Download Diversity and Formation of Endoplasmic Reticulum

Update on Diversity and Formation of Compartments from ER
Diversity and Formation of Endoplasmic
Reticulum-Derived Compartments in Plants. Are
These Compartments Specific to Plant Cells?1
Ikuko Hara-Nishimura*, Ryo Matsushima, Tomoo Shimada, and Mikio Nishimura
Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606–8502, Japan (I.H.-N., R.M.,
T.S.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444–8585, Japan (M.N.)
Unlike animals, plants are not able to escape from
adverse circumstances. To cope with external stresses,
plants modulate the endomembrane systems, especially the endoplasmic reticulum (ER), which is the
most flexible and adaptable organelle (Staehelin, 1997).
Alternatively, plants generate a specialized compartment from the ER to accumulate an enormous amount
of proteins that are actively synthesized on the ER
(Chrispeels and Herman, 2000). Active protein synthesis can be regarded as an internal stress. In response to
the internal and external stresses, the ER differentiates
into various types of compartments, each of which has
its own responsibility.
The diversity of ER-derived compartments has been
getting increased attention in plant cell biology. The ER
networks of membranous tubules and cisternae are
extended throughout the cytosol area surrounding the
vacuoles. The ER membrane, which occupies nearly
one-half the total area of membrane in cells, can be
a source of a variety of compartments. Various ERderived compartments with sizes ranging from 0.1 mm
to 10 mm have been identified in plant cells (Larkins and
Hurkman, 1978; Li et al., 1993; Hara-Nishimura et al.,
1998; Schmid et al., 1998; Toyooka et al., 2000; Hayashi
et al., 2001). All the compartments that have been
characterized share three common features: (1) The
ER-derived compartments accumulate a large amount
of a single protein or only a few different proteins. (2)
The protein components do not act within the compartments, even if they are functional proteins such as
enzymes. (3) The compartments appear in a specific
tissue at a particular time of the life cycle of plants.
These features imply that ER-derived compartments
have distinct responsibilities.
To understand a physiological role of an ER-derived
compartment, it is necessary to identify the protein or
proteins that accumulate in the compartment. The ER-
1
This work was supported by CREST of the Japan Science and
Technology Corporation, and Grants-in-Aid for Scientific Research
(no. 16085203) and for 21st Century COE Research Kyoto University
(A14) from the Ministry of Education, Culture, Sports, Science and
Technology of Japan.
* Corresponding author; e-mail [email protected]; fax
81–75–753–4142.
www.plantphysiol.org/cgi/doi/10.1104/pp.104.053876.
derived compartments are classified into two types
according to their contents: storage-protein type and
hydrolytic-enzyme type. The former type compartments accumulate seed storage proteins in large quantities. The latter type compartments accumulate a
hydrolytic enzyme, although they do not contain substrates for the respective enzyme. We are attempting to
answer several questions. How are enormous amounts
of storage proteins and hydrolytic enzymes accumulated in the compartments? How and where do the
hydrolytic enzymes meet with their own substrate to
fulfill their functions in the cells? How and when are the
compartments formed from the ER? The aim of this
Update is to try to give some answers to these questions.
The mechanisms underlying the protein accumulation
in the compartments and the formation of the compartments are discussed with the two types of ER-derived
compartments.
PAC VESICLE: SELECTIVE UPTAKE OF STORAGE
PROTEIN PRECURSORS
PAC (precursor-accumulating) vesicle is an ERderived compartment that was found in maturing
seeds of pumpkin (Cucurbita maxima) and castor bean
(Ricinus communis; Hara-Nishimura et al., 1998). PAC
vesicles accumulate precursors of storage proteins, 2S
albumin and 11S globulin, to be transported to protein
storage vacuoles. How do the vesicles accumulate the
precursors? The precursor proteins form aggregates
within the ER lumen, and the aggregates develop into
PAC vesicles. PAC vesicles are responsible for an
aggregate sorting of proteins to protein storage vacuoles in a Golgi-independent manner.
Recently, another way of Golgi-dependent uptake of
the storage protein precursors into the PAC vesicles
was discovered. A proteomic analysis of the isolated
vesicles identified a type I integral membrane protein,
PV72, on the membrane of the PAC vesicles (Shimada
et al., 1997). PV72 was specifically and transiently
accumulated in association with the synthesis of storage proteins, and has an ability to bind to the 2S
albumin precursor via the C-terminal region including
a vacuolar targeting signal (Shimada et al., 2002). PV72
has Ca21-binding domains, and the binding of Ca21
stabilizes the receptor-ligand complex even at pH 4.0.
Plant Physiology, November 2004, Vol. 136, pp. 3435–3439, www.plantphysiol.org Ó 2004 American Society of Plant Biologists
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Hara-Nishimura et al.
The association and dissociation of PV72 with the ligand is modulated by the Ca21 concentration (EC50 5
40 mM) rather than the environmental pH (Watanabe
et al., 2002). This indicates protein storage vacuoles that
have an acidic interior pH and high Ca21 concentrations are not an appropriate organelle to dissociate the
precursor proteins from PV72. The questions are where
PV72 traps the precursor molecules and where PV72
releases them. It has been shown that PV72 is localized
not only on the PAC membrane but also on the Golgi
complex (Shimada et al., 2002). PV72 might be recycled
between PAC vesicles and the Golgi complex for
recruiting the storage protein precursor molecules from
the Golgi complex to the PAC vesicles.
The above biochemical and cell biological analyses
suggest the PV72 acts as a vacuolar sorting receptor. For
an in vivo demonstration, a reverse-genetic approach
was used with an Arabidopsis (Arabidopsis thaliana)
mutant lacking the homologous gene to PV72. Arabidopsis has seven homologs of PV72, which are designated AtVSR1 to AtVSR7 (Arabidopsis thaliana vacuolar
sorting receptor). Only the atvsr1 mutant seeds abnormally accumulate the precursors of storage proteins, 2S
albumin and 12S globulin, together with the mature
forms of these proteins (Shimada et al., 2003). The atvsr1
mutant missorts storage proteins by secreting them
from cells, resulting in an enlarged and electron-dense
extracellular space in the seeds. The in planta analysis
demonstrated that AtVSR1/PV72 is a sorting receptor
that sorts storage proteins for protein storage vacuoles.
How does the sorting receptor function? Figure 1A
shows a hypothetical model for the selective uptake of the
storage protein precursors into the PAC vesicles. Most of
the storage protein precursor molecules synthesized on
the ER form an aggregate within the ER. However,
inevitably, some molecules are not incorporated into the
aggregates and remain free. The free molecules leave
the ER for the Golgi complex, where they are trapped by
the vacuolar sorting receptor and recruited to the PAC
vesicles. After releasing the precursor molecules, the
receptor is recycled to the Golgi complex.
Selective uptake of the storage protein precursors
into the PAC vesicles is conducted in two ways: one is
aggregate sorting and the other is receptor-dependent
sorting. The former sorting mechanism is advantageous to maturing seeds that actively synthesized
a large quantity of storage proteins. The latter ensures
proper delivery of the proteins by avoiding the missorting of the escaped molecules. This mechanism can
be comparable to that for the recruitment of the escaped
ER resident proteins by the KDEL receptor that is
localized in the Golgi complex.
ER BODY: ACCUMULATION OF A KDEL-TAILED
HYDROLYTIC ENZYME
Green fluorescent protein (GFP) from luminescent
jellyfish allows us to visualize various organelles in
living cells and in real time. A large ER-derived
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compartment, the ER body, is easily observed as bright
fluorescing spindle-shaped bodies in Arabidopsis cotyledon cells that express an ER-targeted GFP, which is
composed of a signal peptide and GFP followed by
a tetrapeptide sequence, K(H)DEL (Lys/His-Asp-GluLeu) (Haseloff et al., 1997; Ridge et al., 1999; Hawes
et al., 2001; Hayashi et al., 2001).
The shape and size of ER bodies (approximately
1 mm diameter 3 approximately 10 mm long) are completely different from two other hydrolytic enzymetype compartments: KDEL vesicles (F 5 0.2–0.5 mm)
in Vigna mungo cotyledons (Toyooka et al., 2000) and
ricinosomes (F 5 0.2–0.5 mm) in castor bean endosperm (Schmid et al., 1998). Despite these differences,
ER bodies, KDEL vesicles, and ricinosomes share an
accumulation of a hydrolytic enzyme with an ERretention signal (KDEL) at the C terminus. ER bodies
accumulate a KDEL-tailed b-glucosidase (PYK10) as
a major component (Matsushima et al., 2003), while
both KDEL vesicles and ricinosomes accumulate
a KDEL-tailed Cys proteinase (Schmid et al., 1998;
Toyooka et al., 2000).
The question raised is whether the KDEL-tailed
protein PYK10 is selectively transported and accumulated in ER bodies. KDEL and HDEL are typical ER
retention signals that are found at the C terminus of
the ER resident proteins. K(H)DEL-tailed protein molecules that leave the ER for the Golgi complex are
recycled to the ER by the action of a KDEL receptor
(Fig. 1B). In a similar manner, GFP-HDEL is also
accumulated in the ER, resulting in the formation of
bright fluorescent ER bodies in the transgenic Arabidopsis seedlings. Is PYK10 also accumulated in the
ER bodies by this mechanism? Comparison of the
subcellular distributions of GFP-HDEL and PYK10
shows that PYK10 is more concentrated in ER bodies
than the ER, while GFP-HDEL is less concentrated
in the ER bodies than in the ER (Matsushima et al.,
2003).
What is the mechanism responsible for the selective
concentration of PYK10 in the ER bodies? One possibility is that PYK10 mRNA is segregated on the ER
membrane in a similar manner as discussed by Okita
and colleagues in this Focus Issue (Crofts et al., 2004).
This is supported by the presence of ribosomes on the
membrane of ER bodies. Another possibility is that
PYK10, which is abundant in the seedlings, contributes to the formation of the ER bodies by forming
aggregations in the ER cisternae. b-Glucosidase aggregations in plant cells have been reported (Nisius,
1988; Fieldes and Gerhardt, 1994; Kim et al., 2000). The
ER bodies also contain two stress-inducible proteinases, RD21 and gVPE, which have neither KDEL nor
HDEL at their C termini. These proteinases may be
trapped into the ER bodies during the aggregate
formation of the abundant b-glucosidase. A similar
phenomenon is also found in the storage protein-type
compartment. Vacuolar processing enzyme is found in
the PAC vesicles in developing seeds of castor bean
(Hiraiwa et al., 1993).
Plant Physiol. Vol. 136, 2004
Diversity and Formation of Compartments from the ER
Figure 1. Schematic drawing of two types
of ER-derived compartments in plant cells.
Plant cells develop ER-derived compartments, which are classified into two
types according to their contents: storage
protein-type compartments (A) and hydrolytic enzyme-type compartments (B). PAC
vesicles accumulate a large amount of
storage protein precursors (solid triangles),
while ER bodies accumulate a KDELtailed b-glycosidase, PYK10 (blue circles).
Recycling receptors, vacuolar sorting receptor and KDEL receptor, are involved in
the accumulation of the lumenal components in PAC vesicles and ER bodies,
respectively. Open triangles, Mature form
of storage proteins; open circles, ER resident proteins.
INDUCTION OF DIVERSE
ER-DERIVED COMPARTMENTS
ER bodies have a limited distribution in Arabidopsis seedlings. They appear in the epidermal cells of
cotyledons and hypocotyls after seed germination,
and disappear during senescence of the tissues. On
the other hand, the rosette leaves have no ER bodies at
all under normal conditions. However, once rosette
leaves are wounded, many ER bodies are induced
around the wound site of the leaves (Matsushima et al.,
2002). The induction of ER bodies in response to an
environmental stress demonstrates the flexibility of
the ER of plants.
The induction of ER bodies is under hormonal
control (Matsushima et al., 2002). ER bodies are induced throughout the epidermal cells of the rosette
leaves treated with methyl jasmonate (MeJA), which
mediates plant defenses against mechanical wounding
and chewing by insects. On the contrary, ethylene
suppresses the effect of MeJA on the induction of ER
bodies in rosette leaves. The induction of ER bodies is
strictly coupled with the signal transduction of MeJA and
ethylene, both of which have an antagonistic effect on it.
What mechanism is involved in differentiation of
the ER of plants? Recently, another factor that regulates the formation of the ER bodies was identified by
a genetic approach. We isolated an Arabidopsis mutant, nai1, whose seedlings hardly have any ER bodies.
The NAI1 gene encodes a transcription factor that has
a basic-helix-loop-helix domain, and transient expression of NAI1 induced ER bodies in the nai1-1 mutant
(Matsushima et al., 2004). Proteomic analysis showed
that nai1 has no PYK10. The absence of ER bodies in
nai1 mutants is due to the loss of a transcription factor
Plant Physiol. Vol. 136, 2004
(NAI1) that functions upstream of a major component
(PYK10) of the compartment. The transcription factor
regulates the formation of the ER bodies. A similar
transcription factor responsible for the formation of
ER-derived compartments has been identified in
maize (Zea mays). Opaque-2 (O2), a basic Leu zippertype transcription factor, regulates the expression of
a-zein, a major storage protein of maize, which is
accumulated in protein bodies derived from the ER
(Hartings et al., 1989). The protein bodies of the o2
mutant seeds are much smaller (F 5 0.1–0.3 mm) than
normal protein bodies (F 5 1–2 mm). This implies that
both types of ER-derived compartments share a similar
mechanism for their formation.
The ER bodies are inducible in nai1-1 rosette leaves
that have been treated with MeJA, although they
exhibit irregular shapes (Matsushima et al., 2004).
MeJA and ethylene have an antagonistic effect on their
induction in the mutant leaves. MeJA treatment induces the expression of NAI1 and PYK10 genes, and this
induction is suppressed in the presence of ethylene.
The expression pattern of NAI1 and PYK10 genes are
parallel to the induction pattern of the ER bodies.
MeJA-induced NAI1 is needed for the proper formation of ER bodies.
The question is whether the nature of the protein
components determines the shapes of the ER-derived
compartments, such as the large spindle shape of ER
bodies and the small globe shape of KDEL vesicles. A
recent study of the overexpression of KDEL-tailed
proteinase answers this question. As described above,
a hydrolytic enzyme-type compartment, the KDEL
vesicle, accumulates a KDEL-tailed Cys proteinase in
V. mungo (Toyooka et al., 2000). Overexpression of the
proteinase in Arabidopsis plants induces the formation
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Hara-Nishimura et al.
of an ER-derived compartment, which is a spindleshaped structure with a characteristic fibrous pattern
inside (Okamoto et al., 2003). Interestingly, the induced
compartments are more similar to the ER bodies than to
KDEL vesicles. This indicates that a different protein
has an ability to form ER body-like architectures in
Arabidopsis plants. There should be some relationship
between the ER body and the other hydrolytic-type
compartment.
The storage protein-type compartment is also inducible in transgenic Arabidopsis plants by overexpression of a chimeric protein of 2S albumin subunit
and a selectable marker enzyme, phosphinothricin
acetyltransferase (Hayashi et al., 1999). Despite the
limited distribution of PAC vesicles in maturing seeds,
the transgenic Arabidopsis plants induce PAC vesicles
in the vegetative organs, rosette leaves, hypocotyls, and
roots. Aggregates of the expressed proteins may trigger
the formation of PAC vesicles.
PHYSIOLOGICAL FUNCTION OF ER BODY
ER-derived compartments have their own specialized function. The physiological function of each compartment can be estimated by identifying the protein
components and the temporal and spatial development. The ER bodies are abundant in the epidermal
cells of cotyledons and hypocotyls of seedlings and root
cells of Arabidopsis. These cells are most sensitive to
environmental stresses. This implies that the development of ER bodies is linked to environmental stresses.
The induction of ER bodies in the rosette leaves might
also have a defense function against chewing insects
and may be related in some way to other wound
stresses. The development and induction of ER bodies
is a plant strategy that assists the cells under stressed
conditions.
To clarify the ER body-mediated defense system at
the molecular level, it is necessary to identify the natural substrate of b-glucosidase PYK10. b-Glucosidases
acts against pathogens by producing toxic compounds
from the substrates. PYK10 and the substrate should
be stored separately within the cells. PYK10 in the ER
bodies will come into contact with its natural substrates to produce the toxic compounds, when plant tissues are injured or stressed.
The physiological function of b-glucosidases depends on the nature of the aglycon moiety of its natural
substrates, such as glucosinolates for thioglucosidases
and cyanoglucosides for cyanogenic glucosidases. For
identification of the substrate of PYK10, the nai1 mutant
can be an ideal tool. The natural substrates of PYK10
will be identified by comparing the chemical components in wild-type plants with those in the nai1 plants.
Further analysis of the susceptibility of nai1 to herbivore insects or pathogens will provide new insight into
the ER body-mediated plant defense system.
Why do ER bodies develop in seedlings but not in
rosette leaves? The reason may be related to the
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economy of costs of nutrients and energy. ER bodies
are not essential for the fundamental process of plant
life, considering the normal growth of nai1 plants.
Therefore, putting off the synthesis of PYK10 and the
formation of ER bodies in rosette leaves until the need
arises may save costs. On the other hand, cotyledons,
which are most sensitive to wounding and chewing by
insects, may pay the costs for the defense until rosette
leaves develop.
FUTURE PERSPECTIVES
The ER is a subcellular factory producing almost all
the proteins and lipids for the ER itself, the Golgi
complex, endosomes, prevacuolar compartments, vacuoles, lysosomes, secretory vesicles, and the plasma
membrane, which are connected by both forward and
backward vesicle traffic to constitute the endomembrane system. The molecular mechanisms underlying the endomembrane system in plants, animals, and
yeast share common components. ER-derived compartments are closely related to the endomembrane
system. However, most of the compartments are induced in specific organs in response to the internal and
external signals. The induction of ER-derived compartments is controlled in a sophisticated way by the
conditions under which plants grow. Thus, the regulation mechanism should be unique to plants and directly
concerned with the life processes of higher plants.
Although ER-derived compartments vary in their
structures and functions, some molecular mechanisms
underlying their development and formation might be
shared. Proteomic analyses of isolated compartments
should identify key factors playing in the plant system. Isolation of mutants deficient in the formation of
the compartments is another useful approach. For
example, transgenic Arabidopsis that induces PAC
vesicles by overexpressing a chimeric protein of the 2S
albumin subunit followed by a selectable marker
enzyme (described above) are still sensitive to the
respective antibiotics. A mutant resistant to the antibiotics should have a defect in the formation of PAC
vesicles. DNA array analysis with the nai1 mutant
should also identify downstream genes of NAI1.
Identification of such key factors will provide a valuable insight into the induction of the ER-derived
compartments and the strategy that higher plants
have evolved.
Received October 7, 2004; returned for revision October 9, 2004; accepted
October 9, 2004.
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