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Plant Physiol. (1999) 119: 1437-1446
Arabidopsis Sec21p and Sec23p Homologs. Probable Coat Proteins of
Plant COP-Coated Vesicles1
Ali Movafeghi,
Nicole Happel,
Peter Pimpl,
Gui-Hua Tai, and
David
G. Robinson*
Abteilung Strukturelle Zellphysiologie, Albrecht-von-Haller
Institut für Pflanzenwissenschaften, Universität
Göttingen, Untere Karspüle 2, D-37073 Göttingen,
Germany
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ABSTRACT |
Intracellular protein transport
between the endoplasmic reticulum (ER) and the Golgi apparatus and
within the Golgi apparatus is facilitated by COP (coat
protein)-coated vesicles. Their existence in plant cells
has not yet been demonstrated, although the GTP-binding proteins
required for coat formation have been identified. We have generated
antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p
homologs (AtSec21p and AtSec23p, respectively). The former is a
constituent of the COPI vesicle coatomer, and the latter is part of the
Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower
(Brassica oleracea) inflorescence homogenates were
probed with these antibodies and demonstrated the presence of AtSec21p
and AtSec23p antigens in both the cytosol and membrane fractions of the
cell. The membrane-associated forms of both antigens can be solubilized
by treatments typical for extrinsic proteins. The amounts of the
cytosolic antigens relative to the membrane-bound forms increase after
cold treatment, and the two antigens belong to different protein
complexes with molecular sizes comparable to the corresponding nonplant
coat proteins. Sucrose-density-gradient centrifugation of microsomal
cell membranes from cauliflower suggests that, although AtSec23p seems
to be preferentially associated with ER membranes, AtSec21p appears to
be bound to both the ER and the Golgi membranes. This could be in
agreement with the notion that COPII vesicles are formed at the ER,
whereas COPI vesicles can be made by both Golgi and ER membranes. Both
AtSec21p and AtSec23p antigens were detected on membranes equilibrating
at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.
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INTRODUCTION |
Protein transport between the various organelles of the
endomembrane system and the plasma membrane is facilitated by vesicles, many if not all of which possess a protein coat at their cytosolic surface. The first type of coated vesicle to be described was the CCV,
which mediates transport between the plasma membrane and endosomes and
between the trans-Golgi network and the lytic compartments
of the cell (Schmid and Damke, 1995 ; Brodsky, 1997). A different kind
of coated vesicle, discovered in the 1980s as a result of in vitro
studies of the intra-Golgi transport of VSV-G protein and
immunologically distinct from CCV, was the COPI (coat protein I)-coated vesicle (Malhotra et al.,
1989). The components of the coat were later shown to be ARF, a
GTP-binding protein (Serafini et al., 1991), and a heptameric protein
complex called the coatomer (Waters et al., 1991). COPII-coated
vesicles were obtained when yeast ER was incubated in the presence of a
set of five proteins (the GTP-binding protein Sar1p and two
heterodimeric complexes, Sec23/24p and Sec13/31p), GTP, and an
ATP-regenerating system (Barlowe et al., 1994).
CCVs have been recognized in plants for some time. The components of
their coat and their location and function appear to be similar to
those of their counterparts in other eukaryotic cells (Beevers, 1996;
Robinson et al., 1998). In contrast, the evidence for COP-coated
vesicles in plants has remained more conjectural (Staehelin and Moore,
1995; Andreeva et al., 1998b). Except for some algae, such as
Chlamydomonas reinhardtii, in which the ER and the Golgi
apparatus are close together (e.g. Luykx et al., 1997), vesicle-budding
events at the ER in plants are in general difficult to visualize with
the electron microscope (Hawes et al., 1996). Nevertheless, attempts at
generating and isolating ER-derived transport vesicles from higher
plants have been carried out (e.g. Hellgren et al., 1993), albeit
without reference to possible coat proteins. In addition to CCV at the
trans pole, several different types of Golgi-derived
vesicles have been described in higher plants. Some are large,
apparently smooth surfaced, and sometimes with structured contents,
e.g. the slime-containing vesicles of root-cap cells (Mollenhauer and
Morré, 1991) and the storage-protein-containing "dense"
vesicles in developing seed tissues (Robinson et al., 1997, 1998).
Others are small ( 70 nm in diameter) and are usually found at the
peripheries of cis and medial cisternae. When properly fixed
and stained, these small vesicles appear to be coated (see Fig. 2 in
Hawes et al., 1996), but without the appropriate immunogold-labeling
data it remains unclear whether they might represent COP-coated
vesicles.
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| Figure 2.
A, GTP S does not redistribute membrane-bound
and cytosolic AtSec21p and AtSec23p antigens. Equal amounts of
cauliflower inflorescence (10 g) were homogenized in 10 mL of HDKE 10 buffer in the presence or absence of 50 µM GTPS.
Microsomal membranes (m) and cytosolic fractions (c) were isolated as
described in ``Materials and Methods''. The pATPase was used as a
standard intrinsic membrane antigen (20 µg of protein per lane). B,
Cold temperature causes a displacement of AtSec21p and AtSec23p
antigens from the membrane to the cytosolic compartment of the cell.
Whole cauliflower plants were held in a cold room for 14 h before
microsomal membranes and cytosolic fractions were extracted from the
inflorescence (20 µg of protein per lane).
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Two indirect lines of evidence support the existence of COP-coated
vesicles in plants. First, the fungal metabolite brefeldin A is known
to prevent ARF binding in mammalian cells (Dascher and Balch, 1994),
thereby preventing coatomer assembly and the formation of COP-coated
vesicles (Orci et al., 1991). As a consequence, the cisternae of the
Golgi apparatus fuse with one another, tubularize, and are absorbed
into the ER (Klausner et al., 1992). Brefeldin A also targets the Golgi
apparatus in plants, with similar but not identical morphological
effects (Satiat-Jeunemaitre et al., 1996). Second, genes homologous to
ARF and Sar1 have been identified from a number
of higher plants (d'Enfert et al., 1992; Regad et al., 1993; Bar-Peled
and Raikhel, 1997). cDNAs corresponding to other COP coat
proteins were identified in a search of the expressed sequence
tag database (Andreeva et al., 1998b). Finally, AtSec12p, a Sec12p
homolog in Arabidopsis that is an integral ER protein required for
COPII-coated vesicle production in yeast (Nakano et al., 1988), has
been described (d'Enfert et al., 1992; Bar-Peled and Raikhel,
1997). Thus, at the gene level and in terms of sensitivity toward brefeldin A, plants seem to possess the capacity for COP-coated vesicle production.
To explore this possibility further, we generated antisera against
AtSec21p, the Arabidopsis homolog to Sec21p (-COP, a 100-kD subunit
of the coatomer), and AtSec23p, the Arabidopsis homolog to Sec23p (an
85-kD component of the COPII coat), and have probed subcellular
fractions with them. In addition to the binding of AtSec21p and
AtSec23p to Golgi and ER fractions, we report a high-density fraction
that may contain a population of endogenous COP-coated vesicles. Our
experimental organism is the growing inflorescence of cauliflower
(Brassica oleracea), which is genetically closely related to
Arabidopsis (Dozolme et al., 1995), is readily available in large
quantities, and possesses relatively low levels of endogenous proteases, which is advantageous for the detection and characterization of cytosolic coat proteins.
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MATERIALS AND METHODS |
Materials
Cauliflower (Brassica oleracea L. var
botrytis) was grown in a greenhouse. Inflorescences, roughly
10 to 15 cm in diameter, were excised and the woody stalk tissue was
removed. Whole plants were used for all experiments, even for cold
treatments overnight in a refrigerated room at 4°C. Arabidopsis
cell-suspension cultures were established from leaf calli derived from
seedlings grown under sterile conditions and maintained in the log
phase by weekly subculturing into fresh Gamborg's B5 medium (3.86 g
L 1; Sigma G5893) containing 20 g
L1 Glc, 0.5 g L1
Mes, 0.5 mg L1 2,4-D, and 50 µg
L1 kinetin. The cultures were maintained at
24°C on an orbital shaker (110 rpm). Bakers' yeast
(Saccharomyces cerevisiae cv Vital Gold) was purchased
locally (Deutsche Hefewerke, Hamburg, Germany) and taken into
suspension culture at 28°C in Sabouraud's Glc broth (Campbell,
1988). Porcine brain from freshly slaughtered animals was obtained from
a local slaughterhouse and brought to the laboratory under refrigerated
conditions.
Generation and Purification of GST-Fusion Proteins and Preparation
of Antisera
Arabidopsis cDNAs homologous to SEC21 (accession no.
T75984) and SEC23 (accession no. T04245) were obtained from
the Arabidopsis Biological Resource Center (Ohio State University, Columbus) and were cloned by standard procedures into the
SalI-NotI site of the pGEX-4T-3 vector
(Pharmacia). These cDNAs have greater than 50% homology to their yeast
counterparts in the first 200 bp (Robinson et al., 1998). The Sec21
cDNA clone used here corresponds exactly to a fragment of the recently
described full-length sequence for an Arabidopsis genomic sequence
homologous to SEC21 (accession no. AL023094).
Escherichia coli (strain BL21) was transformed with the
ligation product, and cultures containing 100 µg
mL1 ampicillin in Luria-Bertani medium were
grown at 37°C until an A600 of 0.8 was
reached.
Expression of the GST-fusion constructs was then induced by adding
isopropylthio- -D-galactopyranoside (final concentration, 0.2 mM). The temperature was reduced to 28°C, and after
3 h the cells were collected by centrifugation and resuspended in
cold PBST buffer (16 mM
Na2HPO4, 4 mM
NaH2PO4, 150 mM
NaCl, and 1% [v/v] Triton X-100, pH 7.3) containing 0.1%
mercaptoethanol and 0.1 mM PMSF. The cells were lysed in a
sonifier (Branson Ultrasonics, Danbury, CT) operating at 300 W four
times for 30 s each, and the resulting homogenate was centrifuged
at 12,000g for 30 min. The supernatants were mixed with 50%
glutathione-Sepharose beads (Pharmacia) that had been
equilibrated with PBST and shaken at 4°C for 30 min. The beads
were sedimented and washed three times with PBST and twice with 50 mM Tris-HCl, pH 8.0. The fusion proteins were
eluted from the beads with 50 mM Tris-HCl
containing 10 mM GSH and were separated by
SDS-PAGE. After electroelution and dialysis three times for 12 h
each against 10 mM Tris-HCl, pH 7.5, the proteins
were lyophilized. Polyclonal antibodies in rabbits were prepared
commercially (Eurogentec, Seraing, Belgium; Biogentec, Berlin,
Germany).
Preparation of Microsomal Membrane and Cytosol Fractions
Equal weights of cauliflower floret tissue, Arabidopsis cells, pig
brain tissue, and HDKE 10 buffer (40 mM Hepes-KOH, pH 8.0, 1 mM DTT, 10 mM KCl, and 3 mM EDTA)
containing 0.32 M Suc and protease inhibitors (2 µg
mL1 aprotinin, 0.5 µg
mL1 leupeptin, 2 µM pepstatin, 2 mM o-phenanthroline, and 1 µg
mL1
trans-epoxysuccinyl-L-leucylamido-[4-guanidino]butane)
were placed in a blender (Waring) and homogenized three times in 15-s
bursts, and the slurry was passed through a single layer of Miracloth (Calbiochem). After precentrifugation at 18,000g for 20 min,
microsomal membranes were sedimented by centrifugation at
100,000g for 1 h. Yeast cells were homogenized in HDKE
10 buffer, pH 7.5, with Suc and protease inhibitors in a cell mill
(Vibrogen, Bühler, Tübingen, Germany). Cytosol was prepared
by passing an aliquot of the 100,000g supernatant through a
Sephadex G-25 column, equilibrating with HDKE 10 buffer, and collecting
the fractions constituting the void volume.
Fractionation of Cauliflower Cytosol
Floret tissue (25 g) was homogenized in a blender in 25 mL of TDKE
500 buffer (25 mM Tris-HCl, pH 8.0, 1 mM DTT,
500 mM KCl, and 3 mM EDTA) containing a
cocktail of protease inhibitors (see above). After passing through a
single layer of Miracloth, the homogenate was precentrifuged at
18,000g for 20 min and then centrifuged at
100,000g for 60 min. The protein concentration in the
supernatant (cytosol) was adjusted to 5 mg mL1
by appropriate dilution with TDKE 500 buffer, and
(NH4)2SO4
(final concentration, 40% saturated) was added. After being stirred
for 30 min, the precipitated proteins were recovered by centrifugation at 20,000g for 20 min and redissolved in 2 mL of TDKE 150 buffer (150 mM KCl) before being applied to a Sepharose
CL-6B gel-filtration column (1 × 110 cm) and eluted with TDKE 150 buffer at a speed of 0.1 mL min1.
Two-milliliter fractions were collected, from which 100-µL aliquots were taken, and the proteins were precipitated with 8% (w/v) TCA and washed twice with absolute ethanol.
Neomycin Precipitation of Cytosolic Proteins
A 40%
(NH4)2SO4
precipitate of cauliflower cytosol (see above) was redissolved in 25 mM Tris-HCl, pH 7.4, containing 10 mM KCl, to
give a protein concentration of 1.5 mg mL1. To
this, neomycin was added at final concentrations up to 10 mM, followed by centrifugation at 100,000g for
20 min. Equal aliquots of the supernatants were precipitated with 8%
(w/v) TCA, and the proteins were separated by 10% SDS-PAGE.
Subcellular Fractionation
Gel Filtration
Microsomal membranes (100,000g pellet) obtained from
20 g of cauliflower inflorescence were resuspended in 2 mL of TDKE
10 buffer (10 mM KCl), pH 7.0, and 10 mg was applied to a
Sephacryl S-1000 column (100 mL, precoated with lecithin and
equilibrated with TDKE 10 buffer) and eluted with TDKE 10 buffer.
Three-milliliter fractions were collected, and the proteins in 200-µL
aliquots were precipitated with 8% (w/v) TCA.
Suc-Density-Gradient Centrifugation
Microsomal membranes obtained from 5 g of cauliflower
inflorescence homogenized in TDKE 10 buffer, pH 7.0 or in TDKE 10 buffer in which the EDTA was replaced with 3 mM
MgCl2 were recovered on the interface of a 55% (w/w) Suc
solution (dissolved in TDKE 10 buffer), and then loaded onto a linear
20% to 55% (w/w) Suc gradient. After centrifugation at
150,000g in a vertical rotor for 3 h, 1.5-mL fractions
were harvested, and the proteins in 100-µL aliquots from each
fraction were precipitated with 8% (w/v) TCA.
Nycodenz-Density-Gradient Centrifugation
Microsomal membranes prepared under low-Mg2+
conditions as described above were loaded onto a discontinuous
Suc-density gradient (35%, 40%, and 55% [w/w]) and then
centrifuged isopycnically as described above. The membranes collecting
at the 40%/50% interface were diluted 1:1 with 70% (w/w) Nycodenz
(Sigma) dissolved in TDKE 10 buffer and layered under three Nycodenz
solutions of decreasing density (30%, 25%, and 15% [w/w]). After
centrifugation at 240,000g for 3 h, the interfaces were
removed, and the proteins were precipitated with 8% (w/v) TCA.
Membrane Treatments
To determine the nature of the binding of AtSec21/23p to
microsomal membranes, 5-mg aliquots of total membrane pellets were resuspended in HDKE 10 buffer containing either 0.1% or 1% Triton X-100, 0.5 or 2 M urea, 0.1 M
Na2CO3 at pH 11, and 0.25, 0.5, or 1 M NaCl and shaken for 1 h at 4°C before
being centrifuged at 100,000g for 1 h. Microsomal
membranes (200 µg) were also subjected to proteolysis with papain (2 µg in TDKE 10 buffer, pH 7.0, with or without 0.1% Triton X-100) and
incubated on ice for various times. At each time indicated, 10 µg of
leupeptin and 50 µL of hot, 3-fold-concentrated SDS sample buffer
were added, and the mixture was boiled for an additional 5 min.
Solubilized and digested proteins, and residual proteins in extracted
membranes, were separated by SDS-PAGE and probed with antibodies as
described below.
Protein Determinations and Enzyme Assays
Protein concentration was determined by dye binding (Bradford,
1976). Latent IDPase was determined as described by Robinson et al.
(1994).
SDS-PAGE and Immunoblotting
Membrane and cytosolic proteins, as well as
TCA-precipitated proteins, were separated by standard SDS-PAGE
procedures (Holstein et al., 1994) using 10% separating gels.
Polypeptides were electrophoretically transferred to nitrocellulose
filters using a semidry apparatus operating at 2 mA
cm2 for 90 min. Immunoreactive proteins were
detected using an ECL kit (Amersham) according to the manufacturer's
instructions. Marker antibodies and their sources and dilutions were as
follows: BiP (1:8,000), Dr. J. Denecke, University of York, UK; RGP
(1:20,000), Dr. K. Dhugga, Pioneer Hi-Bred International, Johnston, IA;
pATPase (1:1,000), Dr. W. Michalke, University of Freiburg, Germany;
and -adaptin (B1/M6; 1:500), Dr. M.S. Robinson, University of
Cambridge, UK. AtSec21p and AtSec23p antisera were used at a primary
dilution of 1:2,000.
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RESULTS |
AtSec21p and AtSec23p Are Both Membrane and Cytosol Located
Antibodies prepared against the 81-kD GST-fusion protein of
AtSec21p specifically recognized a 100-kD protein in homogenates of
both Arabidopsis and cauliflower inflorescences (Fig.
1). Similarly, antibodies directed
against the 65-kD AtSec23p GST-fusion protein cross-reacted with an
85-kD protein in both plant extracts. For both plants, relatively more
immunoreactive protein was present in the cytosol (per milligram) than
was associated with the total membrane fractions. Whereas AtSec21p
antibodies failed to recognize any protein in yeast or pig brain
homogenates, AtSec23p antibodies cross-reacted with an 85-kD
polypeptide in these two nonplant organisms. Again, this antigen was
more prominent in the cytosol than in the total membrane fraction on a
relative-protein basis.
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| Figure 1.
Cross-reactivities of antisera generated against
GST-fusion proteins (fp) prepared from cDNA clones for AtSec21p and
AtSec23p. Antibodies prepared against the 81-kD AtSec21p fusion protein
recognized a 100-kD protein in the cytosolic fractions (c) and
microsomal membranes (tm) of suspension-cultured Arabidopsis and
cauliflower inflorescences, but not in yeast or pig brain. By contrast,
antibodies against the 65-kD AtSec23p fusion protein recognized an
85-kD protein in the cytosol of all four organisms. Using equal amounts
of protein (20 µg per lane), the signal for the cytosolic fractions
was always stronger.
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Because the isolation of COP-coated vesicles from membrane fractions in
vitro involves the use of nonhydrolyzable GTP analogs, which stabilize
the vesicle coats, we investigated the effect of including GTPS in
the homogenizing medium on the relative amounts of membrane-bound to
cytosol-located AtSec21p and AtSec23p antigens in cauliflower extracts.
As shown in Figure 2A, the presence of 50 µM GTPS did not lead to a significant redistribution
of the two antigens.
It is well known that vesicle-mediated intracellular protein transport
is inhibited at low temperatures (Tartakoff, 1987). As with anoxia,
prolonged exposure to low temperatures leads to an accumulation of
coat-protein complexes in the cytosol of mammalian cells (Goud et al.,
1985; Merisko et al., 1986). In plants, prolonged cold treatment causes
dramatic changes in the endomembrane system (Mollenhauer et al., 1975),
also presumably a consequence of the prevention of vesicle traffic.
When cauliflower plants were held overnight at 4°C, the distribution
of both AtSec21p and AtSec23p antigens was altered; compared with a
true intrinsic membrane protein such as the pATPase (Villalba et al.,
1991), the levels of the membrane-bound forms of the AtSec21p
and AtSec23p antigens were reduced considerably (Fig. 2B). By contrast,
the relative amounts of the two antigens in the cytosol appeared to
increase. This result is similar to that obtained by Bar-Peled and
Raikhel (1997) for AtSar1p with Arabidopsis cell cultures.
Cytosolic AtSec21p and AtSec23p Belong to Different Protein
Complexes
Cytosol from cauliflower inflorescence was subjected to sequential
protein precipitation with increasing concentrations of (NH4)2SO4,
and the fractions were probed with AtSec21p and AtSec23p antisera.
Whereas the AtSec21p antigen was principally found in the 40%
(NH4)2SO4
fraction, the majority of the AtSec23p antigen was precipitated by 20%
to 30%
(NH4)2SO4
(Fig. 3). When a 40% (NH4)2SO4
precipitate was subjected to gel filtration on Sepharose CL-6B, the
AtSec21p antigen eluted in fractions earlier than the 669-kD
calibrating protein thyroglobulin, indicating a molecular mass somewhat
greater than 700 kD. In contrast, the AtSec23p antigen had an elution
profile similar to that of -amylase, i.e. a molecular mass of around
200 kD (Fig. 4). These values correspond
well to those given for the COPI coatomer of mammalian cells (Waters et al., 1991) and the COPII Sec23/24 dimer of yeast cells (Hicke et al.,
1992).
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| Figure 3.
AtSec21p and AtSec23p antigens precipitate
differently with respect to
(NH4)2SO4. Cytosol extracted from
fresh cauliflower inflorescence was subjected to sequential protein
precipitation with increasing concentrations of
(NH4)2SO4 (20 µg of protein per
lane).
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| Figure 4.
Sepharose CL-6B column chromatography of 40%
ammonium-sulfate-precipitated cytosolic proteins obtained from
cauliflower inflorescence. Calibrating proteins are thyroglobulin (th;
669 kD), apoferritin (ap; 443 kD), -amylase (am; 200 kD), and BSA
(bsa; 66 kD).
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A further indication that the AtSec21p antigen in cauliflower cytosol
is part of a plant coatomer complex was found in data obtained with the
aminoglycoside antibiotic neomycin (Fig.
5). Coatomer from mammalian cells is
known to interact with a dilysine (KKXX) motif present in the
cytoplasmic domain of membrane proteins known to travel from the Golgi
to the ER (Letourneur et al., 1994; for review, see Lowe and Kreis,
1998). Neomycin contains paired amino groups at three separate
locations in the molecule, and it has been suggested by Hudson and
Draper (1997) that coatomer has a binding site that accommodates two
amino groups such as those present in neomycin. As a result, neomycin
effectively cross-links coatomer into large, sedimentable aggregates.
We treated cauliflower cytosol with increasing concentrations of
neomycin and found that, in contrast to the AtSec23p antigen, the
cytosol was effectively depleted of the AtSec21p antigen with 4 mM neomycin (Fig. 5).
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| Figure 5.
Neomycin selectively precipitates AtSec21p antigen
from cauliflower cytosol. Increasing concentrations of neomycin were
added to the cytosol proteins, and the sedimentable proteins were
removed by centrifugation at 100,000g (see ``Materials and Methods''). Aliquots of the soluble supernatant were then
subjected to SDS-PAGE, followed by western blotting with AtSec21p and
AtSec23p antisera.
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Membrane-Associated AtSec21p and AtSec23p Are Not Integral Membrane
Proteins
Microsomal membranes from cauliflower cytosol were subjected to
proteolysis with the acid protease papain (Fig.
6). In the absence of detergent, papain
effectively digested AtSec21p and AtSec23p within 15 min, whereas the
ER lumenal protein BiP was only marginally affected by this treatment
(probably indicating a small number of unsealed membrane vesicles). In
the presence of detergent, BiP was fully degraded after 30 min. These
results indicate that AtSec21p and AtSec23p are located at the
surface rather than in the lumen of membranes in cell homogenates.
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| Figure 6.
AtSec21p and AtSec23p antigens are exposed at the
surface of membranes. Microsomal membranes from cauliflower
inflorescence were resuspended in HDKE 10 buffer and subjected to
proteolysis at 1°C with papain in the presence or absence of
detergent (see ``Materials and Methods''). The membrane suspensions
were taken up in sample buffer and subjected to SDS-PAGE followed by
western blotting.
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To determine the nature of the membrane association of the AtSec21p and
AtSec23p antigens, we subjected cauliflower microsomal membranes to a
variety of extraction conditions and analyzed the resulting pellets and
supernatants for the presence of the antigens (Fig.
7). As a standard, we recorded the
response of an intrinsic membrane protein, pATPase, to these
treatments. Resuspension in salt-free buffer alone led to the release
of small amounts of the AtSec21p and AtSec23p antigens. NaCl treatment
solubilized additional quantities of the two antigens, but even with 1 M NaCl, significant amounts remained membrane bound. On the
other hand, 2 M urea, 0.1 M
Na2CO3, and 1% Triton
X-100 effectively removed AtSec21p and AtSec23p from the membranes. By
comparison, pATPase was released from the membranes only through
detergent treatment. Thus, like AtSar1p (Bar-Peled and Raikhel, 1997),
AtSec21p and AtSec23p are tightly associated peripheral membrane
proteins.
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| Figure 7.
AtSec21p and AtSec23p antigens are
membrane-extrinsic proteins. Total membrane fractions from cauliflower
inflorescence were taken up in HDKE 10 buffer alone or with Triton
X-100, urea, Na2CO3, or NaCl at the
concentrations given. After shaking for 1 h at 4°C, the membrane
suspensions were centrifuged at 100,000g to
separate membrane-bound, pelletable (P) from solubilized (S)
proteins.
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Subcellular Localization of AtSec21p and AtSec23p
To identify the membranes responsible for binding AtSec21p and
AtSec23p, we first attempted to separate organelles by gel filtration
on Sephacryl S-1000 (Fig. 8). The elution
profiles for AtSec21p and AtSec23p were identical and showed two peaks: one constituting fractions 14 to 18, and the other constituting fractions 25 to 33. Similar profiles were obtained for markers of the
ER (BiP), the Golgi apparatus (RGP; Dhugga et al., 1997; Delgado et
al., 1998), and the -adaptin of CCV (B1/M6; Holstein et al.,
1994). In contrast, pATPase was found only in fractions 15 to
17. Because the latter is an intrinsic membrane protein (see above),
and all of the other markers, including AtSec21p and AtSec23p antigens,
are soluble or extrinsic membrane proteins, we assumed that the
antigenic proteins detected in fractions 25 to 33 represent
dissociated, membrane-free proteins. Huang and Chiang (1997)
have also used Sephacryl S-1000 to separate organelles from the total
membrane fraction obtained from yeast. Although Sec21p and Sec22p
(COPII) coeluted with integral membrane markers for the ER and Golgi,
their markers for the plasma membrane (yeast pATPase) and endosomes
(Pep12p) had a completely different distribution.
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| Figure 8.
Sephacryl S-1000 gel filtration of microsomal
membranes extracted from cauliflower inflorescence. A microsomal
membrane pellet was resuspended in extraction buffer and applied to the
column, which was then eluted with the same buffer. Each fraction was
monitored by western blotting with antibodies against marker proteins
for the ER (BiP), Golgi apparatus (RGP), CCVs (-adaptin), and plasma
membrane (pATPase), as well as with AtSec21p and AtSec23p antibodies.
The calibrating soluble protein was thyroglobulin (th).
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Because no clear membrane separation could be obtained by gel
filtration, we performed Suc-density-gradient centrifugation of a
cauliflower microsomal membrane fraction prepared under low- (+EDTA)
and high- (+MgCl2) Mg2+
conditions (Fig. 9). In both types
of gradient, small amounts of BiP, RGP, AtSec21p, and AtSec23p antigens
were detected in fractions with densities around 20% (w/w) Suc,
representing solubilized proteins. As expected, the major BiP profiles
showed a typical shift to a higher density when the ER was retained in
its rough, ribosome-attached form. A smaller but still significant
shift (from a peak at 34% [w/w] Suc to a peak at 37% [w/w] Suc in
high-Mg2+ gradients) was also observed for the
Golgi marker RGP, which was confirmed by measurement of latent IDPase
activity. Independent of Mg2+, the pATPase
equilibrated at densities equivalent to 41% (w/w) Suc. The binding
profile for the -adaptin antibody B1/M6 was very similar and likely
represented not only free CCV, but also plasma membrane-bound adaptin,
as reported by Drucker et al. (1995). However, it is also possible that
clathrin-coated elements of the Golgi apparatus, which are often found
detached from the rest of the Golgi stack in situ (Robinson, 1985),
tend to become separated from the rest of the cisternae upon
homogenization and may also equilibrate at Suc concentrations around
41% (w/w).
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| Figure 9.
AtSec21p and AtSec23p antigens are bound to ER and
Golgi membranes. Isopycnic Suc-density gradients of microsomal membrane
fractions from cauliflower inflorescence were prepared under low
(+EDTA) or high (+Mg2+) Mg2+ conditions. The
fractions were probed by western blotting with the antibodies given in
Figure 8 and tested for latent IDPase activity.
|
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Common to the distribution profiles for AtSec21p and AtSec23p,
irrespective of the free Mg2+ concentration, were
peaks at very high densities (43%-46% [w/w] Suc). However, whereas
the other major AtSec23p peak followed the distribution of BiP under
both low- and high-Mg2+ conditions, AtSec21p
seemed to have a double peak under low Mg2+, one
corresponding to the BiP peak at around 28.5% (w/w) Suc and the other
coequilibrating with RGP at around 34% (w/w) Suc. These results
suggest that AtSec23p might be principally associated with ER
membranes, whereas AtSec21p seems to be borne by both ER and Golgi
membranes.
Attempted Isolation of COP-Coated Vesicles
Because COPI-coated vesicles formed in vitro from rabbit liver
Golgi also equilibrate at 40% to 45% (w/w) Suc (Malhotra et al.,
1989), we surmised that the AtSec21p/23p-containing high-density fractions from cauliflower might contain an endogenous population of
COP-coated vesicles. In an attempt to further purify such vesicles, we
subjected the membranes present in the high-Suc-density fractions to
isopycnic centrifugation on a discontinuous Nycodenz gradient, as was
successfully used by Barlowe et al. (1994) for the isolation of yeast
COPII vesicles (Fig. 10). Whereas in
their experiments the COP vesicles became concentrated at the 15%/25%
(w/v) Nycodenz interface, in our case there was no significant
enrichment of AtSec21p in any of the gradient interfaces. Moreover, the
ER and -adaptin markers were also equally distributed among the
three Nycodenz interfaces.
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| Figure 10.
Nycodenz-gradient centrifugation of high-density
AtSec21p antigen-containing membrane fraction from a Suc gradient.
Microsomal membranes from cauliflower inflorescence were layered onto a
discontinuous Suc-density gradient and centrifuged isopycnically. The
membranes collecting on the 40%/55% (w/w) Suc interface (S1) were
layered under a discontinuous Nycodenz gradient (see ``Materials and Methods'') and recentrifuged until equilibrium conditions were
obtained. TCA-precipitable proteins in the fractions collecting at the
three interfaces (N1, 15%/25%; N2, 25%/30%; and N3, 30%/35%) were
monitored by western blotting as described above (20 µg of protein
per lane).
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DISCUSSION |
Like the triskelions and adaptors of CCV, the coat subunits of
COPI and COPII-coated vesicles are also present in the cytosol as
preformed building blocks: the coatomer and the two dimeric complexes,
Sec13/31p and Sec23/24p (Barlowe, 1998; Gaynor et al., 1998). The data
presented here for AtSec21p and AtSec23p antigens in cauliflower
conform with this: Both proteins are part of different cytosolic
complexes that are similar in size to their mammalian and yeast
counterparts. They associate with membranes, but not when vesicle
production is inhibited by low temperature. Moreover, the selective
precipitation of the AtSec21p complex with neomycin can be taken as a
good indicator that this complex is a plant coatomer.
Anterograde protein transport from the ER in yeast and mammalian cells
is accomplished by vesicles that bear a COPII coat (Rowe et al., 1996;
Barlowe, 1998) and is mediated by the GTPase Sar1p (Aridor et al.,
1995). This has been shown by in vitro vesicle-induction experiments
with nuclear or ER membranes, and it has been confirmed in situ by
immunogold-localization studies with antisera against Sec13p, Sec23p,
and Sar1p that recognized budding profiles on transitional
(ribosome-free) ER (Orci et al., 1991, 1994; Shaywitz et al.,
1995). On the basis of our subcellular fractionation data, we suggest
that the AtSec23p antigen in the cauliflower inflorescence is bound
preferentially to ER membranes, suggesting that the principal site of
COPII-coated vesicle production in plants is also the ER.
The site of COPI-coated vesicle production is generally assumed to be
the Golgi apparatus, whether COPI vesicles are considered to operate in
a retrograde direction back to the ER (Letourneur et al., 1994;
Sönnichsen et al., 1996), in an anterograde direction across the
Golgi stack (for review, see Lowe and Kreis, 1998), or simultaneously
in both directions in the same Golgi apparatus (Orci et al., 1997).
Indeed, immunocytochemical studies with various coatomer antisera
(Duden et al., 1991; Oprins et al., 1993; Griffiths et al., 1995; Orci
et al., 1997) invariably reveal a conspicuous labeling of budding
vesicles at the periphery of Golgi cisternae. However, a special
coatomer-binding domain for the ER has been described in mammalian
cells (Oprins et al., 1993; Orci et al., 1994), and both COPI and
COPII-coated vesicles can be formed by yeast ER in vitro (Bednarek et
al., 1995), although the physiological significance of the
latter finding remains unclear (Barlowe, 1998). Our
subcellular-fractionation data could also be interpreted as showing the
binding of the AtSec21p antigen to both ER and Golgi membranes of the
cauliflower inflorescence, but a definitive statement on the site(s) of
COPI-coated vesicle formation in plants must await the results of in
situ immunogold labeling.
To our knowledge, unlike CCV, COP-coated vesicles have been isolated
only after having been induced in vitro (Malhotra et al., 1989; Barlowe
et al., 1994). Vesicle production normally requires GTP, which binds to
either ARF or Sar1p, but such vesicles are unstable and rapidly lose
their coat. However, the nonhydrolyzable analogs GTPS and GMP-PNP
(guanylyl-imidodiphosphate) can also be used to drive vesicle
formation, and the resulting COP-coated vesicles retain their
stability. In vivo, COP-coated vesicles formed with endogenous GTP are
consequently inherently unstable and therefore are difficult to isolate
with an intact coat. This explains why the addition of GTPS to the
homogenization medium is without effect, because the GTP-binding sites
are already occupied. Nevertheless, our data from Suc-density-gradient
centrifugation suggest that an endogenous population of
AtSec21/23p-coated vesicles, equilibrating at densities typical for
COP-coated vesicles derived from mammals and yeast, survives at
least for a short time outside of the plant. Although Golgi membranes
are known to be scarce in this fraction, we were unable to remove
other membranes (including the ER) by subsequent
Nycodenz-density-gradient centrifugation, as was achieved by
Barlowe et al. (1994) for COPII-coated vesicles from yeast. Because
membrane-bound AtSec21p or AtSec23p cannot be precipitated with our
antisera (data not shown), an immunoaffinity purification for
plant COP-coated vesicles is not yet possible. Their successful
isolation, therefore, will be delayed until an effective and convincing
in vitro vesicle-budding system using plant extracts has been
established.
| |
FOOTNOTES |
1
A.M. was the recipient of a scholarship from the
Ministry of Culture and Education of the Government of Iran. G.-H.T.
was a Research Fellow of the Alexander-von-Humboldt Stiftung (Bonn, Germany). This work was also supported by funds from the Deutsche Forschungsgemeinschaft (grant no. SFB 523).
*
Corresponding author; e-mail drobins{at}uni-goettingen.de; fax
49-551-397833.
Received September 14, 1998;
accepted January 6, 1999.
| |
ABBREVIATIONS |
Abbreviations:
BiP, immunoglobulin heavy chain-binding protein
cognate.
B1/M6, monoclonal antibody generated against bovine brain
-adaptin.
CCV, clathrin-coated vesicle.
GST, glutathione-S-transferase.
GTPS, guanosine
5 -O-(3-thiotriphosphate).
pATPase, plasma
membrane-associated ATPase.
RGP, reversibly glycosylated protein.
| |
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Abstract
Introduction