% had never been found in previous proteomic stud-
ies. Only 23% were present in protein databases and all oth-
ers were previously described only at the levels of either
cDNA (67%) or even from genomic sequence (10%). Using
programs for homology search and functional domain pre-
dictions, Marmagne et al. [48] determined that 49% were
known proteins, or paralogues of known proteins, whereas
32% were putative proteins exhibiting low homology with
known proteins and 19% were proteins. These data from
plasma membrane proteome [48] speak for themselves; simi-
lar results could be stated for other membrane proteomes,
pointing out, once more, the proteomic potentialities to iden-
tify new proteins.
In silico analyses of gene and protein structures using all
the diversity of the bioinformatic tools may bring some clues
for identifying the function of a membrane protein and will
lead to a first functional classification. Somehow, the target
membrane worked on is a good criterion for a critical evalu-
ation of the putative function identified in silico. In the best
cases, additional data, like the experimental confirmation of
the membrane compartment where the protein is localized,
will strengthen the identity of the putative function of the pro-
tein. A survey of the different membrane proteomes reported
in the literature (Table 2) shows that the putative biological
roles assigned to most of the proteins are in good agreement
with known or expected cellular functions of the membrane
compartment they were collected from. For instance, Fig. 8
illustrates the different functional classes deduced from the
combination of in silico analyses and data from the literature
958 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962
Fig. 8. Functional classification of plastid envelope, mitochondrial membra-
nes and plasma membrane proteomes. In silico analyses were performed on
proteomics data from plastid envelope [20], mitochondria [8] and plasma
membrane [48]. Predictions for membrane-spanning regions (TM) were
achieved by using the ARAMEMNON database (http://aramemnon.botanik.
uni-koeln.de/); white bars, 0 TM, spotted bars, 1–3 TM and black bars,
4–14 TM. The presence of conserved domain in all proteins was searched on
the NCBI CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).
for proteomes isolated from chloroplast envelope [20], mito-
chondrial membranes [8] and plasma membrane [48]. Differ-
ent functional categories can be distinguished, some being
common to the three compartments. Numerous transport pro-
teins were identified, most of them being specific of the mem-
brane compartment they are originated from. Ion and metabo-
lite transporters as well as subunits of protein import
complexes were identified in both plastid and mitochondrial
membranes. In addition to such transport systems, channels
and H+-ATPase were also found in the plasma membrane pro-
teome. Two sets of proteins are involved in metabolic path-
ways specific to plastids and to mitochondria (Fig. 8). In the
plastid envelope proteome, proteins participating to lipid, car-
bon, vitamin and pigment metabolisms are abundant. In the
mitochondrial membrane proteome, this protein category con-
cerns mainly proteins from the respiratory chain and ATP syn-
thases. Interestingly, chaperones were identified in both com-
partments. A large class, specific to the plasma membrane
proteome, was identified, gathering proteins involved in sig-
naling pathways (stress induced proteins, receptors and GTP-
binding proteins) and in cellular traffic, thus reflecting the
roles of plasma membrane in cell surface and cell exchanges.
Besides these wide protein classes, several other putative cat-
egories can be distinguished, again representative of expected
functions. The proteins pooled in the class “other functions”
are described in the databases by more diverse functions,
mainly associated to metabolism. Proteins whose activity has
been characterized as stress regulated enzymes are more spe-
cifically identified in the plasma membrane proteome. In each
of these proteomes, the high number of proteins displaying
hydrophobic properties is remarkable and shows once more
the contribution of membrane proteomics to enrich protein
repertoires issued from more general proteome approaches
(Fig. 8). Similar percentages (around 30%) of proteins func-
tionally uncharacterized were reported for tonoplast pro-
teome [77,81] and very recently for thylakoid membrane pro-
teins [62]. Finally, these proteins of function are of great
interest as they bring clues for further understanding of the
different membrane systems.
First of all, protein inventories bring confirmation for func-
tions already associated to a given membrane, but more inter-
estingly, they allow identifying the molecular components
involved in these functions. Conversely, they lead to the iden-
tification of unexpected proteins in a membrane system which
offers the double advantage to assign new putative functions
to a membrane compartment and to know, at the same time,
the identity of the molecular entity involved in it. For example,
proteomics help understanding metabolism at the molecular
level and provide a new overview of the biochemical machin-
ery of plastid and mitochondria surrounding membranes, as
illustrated by the identification of several putative phosphate
transporters in the chloroplast envelope proteome [20]. In
other respects, the existence of multigenic families address
the question of the redundancy between the different mem-
bers; crosschecking proteome data may bring part of the
answer, if members are present in distinct compartments. The
small AtVDAC family in Arabidopsis represents a more com-
plex situation. The AtVDAC1 protein, identified as a major
component in both plasma membrane [48] and mitochon-
drial membranes [8], appears as an interesting model to ana-
lyze the mechanisms underlying the multitopological local-
ization of porins and to explore the functions of the channel
proteins in each compartment.
Proteomics data sets are a source for developing new
molecular tools at the different levels, cDNA, mRNA and pro-
 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962
tein, for further functional studies aiming at the molecular
and functional characterization of the protein. At that step, it
should be mentioned again that membrane proteins represent
a special category as they display physico-chemical proper-
ties which often hamper the progress of studies, whatever in
vitro protein handling or in vivo functional approaches. Nev-
ertheless, expression of plant membrane proteins in heterolo-
gous systems (Xenopus oocytes, mammal and insect cells)
has been successfully used for revealing and characterizing
the transport activity of ion and solute transporters. With
regard to these systems, Xenopus oocytes are the more com-
monly used (for a review, see [53]). Functional complemen-
tation of yeast mutants is often helpful to identify or charac-
terize the activity of a protein; numerous papers in the
literature make a report on that strategy, which has been
applied with success on all different kinds of membrane pro-
teins, like ammonium transporters [27], sucrose transporters
[1], metal transporters (for review, see [82]) or other anion
channels, ChLoride Channels (CLC) channels [33] and VDAC
proteins [87] for instance. Recently, Mercier et al. [49] under-
lined the interest of that strategy for functional assays on cyclic
nucleotide gated channel (CNGC), as an alternative when
other heterologous expression systems failed. Progress in
understanding K+-channel functions of the so-called Shaker
family are a good illustration of the complementary contri-
butions of these different approaches. They cover all the steps,
to be short from the functional cloning in yeast [76] to the
characterization of the channel activity in oocytes [13,85],
and more recently, to the isolation of an AKT2 partner by
screening a two-hybrid cDNA library in yeast and the func-
tional characterization of their interaction by co-expressing
both in oocytes and COS cells [14]. Last, reconstitution in a
planar lipid bilayer may, in some cases, overcome the recal-
citrance of plant membrane protein expression in heterolo-
gous systems. A recent paper described the properties of a
chloride anion channel from the chloroplast envelope [86].
Success in all these strategies and/or the physiological inter-
pretation of the results may be limited for several reasons,
the more obvious being possible incorrect maturation, mis-
targeting of the protein and lack of partners essential for the
protein activity.
Therefore, beside protein sequence analyses and cellular
functional characterization of membrane proteins identified
in a proteome, genetic approaches should be developed to get
access to their integrated function in planta. Gene function
can be revealed through the analysis of mutants by either for-
ward or reverse genetics. A highly efficient procedure for iso-
lating mutants in identified genes is insertional mutagenesis.
Several collections of plants or DNA pools and databases of
flanking sequence tags can be screened for the identification
of lines harboring insertions within a gene of interest. Most
collections and databases were developed in A. thaliana; one
can refer to the updated list of Arabidopsis initiatives, avail-
able insertion collections and stock centers published very
recently by Ostergaard and Yanofsky [60]. Since a few years,
genomic and genetic resources in other plants, in particular
959
rice, maize and Medicago truncatula, are considerably grow-
ing. Reverse genetics leads straightforwardly to the isolation
of one or several mutants in the gene of interest, but most of
the time, contrarily to forward genetics, no clear phenotype
characterizes the mutant. Partial or total redundancy, com-
pensation processes or the inability to reveal discrete physi-
ological changes in standard culture conditions may account
for this difficulty. Therefore, generation of revertants from
those mutated lines and production of overexpressing trans-
genic plants provide new tools for a better understanding of
the biological function.
All the approaches cited above apply to one or several can-
didate proteins. Situation is far more complex when address-
ing the question of the function of proteins belonging to a
multigenic family and even more when considering the whole
set of proteins defining a proteome. Recently, high-throughput
reverse genetics systems were developed in Arabidopsis gen-
erating a huge number of genetic tools which facilitate
genomic functional studies. In this post-genomic era, the
bottleneck is shifted to the next step, the identification of the
biological role of every component of the Arabidopsis pro-
teome.
5. Conclusion
We are far from having a complete picture of plant pro-
teomes and proteomics is expected to continue providing new
data in the near future. Furthermore, the extension of pro-
teomic studies is linked to the large diversity between organs,
between close organelles belonging to different cell types (leu-
coplasts and chloroplasts for instance) or displaying differ-
ent functions. Due to the wide variety of physiological situ-
ations, sub-proteomes describing subcellular compartments
should be completed. Situation is even worst when consider-
ing plant membrane proteomes. Numerous membrane-
associated plant proteins are still to be discovered; moreover,
when identified, they are the most poorly characterized. For
further progressing, more powerful bioinformatics tools able
to analyze large protein repertoires have to be developed, espe-
cially devoted to the systematic analysis of membrane pro-
tein structures (i.e. predictions of hydrophobic clusters,
a-helices and b-barrel). In other respects, the functional char-
acterization of the proteomes still lacks in more accurate and
reliable programs for functional annotation, especially in the
case of membrane proteins. Progress should come from com-
bination of proteomic strategies applied to membrane sub-
proteomes. Taking into account the dynamic of the mem-
brane proteomes arose from the diversity between organs,
organelles, the functional specificity of membrane systems,
the post-translational modifications in response to environ-
ment should bring first clues for the function of the protein.
These proteome inventories combined to quantitative pro-
teomics data and transcriptomics studies should provide a
more complete integrated view of cellular processes. The
increasing interest for transcriptome studies dealing with the
960 G. Ephritikhine et al. / Plant Physiology and Biochemistry 42 (2004) 943–962
comparison between different physiological conditions or
wild-type and mutant genotypes is a good indicator. Promis-
ing results are expected from such experiments performed on
knock-out mutants generated by RNA interference, a high-
throughput technology to produce potentially multiple
mutants when it concerns multigene families. Indeed, progress
in proteomics are closely related to the ones in transcriptom-
ics and both are completely dependent on the capacity of the
bioinformatics tools to handle the huge amounts of data gen-
erated by thee approaches.
Acknowledgements
We would like to thank Michel Jaquinod for critical read-
ing of the manuscript. Support by the Genoplante programs
No. 19993663 and No. 2001027 is acknowledged.
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