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Update on Plant Cell Wall Proteome
Straying off the Highway: Trafficking of Secreted Plant
Proteins and Complexity in the Plant Cell Wall Proteome1
Jocelyn K.C. Rose* and Sang-Jik Lee
Department of Plant Biology, Cornell University, Ithaca, New York 14853
From simply skimming through the abstract lists of
more or less any collection of both basic and applied
plant-related journals, it is immediately apparent that
the plant cell wall represents a nexus of many fields of
research: growth and development, plant-pathogen
interactions, abiotic stress, self- and interorganismal
recognition, signaling systems, numerous primary and
specialized metabolic processes, biomaterials and bioproducts, and many others. That said, and to deal with
an issue of semantics, while the term cell wall can refer
specifically to the structural matrix that surrounds all
plant cells, for the purposes of this Update it is used
more broadly, also to include the apoplast, or extracellular environment. Given its multifunctional nature
then, it is not surprising that the apoplast houses a
dynamic and complex proteome, and the compendium of cell wall proteins continues to grow as researchers from disparate disciplines discover new
roles for extracellular proteins. In addition, however,
as cell wall proteomics projects develop and the subcellular localizations of an ever-growing list of plant
proteins are determined, a number of surprises have
been thrown up, both in terms of the identity of
secreted proteins and the trafficking pathways that
they follow. The purpose of this Update is to give some
examples of previously unsuspected aspects of plant
cell wall protein trafficking that are challenging longheld assumptions, rather than to provide an exhaustive review, and to highlight some questions that can
be categorized into the “who, how, where, and when”
of the cell wall proteome.
WHO BELONGS TO THE CELL WALL PROTEOME?
Developing a comprehensive catalog of the cell wall
proteome is generally far more challenging than for
most intracellular organelles, which can be isolated in
highly purified fractions, relatively free from nonspecific protein contamination. Cell wall proteins are not
1
This work was supported by the National Science Foundation
Plant Genome Research Program (grant no. DBI–0606595 to J.K.C.R.)
and the New York State Office of Science, Technology and Academic
Research.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Jocelyn K.C. Rose ([email protected]).
www.plantphysiol.org/cgi/doi/10.1104/pp.110.154872
only spread throughout the apoplastic milieu, but also
show a wide range of affinities for the extracellular
matrix itself, from highly mobile with no apparent
interaction, to covalently bound. In addition, when cell
walls extracts are prepared, by tissue or cell homogenization followed by centrifugation, substantial
amounts of intracellular protein inevitably associate
with the wall pellet, while proteins and peptides that
were not bound to the wall in vivo are lost from the
extract. There are certainly other technical challenges,
such as the fact that most secreted proteins are
glycosylated, which complicates separation and identification, but the major confounding factors are still
contamination and incomplete capture.
Analyses of protein populations from the walls of
many plant organs and tissues have been reported (for
review, see Lee et al., 2004; Jamet et al., 2006, 2008a),
together with detailed methodologies to optimize extraction (Feiz et al., 2006; Watson and Sumner, 2007;
Jamet et al., 2008b). These have resulted in catalogs of
proteins whose identity matches known functions
associated with wall-related processes, such as polysaccharide modification, defense, and signaling, as
well as many with unknown functions. However, a
subset is consistently detected whose localization in
the wall is surprising, based on annotated or even
experimentally established intracellular localization.
These are often simply dismissed as contamination,
and for many this is certainly the case; however, lists of
secreted proteins from extracellular fluids that were
collected under supposedly nondestructive conditions
that would avoid major cell lysis, or from suspension
cell media, also often include proteins that have an
established intracellular role (Isaacson and Rose, 2006).
While contamination can be a major contributing
factor there are other explanations that should be
considered, at least in some cases. First, there are a
growing number of examples of known intracellular
molecules that are also secreted to, or synthesized in,
the apoplast under certain conditions, or in specific
tissues. These include extracellular ATP, which likely
plays a signaling role and is required for maintaining
cell viability (Chivasa et al., 2005; Clark and Roux,
2009), and polymeric DNA that enhances resistances
to fungal infection when secreted at the root tip (Wen
et al., 2009). Similarly, a recent article described an
apoplastic extracellular g-glutamyl transferase, suggesting the existence of an extracellular system to
salvage glutathione derived from the plant or external
sources (Ferretti et al., 2008). Prior to such reports, the
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Rose and Lee
identification of enzymes associated with the biosynthesis or metabolism of these compounds in cell wall
protein extracts would likely have been explained as
contamination, but clearly now their presence could be
seen in a new light, and the same will apply as other
extracellular metabolites/substrates are detected.
Other explanations for the presence of proteins or
peptides in the apoplast with predicted intracellular
functions are that they simply have high homology to
known intracellular proteins but distinct functions, or
that they have more than one function and are localized in multiple cellular compartments. Given that the
cell wall represents the interface with the environment, there are undoubtedly intense selective pressures for existing extracellular proteins to evolve new
functions, or to recruit new proteins to the apoplast
through gene duplication and retargeting. Indeed,
there are already many known examples of secreted
proteins or peptides with more than one function (e.g.
Kuwabara and Imai, 2009; Stotz et al., 2009), almost all
of which have some relationship to abiotic or biotic
stress. As an aside, it should be noted that some
proteins with apparent multiple functions, based on
the presence of both a detectable biological activity
and sequence homology to a known enzyme, may not
necessarily be truly dual function, or moonlighting,
proteins. An example is the family of glucanase inhibitor proteins that are secreted by species of the oomycete Phytophthora, which appear to be derived from an
ancestral Ser protease, and yet have lost the catalytic
triad (Rose et al., 2002). These proteins bind and inhibit
the activity of secreted plant endo-b-1,3-glucanases
and show evidence of evolutionary selection on specific residues from both protein partners at the docking
interface (Damasceno et al., 2008). Such studies hint at
the adoption of new roles in response to evolutionary
pressure, in this case on the part of the pathogensecreted protein, but the principal applies equally to
plant wall proteins.
Another category of unexpected cell wall-localized
proteins has been suggested by the identification of
secreted proteins through individual protein localization studies, or functional screens (Zhu et al., 1994; Lee
et al., 2004, 2006; Kim et al., 2008; Cheng et al., 2009),
which do not apparently have targeting sequences that
would direct to them to the classical secretory pathway. In the next section we discuss the implications for
such observations for compiling a more complete
compendium of the cell wall proteome.
HOW DO PROTEINS REACH THE PLANT
CELL WALL?
The archetypal pathway for secretion of a eukaryotic
protein (Fig. 1) involves an N-terminal region, termed
the signal peptide or leader sequence, which directs
the nascent protein to the endoplasmic reticulum (ER),
whereupon the remainder of the protein is cotranslationally translocated through the Sec61 complex into
the ER lumen (Rapoport, 2007). At this point the
Figure 1. Schematic diagram of the plant classical secretory pathway,
highlighting protein cotranslational translocation from ER-bound ribosomes in the ER lumen and the subsequent major trafficking pathways.
Soluble and membrane-anchored proteins are shown in red and blue,
respectively. N, Nucleus; Chl, chloroplast; TGN, trans-Golgi network;
PVC, prevacuolar compartment; PM, plasma membrane; CW, cell wall,
Vac, vacuole. Adapted from Foresti and Denecke (2008).
protein can also undergo N-linked glycosylation. It
then passes through the endomembrane system, or
secretory pathway, comprising the Golgi apparatus
and trans-Golgi network, where it is packaged into
vesicles that migrate to, and fuse with, the plasma
membrane, releasing the protein cargo into the cell
wall. Many proteins are also retained in the ER or
Golgi, which can include phases of retrograde trafficking, or are targeted to the vacuole or other postGolgi compartments, as described in an excellent
review by Foresti and Denecke (2008).
Several features of the secretory system appear to be
distinctly different in plants and a number of aspects
are still not well understood, such as the identity of the
transport mechanisms that might convey secreted
proteins from the Golgi, trans-Golgi network, or prevacuolar compartment to the plasma membrane,
whether there exist post-Golgi compartments, or if
there are multiple targeting pathways, as has been
suggested in yeast (Saccharomyces cerevisiae; Foresti
and Denecke, 2008; Hwang and Robinson, 2009;
Richter et al., 2009). Additionally, there is evidence
that some proteins can also traffic to the chloroplast
from the Golgi (Villarejo et al., 2005; Kitajima et al.,
2009) or ER (J.K.C. Rose and S.-J. Lee, unpublished
data). The plastid-targeting machinery and protein
structural elements that direct such targeting are not
known, although multiple surface regions of the mature proteins appear to be important (Kitajima et al.,
2009), nor is it known what regulates whether the
proteins are directed to the plastid or to the wall. These
observations suggest a previously unsuspected com-
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Plant Cell Wall Proteome
plexity in interorganellar targeting and provide further
indications of multifunctionality of secreted proteins.
Another major enigma is the growing evidence that
plant proteins can be secreted to the apoplast via
routes that are independent of the ER-Golgi pathway.
Such proteins do not have canonical signal peptides
and treatment with chemical inhibitors, such as brefeldin A, which disrupt vesicle trafficking between the
ER and Golgi, does not perturb secretion. Nonclassical
or leaderless secretion was first described 20 years ago
for two mammalian proteins (Cooper and Barondes,
1990; Rubartelli et al., 1990) and since then considerable progress has been made in characterizing a number of different pathways (Fig. 2) in mammalian and
yeast cells, which will not be described in detail here
(for review, see Nombela et al., 2006; Nickel and
Seedorf, 2008; Prudovsky et al., 2008; Nickel and
Rabouille, 2009). It therefore seems likely that nonclassical secretion is common to all eukaryotes, including plants, although this field is essentially
unexplored. Several algorithms have been developed
to assist with computational prediction of nonclassically secreted proteins (e.g. Bendtsen et al., 2004);
however, these typically use methods that involve
identifying structural features that are conserved or
frequently observed in secreted mammalian proteins,
rather than identifying sequence motifs. Given the
radical differences in the plant and mammalian extracellular environments, it is likely that the respective
protein populations also collectively exhibit very different structural and compositional properties, which
would limit predictive value of such computational
tools with plant proteins. Indeed, in our hands, can-
Figure 2. Schematic diagram of a mammalian cell showing the major
classes of nonclassical secretory pathways that have been identified or
proposed. These pathways are resistant to the inhibitory effects of
brefeldin A, which disrupts ER-Golgi vesicular traffic. N, Nucleus; BFA,
brefeldin A; MVB, multivesicular bodies; PM, plasma membrane.
Adapted from Nickel and Rabouille (2009).
didate proteins that we have identified through functional screens as being secreted by nonclassical
pathways are not predicted to do so using existing
algorithms (data not shown).
It is tempting to ask what sorts of plant proteins are
likely to be secreted by unconventional routes. Some
hints may come from studies of mammalian proteins,
which have been classified into two classes: those that
are mostly secreted and those that usually have an
intracellular function but that are secreted following
specific signals and in specific tissues (Nickel and
Seedorf, 2008). It has also been suggested that such
secretion can be rapid and activated by external
stresses (Keller et al., 2008). It is possible that speed
of secretion might provide a selective advantage,
although other explanations for alternative secretory
routes are the possibility that protein folding in the
environment of the ER and posttranslational modification may not be desirable for some proteins. Moreover, it may be important in some cases to separate
some enzymes from their substrates, or inhibitory and
degradative factors, if they reside in the same secretory
compartments. The super highway for plant cell walllocalized proteins is certainly the classical ER-Golgi
route, and only a small subset is likely to be secreted
by nonclassical pathways; however, this remains an
exciting and entirely uncharacterized area.
WHERE AND WHEN ARE PROTEINS SECRETED TO
THE CELL WALL?
In addition to the questions surrounding the composition of the cell wall proteome and the nature of the
associated trafficking pathways, two other important
and poorly understood issues are the timing and
spatial regulation of protein secretion. A recent review
(Zárský et al., 2009) called into question the preconception that the plant and yeast secretory pathways
are constitutive, while that of animal cells is highly
regulated. There is indeed growing evidence of complex and highly coordinated spatiotemporal protein
secretion in plants. Several cell wall proteins show
evidence of retention in the Golgi or other compartments of the secretory pathway, prior to targeting to
the apoplast by some currently undefined signal,
although the mechanism of release may involve posttranslational proteolytic processing of a proregion of
the protein (e.g. Dal Degan et al., 2001; Wolf et al.,
2009). Interestingly, both these particular proteins act
to degrade pectic polysaccharides and so it may be
that the accumulation of inactive preproteins prior to
the secretion of the processed, and thus enzymatically
activated, mature polypeptides prevents precocious
attack on the cell wall polymers.
Polarized or spatially regulated secretion also provides an additional layer of complexity to the overall
picture. It is known that plasma membrane-localized
proteins, such as the PIN family of auxin efflux carriers, can show clear polar trafficking to specific cell
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Rose and Lee
faces (Richter et al., 2009; Zárský et al., 2009), and
another remarkable example is the spatially coordinated targeting of secretory vesicles to specific regions
of the plasma membrane adjacent to the sites of
infection during microbial challenge (Hückelhoven,
2007). These are particularly well-studied examples,
but it may well be that targeting of secretory vesicles to
plasma membrane domains is more common than is
currently thought.
SUMMARY
The purpose of this Update is to highlight just some
of the burning questions surrounding the plant cell
wall proteome, focusing particularly on the challenges
of determining which proteins truly reside in the wall
and the pathways by which they arrive. There are
certainly many other fascinating, and generally neglected, aspects of cell wall protein research, including
the potential for phosphorylation, the identification of
extracellular protein complexes, and postsecretion
proteolytic and glycanolytic processing, but these are
beyond the scope of this review.
ACKNOWLEDGMENTS
We would like to acknowledge the many researchers who have made
important contributions to the field, but whose work we unfortunately
cannot present in this Update due to length limitations.
Received February 16, 2010; accepted March 10, 2010; published March 17,
2010.
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