The Plant Journal (2004)
doi: 10.1111/j.1365-313X.2004.02167.x
Transport of ricin and 2S albumin precursors to the storage
vacuoles of Ricinus communis endosperm involves the Golgi
and VSR-like receptors
Nicholas A. Jolliffe1,†, Joanna C. Brown1,†, Ulla Neumann2,†, Maı̈te Vicré2, Angela Bachi3, Chris Hawes2, Aldo Ceriotti4,
Lynne M. Roberts1,* and Lorenzo Frigerio1,*
1
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK,
2
Research School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane Campus, Oxford OX3 0BP, UK,
3
Mass Spectrometry Unit, DIBIT-HSR, via Olgettina 58, 20132 Milan, Italy, and
4
Istituto di Biologia e Biotecnologia Agraria, CNR, via Bassini 15, 20133 Milan, Italy
Received 20 May 2004; accepted 11 June 2004.
*
For correspondence (fax þ44 24 765 23701; e-mail [email protected]; [email protected]).
†
These authors contributed equally to this work.
Summary
We have studied the transport of proricin and pro2S albumin to the protein storage vacuoles of developing
castor bean (Ricinus communis L.) endosperm. Immunoelectron microscopy and cell fractionation reveal that
both proteins travel through the Golgi apparatus and co-localize throughout their route to the storage vacuole.
En route to the PSV, the proteins co-localize in large (>200 nm) vesicles, which are likely to represent
developing storage vacuoles. We further show that the sequence-specific vacuolar sorting signals of both
proricin and pro2SA bind in vitro to proteins that have high sequence similarity to members of the VSR/AtELP/
BP-80 vacuolar sorting receptor family, generally associated with clathrin-mediated traffic to the lytic vacuole.
The implications of these findings in relation to the current model for protein sorting to storage vacuoles are
discussed.
Keywords: albumin, ricin, sorting receptor, storage proteins, vacuole.
Introduction
Developing castor bean (Ricinus communis L.) seeds accumulate lipids and storage proteins in their endosperm. The
castor bean endosperm is a living tissue which, upon germination, undergoes programmed cell death to ensure full
mobilization of the storage material to the cotyledons (Gietl
and Schmid, 2001; Schmid et al., 2001). In developing
endosperm, the major storage proteins: 7S lectins [namely
ricin (RCAII) and R. communis agglutinin (RCAI)], 2S albumins and 11S globulins, accumulate in protein storage
vacuoles (PSV, previously named protein bodies or aleurone
grains; Tully and Beevers, 1976; Youle and Huang, 1976).
The R. communis PSV contains phytin globoids and a single
large protein crystalloid within a soluble protein matrix. The
insoluble crystalloid is composed of 11S globulin while the
protein matrix is a mixture of 7S lectins and 2S albumins
(Fukusawa et al., 1988; Tully and Beevers, 1976). Each of
these storage proteins is co-translationally translocated into
ª 2004 Blackwell Publishing Ltd
the lumen of the endoplasmic reticulum (ER) and subsequently transported to the PSV. N-glycosylated lectins travel
through the Golgi apparatus, where their glycans are
modified and, in the case of ricin, specific fucose residues
are added (Lord and Harley, 1985).
We have recently studied the intracellular sorting of both
castor bean ricin and 2S albumin in heterologous tobacco
mesophyll protoplasts (Brown et al., 2003; Frigerio et al.,
1998). Ricin is a heterodimeric glycoprotein composed of a
catalytic, ribotoxic A chain (RTA) disulphide-linked to a
galactose-binding B chain (RTB). It is made as a single
polypeptide precursor in which RTA and RTB are joined by a
12 amino acid linker that is removed upon vacuolar delivery.
2S albumin is also synthesized as a single precursor protein,
subsequently processed in PSV into two, different heterodimeric storage proteins, each composed of a large and a
small subunit (Brown et al., 2003; Irwin et al., 1990). In
1
2 Nicholas A. Jolliffe et al.
transfected protoplasts, as in the native tissue, both proteins
reach the vacuole, where they are processed to their mature
forms (Brown et al., 2003; Frigerio et al., 1998).
We have characterized the vacuolar sorting signals of both
the ricin and albumin precursors. For proricin, the signal
resides within a 12-residue linker propeptide that lies
between the A and the B chain (Frigerio et al., 2001). For
proalbumin, the signal is again contained within an internal
propeptide (PPII), in this case separating small subunit I from
large subunit I (Brown et al., 2003). NMR structural analysis
shows that this propeptide resides in a relatively unstructured loop which is exposed on the surface of the protein
(Pantoja-Uceda et al., 2003). Both the proricin and proalbumin sorting signals appear to be of the sequence-specific
type (ssVSS; Matsuoka and Neuhaus, 1999), which have
previously only been associated with Golgi-mediated traffic
to the lytic vacuole (LV). ssVSS contain a hydrophobic
residue in the context of an NPIR-like sequence which has
been shown to interact with receptor proteins of the
vacuolar sorting receptor (VSR) family (Ahmed et al., 1997;
Kirsch et al., 1994; Paris and Neuhaus, 2002; Shimada et al.,
1997). Accordingly, mutation of a single isoleucine (for
proricin) or leucine (for proalbumin) residue within these
motifs causes the precursors to be secreted by tobacco
protoplasts. Both sorting signals are also sufficient to target
reporter proteins to the vacuole (Brown et al., 2003; Frigerio
et al., 2001; Jolliffe et al., 2003).
A novel transport route for non-glycosylated proteins
including 2S albumin has been described in developing
castor bean endosperm and pumpkin cotyledons (HaraNishimura et al., 1998). In this route, large precursor-accumulating vesicles (PAC) containing proalbumin bud directly
from the ER, seemingly bypassing the Golgi complex to fuse
with PSV. A working model for targeting in castor bean
endosperm has correspondingly been proposed whereby 2S
albumin exits the ER in PAC, which subsequently receive
Golgi-modified glycoproteins, such as proricin, into their
periphery before they reach PSV (Hara-Nishimura et al.,
1998). Indeed, two putative receptors of the VSR class, PV72/
82, have been isolated from the membranes of PAC
(Shimada et al., 1997, 2002). PV72 has been shown to be
present in the Golgi (Shimada et al., 2002), and to interact
with various sequences derived from pumpkin 2S albumin
(Shimada et al., 1997, 2002; Watanabe et al., 2002). These
findings were rationalized by ascribing a role to PV72 in
salvaging any 2S albumin that escapes aggregation in the
ER (Shimada et al., 2002). However, the presence of an
ssVSS in castor bean 2S albumin suggests that transport of
this protein to PSV may be solely receptor-, and thus Golgi-,
mediated. In addition, we have recently provided indirect
evidence for transport of proalbumin through the Golgi in
tobacco protoplasts (Brown et al., 2003). The disparity of
these data prompted us to investigate in more detail the
transport of proricin and proalbumin in their native tissue.
In this work we show that both proricin and proalbumin
indeed co-localize and travel through the Golgi apparatus.
Both proteins subsequently appear in large vesicles which
are likely to represent developing storage vacuoles. These
are distinct from PAC as their lumina contain proteins
carrying Golgi-modified glycans. We further show that the
sequence-specific sorting signals of both proricin and proalbumin bind in vitro to a protein that shares homology with
the VSR/AtELP/BP-80 vacuolar sorting receptor family, previously associated with the clathrin coated vesicle (CCV)mediated pathway to LV (Bassham and Raikhel, 2000; Vitale
and Raikhel, 1999).
Results
We have recently investigated the vacuolar targeting of both
ricin and 2S albumin by transient expression in tobacco
mesophyll protoplasts and wanted to further characterize
the fates of these proteins in their native tissue. To ascertain
whether proricin and proalbumin are transported along the
same, Golgi-mediated route to PSV, we performed immunoelectron microscopy on ultrathin sections of high-pressure frozen developing castor bean endosperm.
Ricin and 2S albumin co-localize in the Golgi and PSV
We isolated developing castor bean endosperm at the stage
of testa maturation corresponding to the peak of synthesis of
all major classes of storage proteins (Lord, 1985b). The
endosperm is characterized by regularly shaped cells containing toluidine blue-reactive structures (Figure 1a). Ultrastructural analysis reveals abundant, electron translucent
lipid bodies (Figure 1b,c) and a number of more electronopaque, spherical structures of variable size corresponding
to the toluidine blue-reactive structures. These were previously identified as PSV (Figure 1) (Tully and Beevers, 1976;
Youle and Huang, 1976). Although both globoids and crystalloids have previously been detected at this stage of
development after chemical fixation (Gifford et al., 1982),
only the globoids were visible in high pressure frozen sections of the same tissue (Figure 1c, arrowheads).
Based on substantial biochemical evidence, ricin and 2S
albumin are classified as storage proteins of the PSV matrix
(Irwin et al., 1990; Lord, 1985b; Tully and Beevers, 1976;
Youle and Huang, 1976). This was confirmed by immunolabelling endosperm sections with anti-ricin A chain (antiRTA) and anti-2S albumin (anti-2SA) antisera. Figure 2
(lanes 1 and 2) shows that both rabbit and sheep anti-RTA
antisera specifically recognized the proricin precursor (grey
arrow) and two glycoforms of ricin A chain (empty arrowheads) (Fulton et al., 1986) from a preparation of total
endosperm proteins. However, it has previously been demonstrated that the rabbit anti-2SA antiserum, in addition to
recognizing 2S albumin (black arrowhead), also recognizes
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x
)
2S
A
α-
α2S
A
(p
(s
ur
he
fiie
ep
it)
bb
(ra
RT
A
α-
RT
A
α-
d)
Vacuolar sorting of ricin and 2S albumin 3
64 -
- 64
36 -
- 36
1
2
3
4
Figure 2. Evaluation of antisera against ricin and 2S albumin.
Total proteins from castor bean endosperm were resolved by SDS-PAGE and
immunoblotted with rabbit (lane 1) or sheep (lane 2) anti-RTA antiserum, or
crude (lane 3) or purified (lane 4) anti-2SA antiserum. The numbers on the left
and right indicate molecular mass markers in kilodaltons. The grey arrow
indicates the ricin precursor, open arrowheads the two immunoreactive ricin
A chain (RTA) glycoforms, and the closed arrowhead the albumin precursor.
the ricin A chain glycoforms (empty arrowheads) (Figure 2,
lane 3; also Brown et al., 2003). We therefore removed the
contaminating antibodies by passage through a column
bearing immobilized ricin, generating an antiserum which
recognized only 2S albumin (Figure 2, lane 4).
To immunologically assess the identity of PSV in developing castor bean endosperm, we initially labelled sections
with antibodies against a-TIP, a well-established marker for
the tonoplast of the PSV (Jauh et al., 1999; Johnson et al.,
1990). Anti-a-TIP coupled with 10 nm gold particles clearly
not only decorated the outer rim of the electron opaque
bodies (Figure 3a), but also the Golgi apparatus (Figure 3b).
This pattern has been previously observed in developing
pea cotyledons (Hillmer et al., 2001). We therefore conclude
that the anti-a-TIP-lined organelles are PSV. Both anti-RTA
(Figure 3c, 10 nm gold) and anti-2SA antisera (Figure 3d)
label the lumen of the electron opaque bodies. Double
immunogold labelling using anti-RTA (coupled with 10 nm
gold) and anti-a-TIP (coupled with 20 nm gold) confirmed
that the matrix of the PSV was the site of ricin and 2S
albumin deposition (Figure 3c, arrowheads indicate a-TIP
labelling). Most importantly, both ricin and 2S albumin are
also found in the Golgi apparatus (Figure 3, panels e and f).
Figure 1. The castor bean endosperm is rich in LB and PSV.
(a) Semi-thin section of high-pressure frozen developing castor bean endosperm stained with toluidine blue and visualized by light microscopy. Bar:
20 lm.
(b, c) Ultrathin, high-pressure frozen sections of castor bean endosperm
visualized by low magnification transmission electron microscopy. LB, lipid
bodies; PSV, protein storage vacuoles; N, nucleus; n, nucleolus; arrowheads,
globoid cavities. Bars: (b), 5 lm; (c), 1 lm.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x
4 Nicholas A. Jolliffe et al.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x
Vacuolar sorting of ricin and 2S albumin 5
Whilst previously reported for ricin, where the acquisition of
Golgi-modified glycans is well documented (Lord, 1985a;
Lord and Harley, 1985), these findings now directly demonstrate that proalbumin is also transported through the Golgi
complex en route to PSV in castor bean endosperm. Indeed,
94% of the Golgi stacks observed in this study (73 of 78) were
labelled with anti-2SA. Double immunogold labelling with
anti-RTA (10 nm gold) and anti-2SA (20 nm gold) confirmed
the co-localization of these two proteins in both the Golgi
and PSV (Figure 4a,b, respectively). Background labelling
was consistently very low (see Table S1). In addition to
reproducibly detecting 2S albumin in the Golgi, we also
observed immunogold labelling in the ER (Figure 4c). Strikingly, the ER in developing endosperm displayed a homogeneous, tubular morphology, in which dense aggregates of
the type postulated to form PAC vesicles were never
observed. Furthermore, 2S albumin labelling within the ER
was always uniformly distributed, in contrast to the clusters
observed in the Golgi cisternae (Figures 3f and 4a). This is
consistent with the sorting event occurring in the Golgi,
rather than the ER. Thus, we conclude that in developing
castor bean endosperm both proricin and proalbumin are
transported from the ER to the Golgi en route to PSV.
Ricin and 2S albumin accumulate in large, post-Golgi
vesicular structures
As well as the Golgi and large PSV, the antibodies raised
against RTA (coupled with 10 nm gold) and 2S albumin
(coupled with 20 nm gold) also label the lumen of large
vesicles of 150–400 nm in diameter (Figure 5a). These vesicles possessed a peripheral electron-translucent layer, and
were often seen close to PSV [Figure 5b(inset),c]. Ribosomes were never observed around the periphery of these
structures, suggesting that they did not originate directly
from rough ER. In addition, anti-complex glycan antiserum
revealed a distribution indistinguishable from anti-RTA
(Figure 5b, 10 nm gold), labelling throughout the lumen of
these vesicles (Figure 5b, main panel and inset, 20 nm gold),
further indicating that the lumenal cargo has travelled via
the Golgi. As these structures appear to have the same distribution of lectins and albumins as PSV, they may therefore
represent precursors to storage vacuoles. In further support
of this, anti-a-TIP labelled the membrane of these vesicles
(Figure 5c). The vesicles were also often found in close
proximity to one another and/or to a large storage vacuole,
suggesting imminent homotypic fusion. Figure 5(c) shows
such an example, where five a-TIP-lined vesicles (black stars)
are clustered together near a PSV.
Ricin and 2S albumin co-fractionate with VSR-like proteins
The sequence-specific nature of the vacuolar sorting signals
of ricin and 2S albumin suggests that both proteins may be
ligands for a sorting receptor(s) of the VSR family (previously named BP-80 or ELP; Ahmed et al., 1997; Kirsch et al.,
1994; Shimada et al., 1997). As the interaction between such
receptors and clathrin adaptor proteins has been demonstrated in vitro (Sanderfoot et al., 1998), this in turn makes
them candidates for recruitment into CCV. As neither the
clathrin heavy-chain antiserum nor the anti-BP-80 antisera
(MAbs 17F9 or 14G7) (Cao et al., 2000) were compatible with
the EM methodology described here (data not shown), we
instead investigated the potential co-localization of these
proteins by subcellular fractionation and Western blotting.
Developing castor bean endosperm was homogenized
and subjected to fractionation on a continuous 15–55%
isopycnic sucrose gradient. Fractions were resolved by
either reducing or non-reducing SDS-PAGE before immunoblotting with a panel of antisera (Figure 6). The majority of
the precursor (Figure 6) and mature (data not shown) forms
of ricin and 2S albumin are detected at the top of the
gradient. This is expected, as fragile PSV break during
homogenization in aqueous buffer. There is also a degree of
breakage of other endomembranes, as testified by the fact
that anti-BiP labels fractions at the top of the gradient, in
addition to the microsome peak. Both the ricin and 2S
albumin precursors, however, also peak at a fraction of
density 1.21 g ml)1. These fractions are clearly distinct from
the ER microsomes, as indicated by the position of the
molecular chaperone BiP (Figure 6). Significantly, proteins
reacting with anti-BP-80 monoclonal antibody 17F9 also
coincide with ricin and 2S albumin. Furthermore, this
1.21 g ml)1 fraction is also labelled by an anti-clathrin heavy
chain antibody. We conclude that a proportion of proricin
and proalbumin is found in cellular fractions that are
enriched in both BP-80-like species and clathrin.
The sorting signals of ricin and 2S albumin bind to VSR-like
proteins in vitro
The cell fractionation data raise the possibility that proricin
and proalbumin may be ligands of VSR (BP-80)-like receptors. To test this hypothesis, we produced affinity columns
Figure 3. Ricin and 2S albumin travel through the Golgi complex and accumulate in PSV.
Ultrathin sections of high-pressure frozen castor bean endosperm were immunolabelled with the following antisera:
(a, b) Polyclonal rabbit anti-a-TIP antibody (Figure 3a,b) showing labelling of the PSV membrane and Golgi.
(c) Polyclonal sheep anti-RTA (10 nm gold) and polyclonal rabbit anti-a-TIP antibody (20 nm gold), showing labelling of the PSV matrix and bounding membrane.
(d) Polyclonal rabbit anti-2SA labelling the PSV matrix.
(e, f) Labelling of a Golgi stack with polyclonal anti-RTA and anti-2SA, respectively.
G, Golgi complex; LB, lipid bodies; PSV, protein storage vacuoles. Bars: (a), 200 nm; (b–f), 100 nm.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x
6 Nicholas A. Jolliffe et al.
carrying immobilized peptides corresponding to the proricin
linker and proalbumin PPII, both in their wild-type form or
with the respective, crucial isoleucine/leucine changed to
glycine (Figure 7a). A potential receptor would be expected
to bind to the wild-type, but not the mutated propeptides. As
a positive control, a column bearing the proaleurain VSS
was also generated, as previously used to select BP-80 from
pea (Kirsch et al., 1994). Clarified CHAPS extracts of membranes prepared from developing castor bean endosperm
were applied to the columns, washed, and eluted at low pH.
Eluates were separated by non-reducing SDS-PAGE and
visualized with Coomassie (Figure 7b). A protein migrating
below the 64 kDa marker bound to the wild-type, but not the
I271G ricin linker peptide column (Figure 7b, empty arrowhead, compare lanes 1 and 2). Likewise, a species of comparable mobility bound to the 2S albumin PPII, but not the
L58G peptide column (Figure 7b, compare lanes 4 and 5). A
protein with an apparent molecular mass of around 50 kDa
was also eluted in every case (Figure 7b, black arrowhead)
as well as from a column prepared without coupled peptide
(data not shown). This protein, identified by mass spectrometry as calreticulin (data not shown), is a contaminant
which binds to the column matrix itself.
Interestingly, the proteins released from the control
proaleurain column were indistinguishable from those
released by the wild-type ricin linker or 2S albumin PPII
columns at the resolution provided by this SDS-PAGE
(Figure 7b, compare lanes 1, 3 and 4). These data suggest
that a protein from castor bean, shown to bind the targeting
signals of these endogenous PSV residents in an isoleucine/
leucine-dependent manner, also bound the ssVSS responsible for targeting proaleurain to LV, and thus may be BP-80like. This possibility was investigated by Western blotting.
Figure 7(c) shows immunoblots of gels loaded with column
eluates from castor bean endosperm as above, using a
primary antibody raised against a non-reduced epitope that
spans the ‘unique’ N-terminal domain of pea BP-80
(MAb17F9) (Cao et al., 2000). The lanes loaded with protein
eluted from the linker-, PPII- and proaleurain-affinity columns yielded immunoreactive bands (Figure 7c, empty
arrowheads), whereas there were no detectable immunoreactive bands in the eluates from either column bearing the
mutated propeptides (Figure 7c, lanes 2 and 5). This concurs
both with an earlier biochemical demonstration of the
Figure 4. Ricin and 2S albumin co-localize in the Golgi and PSV.
(a, b) Ultrathin sections of high-pressure frozen castor bean endosperm were
double-immunolabelled with sheep anti-RTA (10 nm gold) and rabbit anti2SA (20 nm gold). Both the Golgi (a) and the PSV (b) are labelled. G, Golgi
complex; PSV, protein storage vacuoles. Bars: 100 nm. Inset in (b): a detail of
the PSV lumen showing decoration by both 10 and 20 nm gold. Bar: 50 nm.
(c) Sections were immunolabelled with rabbit anti-2SA (20 nm gold). Note the
tubular appearance of the ER. This image is representative of 39 observations
of ER morphology. ER, endoplasmic reticulum; CW, cell wall; LB, lipid bodies.
Bar: 100 nm.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x
Vacuolar sorting of ricin and 2S albumin 7
Figure 5. Ricin and 2S albumin are found in large, post-Golgi vesicular structures.
Ultrathin sections of high-pressure frozen castor bean endosperm were immunolabelled with the following antisera:
(a) Sheep anti-RTA (10 nm gold) and rabbit anti-2SA (20 nm gold). Bar: 50 nm.
(b) Sheep anti-RTA (10 nm gold) and rabbit anti-complex glycans (20 nm gold). Inset shows a large vesicle in close proximity to a protein storage vacuole (PSV).
Bars: 50 nm (main panel) and 100 nm (inset).
(c) Rabbit anti-a-TIP. Stars indicate individual, large vesicles. Bar: 200 nm.
inability of these signals to confer vacuolar targeting in
tobacco protoplasts (Brown et al., 2003; Frigerio et al., 2001),
and with the Coomassie-stained gels presented in
Figure 7(b). In order to provide a size marker for BP-80, total
castor bean and pea (Pisum sativum) extracts were immunoblotted with the same antibody. Figure 7(d) shows that a
single immunoreactive band from pea and three to four
bands from castor bean extracts were revealed that migrated
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x
8 Nicholas A. Jolliffe et al.
(a)
Linker:
RTA
1.21
36 2SA
BP-80 (MAb17F9)
PPIIL58G:
STGEEVGRMPGEDNADVC
Proaleurain:
SSSFADSNPIRPVTDRAASTYC
G
58
PP
II
L
Li
nk
er
PP
II
(b)
-98
-64
1.21
clathrin heavy chain
1.21
-36
to
t.
be
an
to
t.
-50
Pe
a
250 -
C
as
to
r
64 -
STGEEVLRMPGEDNADVC
7
Affinity
column:
1.21
SLLGRPVVPNFNADVC
PPII:
12
64 -
SLLIRPVVPNFNADVC
LinkerI271G:
1G
Pr
oa
le
ur
ai
n
55% Antiserum:
Li
nk
er
15%
BiP
64 -
(c)
(d)
-98 -
1.15
Figure 6. A proportion of proricin and proalbumin is found in cellular
fractions enriched in BP-80-like proteins and clathrin.
Total cell homogenates from developing castor bean endosperms were
subjected to isopycnic centrifugation on a linear 15–55% (w/w) sucrose
gradient. Fractions were resolved on reducing SDS-PAGE (with the exception
of the non-reducing SDS-PAGE for the gel to be immunoblotted with anti-BP80) and immunoblotted with the indicated antisera. Numbers on the left
indicate molecular mass markers in kilodaltons. Numbers below selected
gradient fractions indicate fraction density in g ml)1.
within the range of the castor bean proteins eluted from the
ricin and albumin VSS-bearing columns. To corroborate our
findings, we also performed immunoblots using a different
monoclonal antibody (Mab 14G7) that detects the third EGF
repeat in pea BP-80 (Cao et al., 2000). The 14G7 antibody
recognizes one of the castor bean polypeptides around
64 kDa both in the column eluate (Figure 7e, lane 1) and in
total castor bean extracts (Figure 7f). As two different
monoclonal antibodies detect bands in the same size range,
this confirms that the castor bean species are indeed BP-80like. In most gel systems, BP-80 proteins resolve with an
apparent Mr of approximately 80 kDa. The apparent size
discrepancy detected in our experiment is due to our SDSPAGE system, which is optimized for the separation of
storage globulins and uses a 200:1 acrylamide/bisacrylamide ratio (see Experimental procedures for details). Indeed,
when total castor bean homogenates were resolved on
conventional SDS-PAGE, (acrylamide/bisacrylamide ratio of
37:1), immunoblotting with 17F9 revealed a compact band
migrating at around 80 kDa (see Figure S2).
From the data shown in Figure 7 we conclude that the
ssVSS of both ricin and 2S albumin can bind BP-80-like
receptor(s) in vitro. More than one immunoreactive species
is bound to each wild-type peptide column (Figure 7c, empty
arrowheads), with the number and immunoreactivity of
these species differing between columns. This suggests the
presence of multiple VSR isoforms in castor bean endosperm. We focused on the polypeptide that bound to both
-64 -50 -
-36 3
(e)
1
4
5
-98
-64
98 64 -
-50
50 -
-36
36 -
(f)
2
Figure 7. The sorting signals of proricin and proalbumin bind in vitro to BP80-like proteins.
(a) Sequences of the peptides used for in vitro binding experiments.
(b) Clarified membrane extracts from developing castor bean endosperm
were applied to the indicated peptide columns, washed, and eluted at low pH.
Eluates were separated by non-reducing SDS-PAGE and visualized with
Coomassie.
(c) Gels as for (b) were immunoblotted with monoclonal anti-BP80 17F9
antibody. Immunoreactive bands were visualized by ECL. Numbers on the
right indicate molecular mass markers in kilodaltons.
(d) Total extracts from castor bean endosperm and pea cotyledons were
resolved by non-reducing SDS-PAGE and immunoblotted with monoclonal
anti-BP-80 17F9 antibody. Immunoreactive bands were visualized by ECL.
Numbers on the left indicate molecular mass markers in kilodaltons.
(e) Gels as for (b) were immunoblotted with monoclonal anti-BP-80 14G7
antibody. Immunoreactive bands were visualized by ECL.
(f) Total extract from castor bean endosperm was immunoblotted with
monoclonal anti-BP-80 14G7 antibody. Immunoreactive bands were visualized by ECL.
the ricin linker and 2S albumin PPII peptides and that
produced the strongest Coomassie-stained band (Figure 7b,
lane 1, empty arrowhead). After subjecting the excised
polypeptide to tryptic digestion, the peptide mixture was
analysed by nano-electrospray tandem MS. Several multiple
charged peptides were fragmented in order to resolve their
amino acid sequence. Three peptides presented high
sequence similarity to several known members of the VSR
family (Table 1). Specifically, one peptide was identical to
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x
Vacuolar sorting of ricin and 2S albumin 9
Table 1. Sequence similarity of three peptides from the major proricin linker-binding protein to members of the vacuolar sorting receptor
family
Source
Peptide 1
Peptide 2
Peptide 3
Reference
Ricinus communis
Arabidopsis thaliana (AtVSR1/AtELP)
Cucurbita sp. (PV72)
Pisum sativum (BP-80)
Triticum aestivum
LNLPSALLTK
IPSALITK
IPSALISK
IPSALIGK
IPSVLITK
FVGDGYTHC
FVGDGYTHC
FVGDGYTHC
FKGDGYTTC
FVGDGYTNC
CLGDTEADVDN
CIGDPEADVEN
CIGDPEADVEN
CMGDPNADTEN
CVGDPEADEEN
This work
Ahmed et al. (1997)
Shimada et al. (1997)
Kirsch et al. (1994)
Shy et al. (2001)
the Arabidopsis vacuolar sorting receptor AtELP1 (VSRAt-1;
Ahmed et al., 1997) and the remaining two peptides showed
more than 80% sequence identity with the same protein. The
three peptides span the lumenal domain of the receptor
(Table 1). Intriguingly a fourth peptide, WGGYDC, with no
apparent homology to other known VSR, was also identified.
The deposition of both proricin and proalbumin in the PSV
would therefore appear to be a receptor-mediated event, in a
manner at least analogous to CCV-mediated traffic to LV.
Discussion
We have systematically analysed the transport of two major
storage proteins to the PSV of developing castor bean
endosperm. The data presented here not only confirm the
predictions we made whilst studying the fate of ricin and
albumin in tobacco protoplasts (Brown et al., 2003; Frigerio
et al., 1998), but also provide further insights into the sorting
of seed storage proteins in the native tissue. Namely, we
demonstrate a direct link between the targeting of two
storage protein precursors to PSV and the interaction of their
sorting signals with receptors of the VSR family.
In this study, developing castor bean endosperm was
subjected to high-pressure freezing and freeze-substitution,
to prevent any artefactual alterations to structure and
protein location. The immunogold labelling presented here
clearly shows that castor bean proalbumin is present in the
Golgi complex.
Further, EM data reveal that the distribution of 2S
albumin – a non-glycosylated protein – and the previously
characterized glycoprotein, ricin, is identical using these
methods. In addition to observing these storage proteins
together in the PSV, co-localization was also seen in smaller
structures (150–400 nm, Figure 5). These structures could be
labelled with antibodies raised against a-TIP, a PSV marker,
and were frequently found close to PSV. We believe that the
presence of complex glycan-bearing proteins throughout
the lumen of these vesicles is sufficient to distinguish them
from PAC. Indeed, the data presented here suggest that the
entire content of these large vesicles is derived from the
Golgi. Furthermore, throughout our study, ribosomes were
never seen on their delimiting membranes, again indicating
that these structures did not originate directly from the ER.
Moreover, we never observed aggregates within the ER, or
indeed any evidence of budding from this compartment.
Accordingly, these large vesicles should instead be regarded
as early PSV, or possibly pre-vacuolar compartments,
although further work is required to clarify their precise role
in storage protein deposition.
Our data firmly support the idea that in developing castor
bean endosperm, all 2S albumin is trafficked to PSV via the
Golgi apparatus. The co-localization of 2S albumin and ricin
throughout the secretory pathway indicates that both proteins follow the same route to PSV. Indeed, the evidence
presented here formally rationalizes, in the native tissue, our
previous demonstration that both proteins possess ssVSS
(Brown et al., 2003; Frigerio et al., 2001). A family of VSR has
now been characterized, of which pea BP-80 (Kirsch et al.,
1994) is the prototype. Members of this family are integral
membrane proteins which bind with high specificity to NPIRlike vacuolar sorting signals at the trans-Golgi level and
recruit clathrin coats through an adaptin-binding tyrosine
motif present in their cytosolic tail (Paris and Neuhaus,
2002). VSR have been shown to exist in pea, maize, rice,
pumpkin and Arabidopsis (Paris and Neuhaus, 2002). In
accordance with this model, castor bean proteins recognized
by antibodies raised against BP-80 and clathrin heavy chain
co-localize with the precursors of both ricin and 2S albumin
when resolved on a sucrose gradient (Figure 6). Furthermore, the vacuolar sorting signals of both ricin and 2S
albumin bind in a sequence-specific manner to a BP-80-like
protein. The demonstration of a specific interaction with a
sorting receptor, and co-localization of ricin and 2S albumin
with this receptor and with a clathrin coat protein, may
explain the apparent paradox of two bona fide PSV residents
possessing ssVSS.
Although VSR have been previously implicated in the
transport of other members of the albumin family (Kirsch
et al., 1996; Shimada et al., 1997, 2002; Watanabe et al.,
2002), the roles of the peptides used to bind the receptors
in vitro were not always investigated fully in vivo. Here we
have clearly shown that the well-characterized VSS of two
castor bean storage proteins (Brown et al., 2003; Frigerio
et al., 2001; Jolliffe et al., 2003) interact with a novel member
of the recognized VSR family, thus adding R. communis to
the growing list of plants in which these receptors have been
identified, and fuelling the debate as to the breadth of their
function. Indeed a recent report by Shimada et al. (2003) has
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x
10 Nicholas A. Jolliffe et al.
shown that genetic knockout of one of the seven Arabidopsis
isoforms of VSR, namely VSRat-1, led to partial secretion of
the precursors of all major classes of storage proteins into
the apoplast of mature Arabidopsis seeds. This new finding
further strengthens the link between VSR-mediated transport pathways and the sorting of proteins to the PSV
in seeds. Together, these findings potentially call for a
re-evaluation of the working model for seed storage protein
deposition and for the role of VSR in this process. In this
respect it will be important to characterize the transport of
the remaining class of storage proteins – the 11S globulins –
to the PSV of Ricinus endosperm.
Experimental procedures
Plant material
Ricinus communis L. plants were grown from seed in a greenhouse at 15C and under a 16 h/8 h light/dark cycle. Endosperm
tissue was excised from the ripening seeds during testa formation
(typically 8 weeks after flowering) at a developmental stage when
the lectins and storage proteins are rapidly synthesized (Lord,
1985b).
Antisera
The following antibodies were used for biochemical analysis and
immunoelectron microscopy: mouse monoclonal anti-clathrin
heavy chain (BD Biosciences, Oxford, UK), mouse monoclonal antiBP-80 antibody (MAb17F9, Cao et al., 2000); rabbit polyclonal antibean a-TIP (Johnson et al., 1990); rabbit polyclonal anti-tobacco BiP
(Pedrazzini et al., 1997), rabbit anti-recombinant ricin A-chain (antiRTA, Frigerio et al., 1998); sheep anti-recombinant RTA, rabbit
polyclonal anti-castor bean 2S albumin (anti-2SA); rabbit polyclonal
anti-complex glycans (Lainé et al., 1991).
2S albumin antibody purification
Crude rabbit polyclonal antisera raised against 2S albumin (Brown
et al., 2003) was purified to remove contaminating antibodies
recognizing ricin agglutinins by passage through a column bearing
1 ml of agarose-bound R. communis agglutinin II (Vector Laboratories, Peterborough, UK). The column was washed with PBS containing 0.1 M lactose, and the antisera applied in 0.1 M lactose to
prevent interaction of antibody glycans with the lectin subunit of the
immobilized ricin. Flow-through fractions, which contained the anti2SA antibodies, were collected.
Sucrose gradient fractionation
Endosperm isolated from maturing castor bean seeds was homogenized in an ice-cold mortar with ice-cold 100 mM Tris-Cl, pH 7.8,
10 mM KCl, containing 12% (w/w) sucrose and 2 mM MgCl2, using
6 ml of buffer per gram of fresh leaf tissue. The homogenate was
centrifuged for 10 min at 1000 g at 4C; 600 ll of the supernatant
was loaded on a 11-ml linear 15–55% (w/w) sucrose gradient. After
centrifugation at 35 000 rpm, 4C for 2 h in a Beckman SW40 rotor
(Beckman Coulter, High Wycombe, UK) (154 400 g average), fractions of 600 ll were collected. Immunoblot analysis of 60 ll aliquots
of these fractions was performed as described (Pedrazzini et al.,
1997), using the indicated antisera.
Electrophoretic separation of proteins
Proteins were separated by sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis using the method described by
Laemmli (1970), and using an acrylamide-bisacrylamide ratio in the
separating gel of 200:1, which has been found to be optimal for the
resolution of certain storage globulins (Bollini and Chrispeels,
1979).
Affinity chromatography
A 2.5 ml volume [2 mg ml)1 in coupling buffer (50 mM Tris;
5 mM EDTA, pH 8.5)] of sulphydryl-containing peptides (synthesized by Alta Biosciences, University of Birmingham, UK)
were immobilized on ‘SulfoLink’ coupling gel columns (Pierce,
Perbio Science UK, Tattenhall, UK), and the non-specific binding
sites blocked, according to the manufacturer’s instructions.
Endosperm was homogenized in a Petri dish on ice by continuous chopping for 15 min using a hand-held razor blade, after
addition of cold grinding buffer (150 mM Tricine, pH 7.5; 1 mM
EDTA-Na, pH 7.5; 10 mM KCl; 1 mM MgCl2; 100 mM lactose),
supplemented immediately before use with ‘complete’ protease
inhibitor cocktail (Roche Diagnostics, Lewes, UK). Pea homogenates were prepared using an ice-cold pestle and mortar by
grinding in the same buffer. The resulting suspensions were filtered through four layers of mira cloth, and centrifuged at
10 000 g for 15 min at 4C. The floating lipid layers were discarded and the underlying supernatants carefully removed to a
clean tube. Membranes were pelleted from post-mitochondrial
supernatants by centrifugation in 3 ml thick-walled polycarbonate
tubes at 100 000 g (Beckman TL-100 ultracentrifuge, TLA-100.3
rotor) at 4C for 30 min, and the supernatant discarded. Pellets
were resuspended in CHAPS buffer (Kirsch et al., 1994: 20 mM
Hepes NaOH, pH 7.1; 150 mM NaCl; 1 mM MgCl2; 1 mM CaCl2;
1% (w/v) 3-[(3-cholramidopropyl) dimethylammonio]-1-propanesulphonate (CHAPS)], and incubated at room temperature for
30 min to solubilize integral membrane proteins. Residual
membranes were removed by re-centrifugation of the same
tubes, as in the first step, and the supernatant was carefully
removed to a clean tube. Two-millilitre aliquots of membrane
protein extracts were applied to the affinity columns, previously
brought to room temperature and equilibrated according to the
manufacturer’s instructions. Column gel beds were resuspended,
and incubated with gentle agitation for 30 min, before being
allowed to settle for 30 min. Columns were washed according to
the manufacturer’s instructions. Bound proteins were released by
addition of 10 ml elution buffer [25 mM NaOAc, pH 4.0; 150 mM
NaCl; 1 mM EGTA; 1% (w/v) CHAPS], and the eluate collected in
1 ml aliquots. Protein was precipitated from fractions by the
addition of an equal volume of cold 30% (w/v) TCA. After mixing,
the sample was incubated on ice for a minimum of 30 min. The
precipitant was harvested by centrifugation at 17 000 g for
15 min at 4C, the supernatant discarded, and the pellet washed
twice with 1 ml of cold acetone, centrifuging between each wash
for 5 min as before. The acetone-washed pellet was then dried in
a vacuum desiccator, resuspended in non-reducing loading
buffer, and separated by SDS-PAGE. Subsequent protein detection was either by Coomassie-blue staining, or immunoblot as
described (Pedrazzini et al., 1997), using monoclonal BP-80 antibody.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x
Vacuolar sorting of ricin and 2S albumin 11
Peptide sequencing
The band of interest was excised from a Coomassie-stained gel and
digested overnight with trypsin as described (Anelli et al., 2002).
The unseparated peptide mixture was concentrated and desalted
over a capillary column packed with POROS R2 material and eluted
directly into the electrospray needle. Nanoelectrospray tandem MS
experiments were performed on a Q-Star Pulsar (PE SCIEX Instruments, Toronto, Canada). Several multiple charged peptides were
fragmented in order to read the amino acid sequence. One peptide
(FVGDGYTHCK) was identical to AtELP (Accession number
sptrembl: P93026). Two other sequenced peptides, namely CIGDTEADVDNPVLK and LNIPSALITK, showed high homology to
CIGDPEADVENPVLK and IPSALITK, also present in P93026.
Transmission electron microscopy
High-pressure freezing. Samples for transmission electron
microscopy observation were chosen from castor bean endosperm
at the testa maturation stage. Thin slices of endosperm (<0.5 mm)
were dissected, then 2 mm diameter discs were cut out of the slices
using a disposable biopsy punch (Stiefel Laboratories Ltd, Wooburn
Green, UK), and placed in an aluminium sample holder. Prior to
capping the sample holder, the sample was covered with hexadecene in order to remove any remaining air between the sample
and the holder. Pairs of holders were clamped together, and samples were immediately frozen using a BAL-TEC HPM 010 highpressure freezer (BAL-TEC AG, Balzers, Liechtenstein).
Freeze-substitution
Freeze-substitution was carried out in a Reichert AFS (Leica, Vienna,
Austria) freeze-substitution system. Sample holders were split open
under liquid nitrogen and placed into plastic porous specimen pots
containing the substitution medium previously frozen in liquid
nitrogen. For standard ultrastructural observations, 2% osmium
tetroxide in acetone dried over molecular sieve was used. Plastic
specimen pots were put into an universal aluminium container onto
the surface of the frozen substitution medium and transferred into
the Reichert AFS pre-cooled to )160C. Sample temperature was
increased to )85C over 5 h. Freeze-substitution was carried out as
follows: )85C for 26 h, 2C h)1 temperature increase over 12.5 h,
)60C for 8 h, 2C h)1 temperature increase over 15 h, )30C for 9 h
(Steinbrecht and Müller, 1987; Studer et al., 2001). At the end of the
freeze-substitution run, samples were warmed up to 20C with a
temperature increase of 4C h)1. All subsequent steps were carried
out at room temperature.
For immunogold labelling, samples were subjected to the same
treatment except that the freeze-substitution medium was 0.5%
uranyl acetate in ethanol dried over molecular sieve and after the
freeze substitution run, samples were warmed up to )20C with a
temperature increase of 1C h)1. All subsequent steps were carried
out at this temperature.
stepwise in LR White medium resin (Agar Scientific, Stansted, UK)
over 1 week. Polymerization was under UV light for 24 h at )20C
and for another 24 h at 0C.
Ultrathin sections were cut with an RMC MTXL ultramicrotome
(Boeckeler Instruments, Tucson, AZ, USA) and collected on formvar-coated 300 mesh hexagon copper grids (Agar Scientific).
Immunogold labelling, staining and observation
For immunogold labelling, sections were first blocked for 30 min
with a 1:30 dilution of pre-immune serum in TRIS (20 mM TRIS,
15 mM NaN3, 225 mM NaCl), pH6.9, supplemented with 1% BSA
(TRIS-BSA). They were then treated with 0.1% Tween20 in TRIS-BSA
and 0.02 M glycine in TRIS-BSA, each for 15 min, washed three
times for 10 min in TRIS-BSA, and incubated in a dilution of primary
antibody at 4C overnight. The following primary antibodies were
used in this study: anti-a-TIP (dilution 1:1000, 1:2000), rabbit antiRTA (dilution 1:200), sheep anti-RTA (dilution 1:2000, 1:4000), rabbit
anti-2SA (dilution 1:2000, 1:8000), rabbit anti-complex glycans
(dilution 1:2000, 1:4000).
After washing in TRIS-BSA (three times 15 min), sections were
treated with a 1:20 dilution (in TRIS-BSA) of the corresponding
secondary antibodies (goat anti-rabbit or donkey anti-sheep) conjugated to 10 nm gold (BBInternational, Cardiff, UK) for 1.5 h at
room temperature. Finally, after thorough washing in TRIS-BSA and
ultrapure water, sections were dried on filter paper. Occasionally,
1% fish gelatin (Sigma, Dorset, UK) was added to the primary and
secondary antibody solutions as well as to the TRIS-BSA washings
as an additional blocking agent.
For simultaneous double immunogold labelling, the same protocol was applied except that sections were exposed to mixtures of
pre-immune serum (both at a 1:30 dilution in TRIS-BSA) and to
mixtures of the primary antibodies (for dilutions see above).
Secondary antibodies were also used as a mixture (both at a 1:20
dilution in TRIS-BSA) with one conjugated to 10 nm gold particles
and the other one to 20 nm gold particles (see Figures 3–5).
For controls of both single and double immunogold labelling,
samples were subjected to the same treatment except that the
corresponding pre-immune serum substituted the primary antibody.
All sections were stained with 2% uranyl acetate in 70% ethanol
for 15 min, followed by lead citrate for 15 min, and examined with a
JEOL JEM-1200EXII (JEOL UK, Hertfordshire, UK) at 80 or 120 kV.
Acknowledgements
We thank Barry Martin for technical assistance with high-pressure
freezing and Angela Cattaneo for tandem MS peptide sequencing.
We are grateful to Martin Chrispeels for the gift of the anti-a-TIP
antiserum and John Rogers for the 17F9 and 14G7 MAbs. This work
was supported by the BBSRC (grants 88/C17404 to LF/LMR, C15728
to CH and a studentship to JCB).
Supplementary material
Embedding and sectioning
Samples for standard ultrastructural observations were rinsed in
anhydrous acetone, carefully removed from the sample holders,
embedded stepwise in Spurr’s medium resin (TAAB Laboratories
Equipment Limited, Aldermaston, Berkshire, UK) (Spurr, 1969), and
polymerized at 70C for 9 h.
Samples for immunogold labelling were rinsed in cold ethanol
and, after careful removal from the sample holders, embedded
The following material is available from http://www.
blackwellpublishing.com/products/journals/suppmat/TPJ/TPJ2166/
TPJ2166sm.htm
Table S1 Analysis of background immunogold labelling
Sets of electron micrograph negatives were digitalized and the
areas of Golgi stacks, protein storage vacuoles (PSV), and surrounding cytoplasm were calculated using the ImageJ analysis
package (publicly available at http://www.rsb.info.nih.gov/ij/
index.html). Labelling of the Golgi and/or PSV was compared with
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x
12 Nicholas A. Jolliffe et al.
background labelling and is shown as the average number of gold
particles per square micrometre standard deviation. (*) number
of gold particles for a-TIP was calculated for the areas of both Golgi
and PSV
Figure S1 Gel mobility of castor bean VSR-like proteins. Total
extract from castor bean endosperm was resolved by SDS-PAGE
either using either standard (37:1) or 200:1 acrylamide/bisacrylamide ratio and immunoblotted with monoclonal anti-BP-80 17F9
antibody. Immunoreactive bands were visualized by ECL. Numbers
on the left indicate molecular mass markers in kilodaltons. Note the
gel-type-dependent difference in mobility of the immunoreactive
polypeptides.
References
Ahmed, S.U., Bar-Peled, M. and Raikhel, N.V. (1997) Cloning and
subcellular location of an Arabidopsis receptor-like protein that
shares common features with protein-sorting receptors of eukaryotic cells. Plant Physiol. 114, 325–336.
Anelli, T., Alessio, M., Mezghrani, A., Simmen, T., Talamo, F., Bachi,
A. and Sitia, R. (2002) ERp44, a novel endoplasmic reticulum
folding assistant of the thioredoxin family. EMBO J. 21, 835–844.
Bassham, D. C. and Raikhel, N. V. (2000) Unique features of the plant
vacuolar sorting machinery. Curr. Opin. Cell Biol. 12, 491–495.
Bollini, R. and Chrispeels, M.J. (1979) The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing
Phaseolus vulgaris cotyledons. Planta, 146, 487–501.
Brown, J.C., Jolliffe, N.A., Frigerio, L. and Roberts, L.M. (2003)
Sequence-specific, Golgi-dependent targeting of the castor bean
2S albumin to the vacuole in tobacco protoplasts. Plant J. 36, 711–
719.
Cao, X., Rogers, S.W., Butler, J., Beevers, L. and Rogers, J.C. (2000)
Structural requirements for ligand binding by a probable plant
vacuolar sorting receptor. Plant Cell, 12, 493–506.
Frigerio, L., Vitale, A., Lord, J.M., Ceriotti, A. and Roberts, L.M.
(1998) Free ricin A chain, proricin and native toxin have different
cellular fates when expressed in tobacco protoplasts. J. Biol.
Chem. 273, 14194–14199.
Frigerio, L., Jolliffe, N.A., Di Cola, A., Hernández Felipe, D., Paris, N.,
Neuhaus, J.-M., Lord, J.M., Ceriotti, A. and Roberts, L.M. (2001)
The internal propeptide of the ricin precursor carries a sequencespecific determinant for vacuolar sorting. Plant Physiol. 126, 167–
175.
Fukusawa, T., Hara-Nishimura, I. and Nishimura, M. (1988) Biosynthesis, intracellular transport and in vivo processing of 11S
globulin precursor proteins of developing castor bean endosperm. Plant Cell Physiol. 29, 339–345.
Fulton, R.J., Blakey, D.C., Knowles, P.P., Uhr, J.W., Thorpe, P.E. and
Vitetta, E.S. (1986) Purification of ricin A1, A2, and B chains and
characterization of their toxicity. J. Biol. Chem. 261, 5314–5319.
Gietl, C. and Schmid, M. (2001) Ricinosomes: an organelle for
developmentally regulated programmed cell death in senescing
plant tissues. Naturwissenschaften, 88, 49–58.
Gifford, D., Greenwood, J. and Bewley, J. (1982) Deposition of
matrix and crystalloid storage proteins during protein body
development in the endosperm of Ricinus communis L. - cv Hale
seeds. Plant Physiol. 69, 1471–1478.
Hara-Nishimura, I., Shimada, T., Hatano, K., Takeuchi, Y. and Nishimura, M. (1998) Transport of storage proteins to protein storage vacuoles is mediated by large precursor-accumulating
vesicles. Plant Cell, 10, 825–836.
Hillmer, S., Movafeghi, A., Robinson, D.G. and Hinz, G. (2001) Vacuolar storage proteins are sorted in the cis-cisternae of the pea
cotyledon Golgi apparatus. J. Cell Biol. 152, 41–50.
Irwin, S.D., Keen, J.N., Findlay, J.B.C. and Lord, J.M. (1990) The
Ricinus communis 2S albumin precursor: a single preproprotein
may be processed into two different heterodimeric storage proteins. Mol. Gen. Genet. 222, 400–408.
Jauh, G.-Y., Phillips, T.E. and Rogers, J.C. (1999) Tonoplast intrinsic
protein isoforms as markers for vacuolar functions. Plant Cell, 11,
1867–1882.
Johnson, K.D., Höfte, H. and Chrispeels, M.J. (1990) An intrinsic
tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GlpF). Plant Cell, 2,
525–532.
Jolliffe, N.A., Ceriotti, A., Frigerio, L. and Roberts, L.M. (2003) The
position of the proricin vacuolar targeting signal is functionally
important. Plant Mol. Biol. 51, 631–641.
Kirsch, T., Paris, N., Butler, J.M., Beevers, L. and Rogers, J.C. (1994)
Purification and initial characterization of a potential plant
vacuolar targeting receptor. Proc. Natl Acad. Sci. USA, 91, 3403–
3407.
Kirsch, T., Saalbach, G., Raikhel, N. V. and Beevers, L. (1996) Interaction of a potential vacuolar targeting receptor with amino- and
carboxyl-terminal targeting determinants. Plant Physiol. 111,
469–474.
Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature, 227, 680–685.
Lainé, A.-C., Gomord, V. and Faye, L. (1991) Xylose-specific antibodies as markers of subcompartmentation of terminal glycosylation in the Golgi apparatus of sycamore cells. FEBS Lett. 295,
179–184.
Lord, J.M. (1985a) Precursors of ricin and Ricinus communis
agglutinin. Glycosylation and processing during synthesis and
intracellular transport. Eur. J. Biochem. 146, 411–416.
Lord, J.M. (1985b) Synthesis and intracellular transport of lectin and
storage protein precursors in endosperm from castor bean. Eur.
J. Biochem. 146, 403–409.
Lord, J. and Harley, S. (1985) Ricinus communis agglutinin B-chain
contains a fucosylated oligosaccharide side-chain not present on
ricin B-chain. FEBS Lett. 189, 72–76.
Matsuoka, K. and Neuhaus, J.-M. (1999) Cis-elements of protein
transport to the plant vacuoles. J. Exp. Bot. 50, 165–174.
Pantoja-Uceda, D., Bruix, M., Gimenez-Gallego, G., Rico, M. and
Santoro, J. (2003) Solution structure of RicC3, a 2S albumin
storage protein from Ricinus communis. Biochemistry, 42,
13839–13847.
Paris, N. and Neuhaus, J.M. (2002) BP-80 as a vacuolar sorting
receptor. Plant Mol. Biol. 50, 903–914.
Pedrazzini, E., Giovinazzo, G., Bielli, A., de Virgilio, M., Frigerio, L.,
Pesca, M., Faoro, F., Bollini, R., Ceriotti, A. and Vitale, A. (1997)
Protein quality control along the route to the plant vacuole. Plant
Cell 9, 1869–1880.
Sanderfoot, A.A., Ahmed, S.U., Marty-Mazars, D., Rapoport, I.,
Kirchhausen, T., Marty, F. and Raikhel, N.V. (1998) A putative
vacuolar cargo receptor partially colocalizes with AtPEP12p on a
prevacuolar compartment in Arabidopsis roots. Proc. Natl Acad.
Sci. USA, 95, 9920–9925.
Schmid, M., Simpson, D.J., Sarioglu, H., Lottspeich, F. and Gietl, C.
(2001) The ricinosomes of senescing plant tissue bud from the
endoplasmic reticulum. Proc. Natl Acad. Sci. USA, 98, 5353–
5358.
Shimada, T., Kuroyanagi, M., Nishimura, M. and Hara-Nishimura, I.
(1997) A pumpkin 72-kDa membrane protein of precursor accumulating vesicles has characteristics of a vacuolar sorting
receptor. Plant Cell Physiol. 38, 1414–1420.
Shimada, T., Watanabe, E., Tamura, K., Hayashi, Y., Nishimura, M.
and Hara-Nishimura, I. (2002) A vacuolar sorting receptor PV72 on
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x
Vacuolar sorting of ricin and 2S albumin 13
the membrane of vesicles that accumulate precursors of seed
storage proteins (PAC vesicles). Plant Cell Physiol 43, 1086–1095.
Shimada, T., Fuji, K., Tamura, K., Kondo, M., Nishimura, M. and
Hara-Nishimura, I. (2003) Vacuolar sorting receptor for seed
storage proteins in Arabidopsis thaliana. Proc. Natl Acad. Sci.
USA, 100, 16095–16100.
Shy, G., Ehler, L., Herman, E. and Galili, G. (2001) Expression
patterns of genes encoding endomembrane proteins support
a reduced function of the Golgi in wheat endosperm during
the onset of storage protein deposition. J Exp Bot 52, 2387–
2388.
Spurr, A.R. (1969) A low-viscosity epoxy resin embedding medium
for electron microscopy. J. Ultrastruct. Res. 26, 31–43.
Steinbrecht, R. and Müller, M. (1987) Freeze-substitution and freezedrying. in: Freeze-substitution and freeze-drying, Springer Verlag,
Berlin, 149–172.
Studer, D., Graber, W., Al-Amoudi, A. and Eggli, P. (2001) A new
approach for cryofixation by high-pressure freezing. J. Microsc.
203, 285–294.
Tully, R.E. and Beevers, H. (1976) Protein bodies of castor bean
endosperms. Isolation, fractionation and characterisation of
protein components. Plant Physiol. 58, 710–716.
Vitale, A. and Raikhel, N. V. (1999) What do proteins need to reach
different vacuoles? Trends Plant Sci. 4, 149–155.
Watanabe, E., Shimada, T., Kuroyanagi, M., Nishimura, M. and
Hara-Nishimura, I. (2002) Calcium-mediated association of a
putative vacuolar sorting receptor PV72 with a propeptide of 2S
albumin. J Biol Chem 277, 8708–8715.
Youle, R.J. and Huang, A.H.C. (1976) Protein bodies from the
endosperm of castor bean. Subfractionation, protein components, lectins and changes during germination. Plant Physiol. 58,
703–709.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), doi: 10.1111/j.1365-313X.2004.02167.x