Download Protein Quality Control along the Route to the Plant Vacuole

The Plant Cell, Vol. 9, 1869-1880, October 1997 O 1997 American Society of Plant Physiologists
Protein Quality Control along the Route to the Plant Vacuole
Emanuela Pedrazzini,a,l Giovanna Giovinazzo,a,2Anna Bielli,a Maddalena de Virgilio,a Lorenzo Frigerio,a,b
Míchela Pesca,a F r a n c o Faoro,c Roberto Bollini,aAldo Ceriotti,a and A l e s s a n d r o Vitaleas3
alstituto Biosintesi Vegetali, Consiglio Nazionale delle Ricerche, via Bassini 15, 201 33 Milan, ltaly
bDepartment of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom
=Centro Miglioramento Sanitario Colture Agrarie, Consiglio Nazionale delle Ricerche, via Celoria 2, 201 33 Milan, ltaly
To acquire information on the relationships between structural maturation of proteins in the endoplasmic reticulum
(ER) and their transport along the secretory pathway, we have analyzed the destiny of an assembly-defective form of
the trimeric vacuolar storage glycoprotein phaseolin. In leaves of transgenic tobacco, where assembly-competent
phaseolin is correctly targeted t o the vacuole, defective phaseolin remains located in the ER or a closely related compartment where it represents a major ligand of the chaperone BiP. Defective phaseolin maintained susceptibility to
endoglycosidase H and was slowly degraded by a process that is not inhibited by heat shock or brefeldin A, indicating
that degradation does not involve transport along the secretory pathway. These results provide evidence for the presente of a quality control mechanism in the ER of plant cells that avoids intracellular trafficking of severely defective
proteins and eventually leads t o their degradation.
INTRODUCTION
Vacuolar storage proteins reach their oligomeric structure
mainly before they exit from the endoplasmic reticulum (ER),
where they have been cotranslationally inserted to be transported via the Golgi complex to their destination along the
secretory pathway (Shewry et al., 1995; Vitale and Bollini,
1995). Quaternary structure is the major determinant of the
resistance of 7 s and 11s storage proteins to proteolysis
(Deshpande and Damodaran, 1989; Dickinson et al., 1989;
Ceriotti et al., 1991). Such resistance is probably necessary
for their efficient accumulation in protein storage vacuoles:
in fact, these compartments also accumulate proteolytic enzymes, although their hydrolytic activity is lower than that of
vacuoles of vegetative tissues (Okita and Rogers, 1996).
In this study, we used a storage protein to investigate
whether plant cells possess a mechanism that senses the
leve1 of structural maturation of newly synthesized proteins
and allows their intracellular traffic only upon acquisition of
their correct conformation. There is much evidence that
such a mechanism operates during the synthesis of many
oligomeric proteins in mammalian cells and of at least two
vacuolar proteins in yeast cells (Finger et al., 1993; Hammond
and Helenius, 1995).The details of the molecular interactions
that allow this process to occur are not yet fully understood;
’ Current address: Centro di Farmacologia Cellulare e Molecolare,
Consiglio Nazionale delle Ricerche, via Vanvitelli 32, 20129 Milan, Italy.
* Current address: lstituto di Ricerca sulle Biotecnologie Agroalimen-
tari, Consiglio Nazionale delle Ricerche, via prov. Lecce-Monteroni,
731O0 Lecce, Italy.
To whom correspondence should be addressed. E-mail vitale@
icm.mi.cnr.it; fax 39-2-23699411.
however, interactions of newly synthesized polypeptides with
ER resident proteins, whose roles are to assist correct structural maturation, seem to be important in the process of ER
retention. Proteins that fail to reach the correct maturation
because of permanent structural defects are eventually degraded by the system, in some cases via retrotranslocation
from the ER into the cytosol (Lord, 1996).Therefore, the system described for mammalian and yeast cells, termed quality control, has two functions, retention and degradation.
The major storage protein of common bean, phaseolin, is
a homotrimeric protein of the 7s class (Lawrence et al.,
1994). We have shown that one of the ER residents, the
binding protein (BiP), is found in association with phaseolin
before this assembles into trimers in the ER (Vitale et al.,
1995). BiP is a molecular chaperone of the heat shock 70
class and assists protein maturation in the ER, probably by
inhibiting hydrophobic interactions that may lead to permanent misfolding of folding and assembly intermediates
(Pedrazzini and Vitale, 1996).We have also found that an assembly-defective form of phaseolin, in which a C-terminal
domain involved in assembly has been deleted, shows more
extensive association with BiP than does wild-type PHSL,
when transiently expressed in tobacco protoplasts (Pedrazzini
et al., 1994).This truncated phaseolin is impaired in intracellular transport along the secretory pathway when expressed
in Xenopus oocytes, indicating that it is subjected to quality
control in animal cells (Ceriotti et al., 1991). We show here
that this defective phaseolin expressed in transgenic tobacco remains confined to the ER or a closely related compartment and is degraded independently of the Golgi
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complex-mediated route of protein transport to the vacuole.
These results provide direct evidence for quality control in
the plant secretory pathway.
RESULTS
Assembly-Defective Phaseolin Is Located in the ER or a
Closely Related Compartment
The assembly-defective A363 phaseolin, schematically represented in Figure 1, was subcloned into plasmid pDHA to
allow constitutive expression driven by the cauliflower mosaic virus 35S promoter (Pedrazzini et al., 1994; Tabe et al.,
1995) and was stably expressed in tobacco by Agrobacterium-mediated transformation. We previously determined that
A363 expressed transiently in tobacco mesophyll protoplasts
is correctly translocated into the ER lumen and glycosylated
but remains monomeric and shows extensive interaction
with BiP before being degraded (Pedrazzini et al., 1994). We
first determined the banding pattern, level of accumulation,
and intracellular location of A363 in leaves of transgenic tobacco; in subsequent experiments, leaves were used as a
source of protoplasts for pulse-chase analysis of the stability and intracellular traffic of the defective phaseolin.
Leaves were homogenized in the presence of nonionic
detergent, and the solubilized proteins were separated by
SDS-PAGE and immunodetected on gel blots by use of an
anti-phaseolin antiserum. As shown in Figure 2, no signal
was detectable in leaf extracts from transgenic plants transformed with plasmid pDHA without inserts (Figure 2, lane 7).
A363 accumulated as a single polypeptide with an M, of 46
kD (lanes 8 and 9, two independent transformants). This is
the size expected for fully glycosylated A363 phaseolin
(Pedrazzini et al., 1994; phaseolin contains two N-glycosylation sites, Asn-252 and Asn-341). Extraction of phaseolin in
the presence of nonionic detergent was quantitative, be-
wild-type
A363
T343FA360
L_L
V//////////M
FT
Figure 1. Schematic Representation of Phaseolin Polypeptides Encoded by the DMA Constructs Used in This Work.
The signal peptide for insertion into the ER (stippled rectangles), the
p-barrel domains (hatched rectangles), the a-helical domains involved in assembly (black rectangles), and the glycosylation sites (M')
are highlighted. White rectangles represent interconnecting and terminal regions.
1
2 3
4 5 6
7 8 9
Figure 2. Accumulation of Phaseolin Constructs in Leaves of Transgenic Tobacco.
Proteins were detected by SDS-PAGE and protein gel blot analysis
using the anti-phaseolin antiserum. Lanes 1 to 3 contain 1 ^.g, 100
ng, and 10 ng, respectively, of phaseolin purified from bean cotyledons as markers for quantitation; lanes 4 to 9 contain 50 ^g each of
total protein extracts from leaves of transgenic tobacco plants.
Plants were transformed with plasmid without inserts (Co), wild-type
phaseolin (wt), T343F (two independent transformants), or A363 (two
independent transformants). The arrowhead indicates the position of
A363; the vertical bar indicates phaseolin fragmentation products.
cause further treatment of the insoluble material with SDScontaining buffer at 90°C did not result in the recovery of additional amounts of the recombinant protein (data not
shown). A363 constitutes ~0.05 to 0.1 % of total leaf protein
in the transgenic plants, as indicated by quantitative comparisons with known amounts of phaseolin extracted from
bean cotyledons and loaded on the same gel (Figure 2,
lanes 1 to 3; the banding pattern of natural phaseolin is
complex because the protein is produced by a multigene
family and has different extents of glycosylation; see also
below).
For controls, we also produced transgenic tobacco expressing two assembly-competent forms of phaseolin: the
wild-type (3-31 phaseolin (Slightom et al., 1985), from which
A363 was derived, and a mutant of (3-31 phaseolin, T343F,
in which Thr-343 was mutated to Phe, and thus, the consensus for N-glycosylation of Asn-341 was destroyed (Figure 1).
Although it is a mutated protein, T343F can be considered
to be very similar to a natural form of phaseolin, because
normally in bean cotyledons, glycosylation at Asn-341 is inefficient and a significant proportion of polypeptides has
therefore only a single glycan on Asn-252 (Sturm et al., 1987).
Singly glycosylated phaseolin is fully competent for trimerization and delivery to protein storage vacuoles.
Polypeptides of the expected molecular masses for fully
glycosylated wild-type phaseolin and singly glycosylated
T343F (51 and 46 kD, respectively), together with fragments
in the 20- to 25-kD range, were detected in the transgenic
plants (Figure 2, lanes 4 to 6). Fragmentation into polypeptides corresponding roughly to subunit halves is a common
event when phaseolin is expressed in transgenic plants, al-
Quality Control in the Secretory Pathway
As shown in Figure 3A, A363 was located exclusively in
sucrose gradient fractions that also contained BiP in organelles with a density of ~1.17 g/mL. The top fractions of
the gradient contain soluble cytosolic and vacuolar proteins,
because vacuoles break during homogenation; consistently,
the fragments of assembly-competent phaseolin, which are
vacuolar because fragmentation occurs in the vacuole (Pueyo
et al., 1995), are recovered entirely in the top fractions of
these gradients (data not shown). A very small proportion of
A363 was recovered on top of the gradients, but this was
less than the proportion of BiP, an ER resident, in the same
fractions. We cannot exclude that this proportion of A363 is
vacuolar; however, the presence of BiP suggests that it might
result from the release of part of the soluble lumenal content
of the ER or other compartments during homogenation.
When homogenation and centrifugation were performed
in the presence of EDTA rather than magnesium to release
ribosomes from the ER and therefore lower its density but
not that of the other organelles, both BiP and A363 phaseolin shifted to fractions of lighter density (Figure 3B).
The results presented in Figure 3 indicate that the bulk of
A363 is not located in the vacuole and is possibly located in
though it does not occur in bean seeds (Bagga et al., 1992;
Pueyo et al., 1995). The results shown in Figure 2 indicate
that A363 does not give rise to immunoreactive fragmentation products and that its accumulation is higher with respect to unfragmented assembly-competent phaseolin. The
overall amount of phaseolin, including the fragments, does
not seem very different between assembly-competent and
defective constructs.
To obtain information on the subcellular location of A363,
leaves from transgenic plants were homogenized in the
presence of sucrose and absence of detergent, and organelles were separated on isopycnic sucrose gradients.
Gradient fractions were then analyzed using a mixture of
anti-phaseolin and anti-BiP antisera. The ER-resident chaperone BiP was used as a marker for the ER. The anti-BiP antiserum was produced against a recombinant fusion protein
containing a C-terminal portion of tobacco BiP. This anti-BiP
antiserum recognizes a band of the expected size (78 kD) on
protein blots or by immunoprecipitation of in vivo radioactively labeled proteins (see below) and does not immunoprecipitate other members of the heat shock protein 70 family
(data not shown).
1.11
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1.17
1.24
total
Mg:2+
B
EDTA
Figure 3. Subcellular Distribution of Assembly-Defective Phaseolin.
Leaves from transgenic tobacco expressing A363 phaseolin were homogenized, and the homogenate was fractionated on isopycnic sucrose
gradient.
(A) Magnesium-containing buffer.
(B) EDTA-containing buffer.
Gradient fractions were analyzed by SDS-PAGE followed by protein gel blotting and immunodetection with a mixture of anti-phaseolin and antiBiP antisera. In (A) and (B), the lane at right contains total unfractionated homogenate. The positions of BiP (asterisks) and A363 phaseolin
(arrowheads) are indicated. Numbers at top indicate density (grams per milliliter).
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the ER. However, it is possible that other organelles aggregate with the ER during homogenation and therefore also
shift their position in the presence of EDTA. To test this possibility, we performed immunodetection analysis of isopycnic gradients using an antiserum that recognizes complex
N-linked glycans of plant glycoproteins (Laine et al., 1991).
Because N-linked oligosaccharide chains acquire a complex
structure in the Golgi complex, immunoreactive polypeptides should not shift their position in the presence of EDTA
if the Golgi complex and post-Golgi complex compartments
1.24
T
and vesicles of the secretory pathway do not aggregate with
the ER. The antiserum recognizes many polypeptides in
plant cells (Laine et al., 1991). Part of the immunoreactive
polypeptides remains on top of the gradients and most
probably represents proteins that have reached the vacuole
or the cell wall, whereas others are associated with membrane-bound compartments (Figure 4). The position of these
along gradients containing magnesium or EDTA was very
similar (compare Figures 4A and 4B). We conclude that the
subcellular location of A363 corresponds to the ER or a
closely related compartment.
B
EDTA
A363 Phaseolin Is Degraded Independently of Golgi
Complex-Mediated Vesicular Traffic
The location of A363 indicates that the defective phaseolin
is either not competent for transport beyond the ER or very
rapidly degraded when it arrives at the vacuole or some
other post-ER compartment. Therefore, we investigated its
stability by pulse-chase labeling experiments under normal
conditions or under conditions that inhibit vesicular trafficking. Protoplasts were prepared from leaves of transgenic
plants expressing A363 or T343F and were subjected to 2 hr
of pulse labeling in the presence of 35S-labeled amino acids
and to 4 or 9 hr of chase. Protoplasts were homogenized at
the end of the pulse or the chase time points. Phaseolin was
immunoprecipitated and analyzed by SDS-PAGE and fluorography. The results are shown in Figure 5.
The polypeptides with molecular masses of ^60 and 40
kD, present in all of the lanes of the gel, were also selected
from pDHA-transformed plants (data not shown) and therefore represent endogenous immunoreactive proteins. Newly
synthesized A363 consists of a major polypeptide with an M,
of 46 kD and a minor polypeptide with an M, of 43 kD (Figure
5, lane 7). When synthesis was allowed to occur in the presence of the N-glycosylation inhibitor tunicamycin, A363 was
synthesized as a single polypeptide with an M, of 40 kD,
confirming that the two polypeptides synthesized in normal
conditions represent fully and singly glycosylated A363
(data not shown). During the chase, there was a decrease in
the recovery of A363 (Figure 5, lanes 7 to 9). This was not
due to secretion, because A363 was not detectable in the
protoplast incubation medium at any pulse-chase time point
(data not shown). No fragmentation products could be detected, indicating that either full degradation or fragmenta-
Figure 4. Subcellular Distribution of Glycoproteins with Complex
Glycans.
Leaves from transgenic tobacco plants expressing A363 phaseolin
were homogenized, and the homogenate was fractionated on isopycnic sucrose gradient.
(A) Magnesium-containing buffer.
(B) EDTA-containing buffer.
Gradient fractions were analyzed by SDS-PAGE followed by protein
gel blotting and immunodetection with the anti-complex glycan antiserum. Numbers at top indicate density (grams per milliliter).
tion into nonimmunoreactive peptides occurs without the
formation of long-lived intermediates. This is probably related to the high susceptibility of monomeric phaseolin to
degradation once attacked by proteases, as shown by in
vitro experiments (Ceriotti et al., 1991).
Because fragmentation of assembly-competent phaseolin
in heterologous cells occurs in the vacuole, it can indicate a
lower limit for the rate of phaseolin transport to this compartment. Assembly-competent T343F has an Mr of ~46 kD,
and as expected, upon the chase, it was fragmented into
polypeptides with molecular masses of 20 to 25 kD (Figure
5, lanes 1 to 3). The rate of fragmentation of T343F was
much higher than was the rate of degradation of A363 (Figure 5, compare the disappearance of intact phaseolin in
lanes 1 to 3 and 7 to 9). Figure 6 shows a detailed analysis
of the kinetics of degradation of the two constructs, performed by labeling protoplasts for 1 hr followed by 2, 4, 8, or
16 hr of chase. At the normal temperature of 25°C, fragmen-
Quality Control in the Secretory Pathway
tation of T343F was almost twice as fast as degradation of
A363. These results indicate that despite the higher susceptibility of monomeric phaseolin to in vitro proteolytic attack
(Ceriotti et al., 1991), in vivo degradation of A363 is a slow
process, slower than fragmentation of trimeric phaseolin,
which requires transport to the vacuole. This suggests that
A363 is not competent for rapid transport to the vacuole.
To determine whether vesicular transport along the secretory pathway is necessary for the degradation of assemblydefective phaseolin, we performed pulse-chase labeling under conditions that inhibit activity of the secretory pathway.
Brefeldin A is a fungal antibiotic that inhibits Golgi-mediated
vesicular traffic. In plant cells, brefeldin A blocks both protein secretion and transport of soluble proteins to the vacuole (Gomez and Chrispeels, 1993; Jones and Herman, 1993).
In the presence of brefeldin A, assembly-competent and assembly-defective phaseolin had strikingly different behaviors during the chase: fragmentation of T343F was inhibited
(Figure 5, compare lanes 4 to 6 and 1 to 3), as expected if a
block in transport to the vacuole occurs, whereas degrada-
T343F
A363
BFA:
h r o f chase:
0
4
9
0
4
9
0
4
9
0
4
9
976846-
30-
143
4
5
6
8
9
10
11 12
Figure 5. Synthesis and Stability of Phaseolin Constructs under Different Conditions.
Protoplasts prepared from transgenic tobacco plants expressing
T343F or A363 phaseolin were pulse-labeled for 2 hr with 35Smethionine and 35S-cysteine and subjected to chase for 0, 4, or 9 hr.
The pulse-chase experiment was performed in the presence (+) or
absence (-) of brefeldin A (BFA). Phaseolin was immunoprecipitated
and analyzed by SDS-PAGE and fluorography. The positions of BiP
(asterisk), fully and singly glycosylated A363 (arrowheads), and endogenous tobacco polypeptides (dots) are indicated at right. Numbers at left indicate the positions of molecular mass markers in
kilodaltons.
1873
150
10
12
14
16
18
20
hours of chase
Figure 6. Rates of Degradation of Phaseolin Constructs.
Protoplasts prepared from transgenic tobacco plants expressing
T343F or A363 phaseolin were pulse-labeled at 25°C for 1 hr with
35
S-methionine and 35S-cysteine and subjected to chase for 0, 2, 4,
8, or 16 hr at 25°C or for 4 hr at 42°C. Phaseolin was immunoprecipitated and analyzed by SDS-PAGE and fluorography. The fluorograph was subjected to densitometric analysis, and intact
phaseolin at each chase point is expressed as the percentage of the
amount measured at the end of the pulse. Error bars indicate the
standard deviation of results from two independent experiments.
tion of A363 was unaffected (Figure 5, compare lanes 10 to
12 and 7 to 9). Heat shock inhibits trafficking of the vacuolar
storage protein phytohemagglutinin beyond the ER in bean
cbtyledons (Chrispeels and Greenwood, 1987) and severely
decreases protein secretion in gibberellin-treated barley aleurone layers (Grindstaff et al., 1996). As shown in Figure 6,
when protoplasts pulse-labeled for 1 hr at normal temperature (25°C) were subjected to chase for 4 hr at 42°C, fragmentation of T343F was inhibited, whereas degradation of
A363 was slightly accelerated.
From the results shown in Figures 5 and 6, we conclude
that both constructs undergo proteolysis. Fragmentation of
assembly-competent phaseolin requires vesicular transport
to the vacuole; however, degradation of assembly-defective
phaseolin is independent of Golgi complex-mediated vesicular traffic and occurs at a rate that is markedly slower than
is fragmentation of trimeric phaseolin.
Retention of Assembly-Defective Phaseolin Occurs
before the Medial frans-Golgi Complex
To investigate in more detail the pathway followed by assembly-defective phaseolin, we determined its resistance to
endoglycosidase H. The enzyme removes from glycoproteins the N-linked glycans of the high-mannose type but not
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of the complex type. Because the complex structure is acquired when acted on by glycosidases and glycosyltransferases located in the medial and frans-Golgi complex,
endoglycosidase H resistance indicates that a glycoprotein
has reached these compartments of the secretory pathway
(Vitale and Chrispeels, 1984; Fitchette-Laine et al., 1994). As
mentioned above, wild-type phaseolin exists in bean cotyledons in two glycoforms. During passage of phaseolin
through the Golgi complex, the glycan of the singly glycosylated form acquires a complex structure, but the glycans of
the fully glycosylated form remain of the high-mannose type
(Sturm et al., 1987). Interactions between the two glycans
probably make them inaccessible to the processing enzymes of the Golgi complex. Glycan processing can therefore be used as a marker for intracellular transport of singly
glycosylated but not fully glycosylated phaseolin.
We have shown in Figure 5 that A363 is mainly glycosylated at both sites: it is very unlikely to acquire endoglycosidase H resistance even if transported through the Golgi
complex. Therefore, we introduced a stop codon at position
360 in T343F to produce an assembly-defective, singly glycosylated construct (T343FA360, schematically represented in
Figure 1). The construct was expressed transiently in tobacco mesophyll protoplasts, where, as expected, it remained monomeric (data not shown), and processing of its
glycan was analyzed by endoglycosidase H digestion, as
shown in Figure 7. The polypeptides with molecular masses
larger than T343FA360 (migrating above the position indicated by the arrowhead in Figure 7) are contaminants selected also from protoplasts transiently transformed with the
plasmid without insert (data not shown). T343FA360 remained
fully susceptible to endoglycosidase H during the chase,
when protoplasts were cultured under normal conditions (Fig-
BFA:
hr of chase:
endo H:
1
3
4
5
6
7
8
Figure 7. Processing of the Oligosaccharide of T343FA360 Phaseolin.
Tobacco leaf protoplasts transiently expressing T343FA360 phaseolin were pulse-labeled for 2 hr with 35S-methionine and 35S-cysteine
and subjected to chase for 0 or 5 hr. The pulse-chase was performed in the presence (+) or absence (-) of brefeldin A (BFA).
Phaseolin was immunoprecipitated and incubated in the presence
of endoglycosidase H (endo H [+]) or in the absence (-) of the enzyme as a control. Proteins were analyzed by SDS-PAGE and fluorography. The positions of intact (arrowhead) or deglycosylated
(arrow) T343FA360 are indicated.
ure 7, lanes 1 to 4), but it acquired complete resistance to
the enzyme in brefeldin A-treated protoplasts (Figure 7,
lanes 5 to 8). Therefore, in plant cells, brefeldin A not only
blocks transport beyond the Golgi complex but also either
promotes transport from the ER to the Golgi complex or,
perhaps more likely, retains and relocates enzymes of the
Golgi complex into the ER. The results indicate that the conformation of T343FA360 allows access to Golgi complexprocessing enzymes, but under normal conditions, such
enzymes are not encountered by T343FA360. We conclude
that the mechanism retaining assembly-defective phaseolin
from trafficking does not involve recycling from the medial
frans-Golgi complex.
Assembly-Defective Phaseolin Is a Major BiP Ligand
in Transgenic Plants, and Association with the
Chaperone Is on the Pathway for Degradation
of the Defective Protein
We have previously shown that A363 interacts with BiP during transient expression in tobacco protoplasts (Pedrazzini
et al., 1994). We have used transgenic tobacco plants expressing A363 to analyze more precisely the extent of its interactions with BiP.
When protoplasts were labeled and immunoprecipitation
was performed using anti-phaseolin antiserum, BiP was
coselected with A363. This is visible in Figure 5. BiP has a
very slow turnover, and the amount of radioactive BiP coselected is constant throughout the pulse-chase experiment.
This strongly suggests that radioactive BiP synthesized during the pulse remains available for association with unlabeled
A363 synthesized during the chase. Assembly-competent
phaseolin rapidly forms trimers in tobacco protoplasts, and
trimerization conceals BiP binding sites of phaseolin (Pedrazzini
et al., 1994; Vitale et al., 1995). Consistently, there is almost
no BiP associated with T343F (Figure 5).
BiP has ATPase activity, and the association between BiP
and its ligands is ATP sensitive (Pedrazzini and Vitale, 1996).
Figure 8, lanes 1 and 2, shows that BiP coimmunoprecipitated with A363 expressed in transgenic tobacco is released
when the complex is treated with ATP, indicating that the
coselection is entirely related to the chaperone function of
BiP. Immunoselection of labeled protoplasts using antiserum
raised against tobacco BiP resulted in the ATP-sensitive
coselection of A363, whereas no polypeptides were selected using the preimmune serum (Figure 8, lanes 9 to 11).
A363 is the most intensely labeled polypeptide coselected
with BiP in an ATP-sensitive fashion after pulse-labeling.
Taking into consideration that A363 has only three methionine and no cysteine residues, we conclude that the assembly-defective phaseolin is a major BiP ligand in tobacco leaf
protoplasts. The 60-kD polypeptide of unknown identity,
which was selected by the anti-phaseolin antiserum, was
also selected by the anti-BiP antiserum (Figure 8, lanes 3 to
Quality Control in the Secretory Pathway
antiserum:
h r o f chase:
ATP:
cxPHSL
aBiP
P
aPHSL
00
0 3 6 0 3 6
- +
. . . . . .
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aBiP
686846-
3030-
12
3 4 5 6 7 8
9
10 11
Figure 8. Association between Assembly-Defective Phaseolin and BiP.
Protoplasts from transgenic tobacco plants expressing A363 phaseolin were pulse-labeled for 1 hr with 35S-methionine and 35S-cysteine and subjected to chase for 0, 3, or 6 hr. The homogenates were selected by using anti-phaseolin (uPHSL), anti-BiP (aBiP), or preimmune (P) antiserum.
After immunoselection, the material in lanes 1,2, 10, and 11 was treated with buffer supplemented with magnesium and ATP (+) or magnesium
alone (-) as a control. Analysis was by SDS-PAGE and fluorography. The arrowheads and the asterisks mark the positions of fully glycosylated
A363 phaseolin and of BiP, respectively. Numbers at left of gels indicate the positions of molecular mass markers in kilodaltons. Lanes 1 and 2,
3 to 8, and 9 to 11 are from independent experiments.
8; see also Figure 5). However, the significance of this coselection is unclear because it varies between different experiments (Figure 8, compare lanes 3 and 10) and is insensitive
to ATP (Figure 8, compare lanes 10 and 11).
In the experiment whose results are shown in lanes 3 to 8
of Figure 8, equal amounts of A363-expressing protoplasts
were selected with anti-BiP (lanes 3 to 5) or anti-phaseolin
(lanes 6 to 8) antiserum. The results indicate that only a proportion of defective phaseolin is coselected with BiP and
vice versa. Quantitation by densitometry of the fluorographs
indicates that at the end of the pulse, ~10% of total newly
synthesized A363 is associated with BiP and ~15% of total
newly synthesized BiP is associated with A363. The results
shown in lanes 3 to 5 of Figure 8 also indicate that BiP-associated A363 decreases during the chase, as total A363 is
degraded. A detailed comparison between the rates of disappearance of total and BiP-bound A363 was performed,
with protoplasts subjected to 1 hr of pulse followed by 0, 2,
4, or 8 hr of chase. As shown in Figure 9, there is an initial
decrease in the ratio between BiP-associated and total A363;
however, subsequently, the time course of degradation of
total A363 is similar to that of release of BiP-associated
A363 from the chaperone. This indicates that association to
BiP is on the pathway for A363 degradation and suggests
that BiP-bound A363 is not a subset of molecules with a distinct destiny but rather that there is only one pool of A363
that may undergo cycles of binding and release from BiP
before being degraded.
120
100
4
6
hours of chase
Figure 9. Rates of Degradation of Total and BiP-Associated A363
Phaseolin.
Protoplasts from transgenic tobacco plants expressing A363
phaseolin were pulse-labeled for 1 hr with 35S-methionine and 35S-cysteine and subjected to chase for 0, 2, 4, or 8 hr. Phaseolin or BiP
was immunoprecipitated from equal numbers of protoplasts and analyzed by SDS-PAGE and fluorography. Total phaseolin immunoprecipitated with the anti-phaseolin antiserum or phaseolin coselected
with the anti-BiP antiserum was quantified by densitometry of the
fluorograph, and phaseolin at each chase point is expressed as a
percentage of the amount measured at the end of the pulse.
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The Plant Cell
DlSCUSSlON
The data presented here indicate that a defect in the assembly of phaseolin results in an alteration of its intracellular
transport. The following evidence indicates that intracellular
traffic of assembly-defective phaseolin along the secretory
pathway is blocked at some stage before the media1 tmnsGolgi stacks: (1)the defective protein is located in a subcellular compartment that is the ER or is closely associated
with it; (2) its degradation is not inhibited by brefeldin A or
heat shock, whereas both treatments block transport of assembly-competent phaseolin to the vacuole; (3)T343FA360
remains sensitive to endoglycosidase H after a chase; (4)
A363 is a major BiP ligand in transgenic tobacco and has
prolonged interactions with this ER-located chaperone; and
(5) despite the fact that monomeric phaseolin is much more
susceptible to in vitro proteolysis than is the assembled
protein (Ceriotti et al., 1991),the rate of degradation of A363
is approximately half the rate of fragmentation of trimeric
phaseolin, which occurs when transported to the vacuole,
and the levels of accumulation of A363 and trimeric phaseolin are similar.
Quality Control in the Secretory Pathway of Plant Cells
We have presented compelling evidence for a causal relationship between the acquisition of quaternary structure and
trafficking along the secretory pathway to the vacuole of
plant cells. Acquisition of the correct conformation is also a
prerequisite for the intracellular trafficking of many secreted
and plasma membrane mammalian proteins and at least two
yeast-soluble vacuolar proteins (Finger et al., 1993;Hammond
and Helenius, 1995).Therefore, it appears that a conserved
quality control mechanism or severa1 mechanisms controlling similar processes exist in the secretory pathway of eukaryotic cells.
To date, evidence for ER quality control in higher plants
has been only indirect and not definitive. lndirect evidence
includes the discovery that the ER of all eukaryotes is provided with a set of very abundant resident proteins, such as
BiP, protein disulfide isomerase, calnexin, calreticulin, and
endoplasmin, that are involved in folding and assembly of
newly synthesized polypeptides (Hammond and Helenius,
1995; Denecke, 1996). It is thought that interaction with
such helpers may be part of the quality control: clearly,
newly synthesized polypeptides must be confined to the ER
when bound to ER residents, and plant proteins undoubtedly should not be an exception.
Studies on the expression of subunits of immunoglobulins
and of the maize storage proteins in transgenic plants
showed that accumulation is enhanced when partner subunits are expressed together, also suggesting the presence
of ER quality control in plants (Hiatt et al., 1989;Coleman et
al., 1996). It has also been observed that certain mutated
constructs of barley aleurain, a vacuolar protein, fail to reach
the vacuole or the cell surface in transformed tobacco protoplasts (Holwerda et al., 1992).Finally, an assembly-defective mutant of pea vicilin, a storage protein closely related to
phaseolin, accumulates at particularly low levels and is not
located in the vacuoles in transgenic tobacco (Kermode et
al., 1995).The pathways for degradation of assembly-defective phaseolin and of the above-mentioned proteins could
be similar.
Retention
Folding and assembly usually occur in the compartment of
residence of a protein, and in this sense, the ER is an exception. It has the unique characteristic of taking care of these
processes for proteins that are destined for other locations.
By retaining proteins in the ER, quality control probably both
optimizes folding and assembly and avoids expression of
defective proteins in their compartment of destination. We
have shown here that unassembled phaseolin is retained in
an early compartment of the secretory pathway that has low
hydrolytic activity rather that being transported to the vacuole, where it would most likely be degraded quickly. Trimerization of wild-type phaseolin both in bean cotyledonary
cells and in transfected tobacco protoplasts is a very rapid
and efficient process, much faster than vesicular transport
out of the ER (Pedrazini et al., 1994;Vitale et al., 1995).For
wild-type phaseolin, it is impossible to establish whether
some form of ER retention of monomers occurs before assembly. The results shown here, however, suggest that such
retention is likely to occur and might favor efficient assembly.
Assembly-defective phaseolin is a major newly synthesized BiP ligand in the transgenic plants that we have produced. However, we have only been able to detect 40% of
A363 in association with BiP. Because we also found that
6iP-bound and total A363 have very similar degradation kinetics, we conclude that BiP binding is on the pathway to
A363 degradation and that degradation probably occurs after cycles of binding and release from the chaperone. If we
take into consideration that other chaperones are present
in the ER and probably act on phaseolin (Lupattelli et al.,
1997) and that assembly-defective phaseolin is found in a
monomeric state and not as a large aggregate that might
physically be unable to enter vesicular trafficking, then the
prolonged interaction with chaperones could be the most
reasonable explanation for retention, and a mechanism
sensing such prolonged interaction could be the system
leading to degradation.
Degradation
When defects in the newly synthesized proteins occur or
when stress conditions do not allow proper structural maturation to take place, quality control eventually degrades the
Quality Control in the Secretoty Pathway
defective proteins, allowing the cell to maintain homeostasis
(Hammond and Helenius, 1995). Similarly, we have shown
that the accumulation of A363 phaseolin is not indefinitely
prolonged and that the defective protein is slowly degraded.
In most cases, it is still unclear which features of defective
proteins make them selected substrates for degradation. A
cysteine residue that mediates assembly of immunoglobulin
M also promotes retention and degradation of the excess
of unassembled subunits that is normally synthesized in
plasma cells (Fra et al., 1993). Phaseolin, however, does not
contain cysteine residues. The mechanism of degradation
must sense either the prolongation of the unassembled
state beyond a “physiological” period of time or the structural features of the mutant we have produced that are not
present in the assembly-competent protein. This degradation is not simply caused by prolonged retention in the ER or
in some undefined pre-Golgi complex compartment but requires a structural defect in phaseolin, because in the presente of brefeldin A, T343F is stable. The defect must be
severe to allow for degradation: T343F can be considered a
form of wild-type phaseolin, because indeed many wild-type
phaseolin polypeptides do not acquire a glycan on Asn-341
(Sturm et al., 1987); however, when we treated protoplasts
with tunicamycin and brefeldin A at the same time, the absence of the glycan at Asn-252, which is always present in
natural phaseolin, did not affect the stability of T343F (data
not shown).
It has been shown recently that at least in some cases,
degradation by quality control involves retrotranslocation
into the cytosol and the action of the cytosol-located ubiquitin-proteasome system (Lord, 1996). This suggests that
defective proteins must be unfolded, possibly with the help
of chaperones, to make their pathway backward into the cytosol through a channel that could be the same channel used
during synthesis and translocation into the ER or another
channel not yet discovered. However, at present, we have
no proof that A363 is retrotranslocated into the cytosol. An
alternative pathway cannot be excluded; this could involve
degradation in the ER itself or in the vacuole, reached by a
mechanism that does not involve the Golgi complex. The
wheat storage protein gliadin forms protein bodies in the
ER, and these are then in part directly internalized into storage vacuoles by autophagy (Galili et al., 1996). The tonoplast intrinsic protein TIP expressed in tobacco mesophyll
protoplasts reaches its destination through a brefeldin Ainsensitive process (Gomez and Chrispeels, 1993). However,
it is not yet known whether the Golgi complex-independent
pathways of gliadin and TIP to the vacuole are also insensitive to heat shock, as is the case with A363 degradation.
1877
port, but the mutated phaseolin is much more unstable than
is the wild-type counterpart once it reaches the storage vacuoles of transgenic tobacco seeds, suggesting that its conformation has been altered (Hoffman et al., 1988; Pueyo et
al., 1995). In addition, fully unglycosylated phaseolin is considerably less stable than is glycosylated phaseolin when
expressed in transgenic tobacco seeds, and again, instability is due to degradation in protein storage vacuoles (Bustos
et al., 1991). Trimerization of phaseolin is unaffected by the
lack of glycans (Vitale et al., 1995) and is only partially affected by the introduction of the methionine-rich peptide
(Hoffman et al., 1988). Clearly, quality control fails to recognize these phaseolin mutants as defective, although they are
defective in their function of stable storage material. Again,
we can reason that trimer formation is a key step that
conceals most if not all interactions with the retention mechanism, which may be constituted by the chaperone machinery. This would explain why defective phaseolins that can
trimerize escape quality control. We have shown previously
that the BiP binding sites in phaseolin are concealed upon
assembly, independent of the presence of the glycans
(Vitale et al., 1995). We are currently attempting to identify
regions necessary for BiP binding in phaseolin monomers. If
we succeed, the hypothesis that binding to chaperones mediates retention can be tested by fusing such regions to reporter secretory proteins.
METHODS
Phaseolin Mutants
All phaseolin constructs derive from the cDNA clone p31 (Slightomet
al., 1985). The constructions of A363 and T343F have been described by Ceriotti et al. (1991) and Lupattelli et al. (1997), respectively. To produce T343FA360, we introduced a stop codon at
position 360 of T343F by site-directed mutagenesis using the antisense oligonucleotide 5’-ATTGTCCGTeCCTGCAAG-3’ (where
underlining indicates the antisense for the stop codon). Mutagenesis
was performed with a fragment subcloned in the vector M13mp18.
The Sculptor in vitro mutagenesis system (Amersham) was used for
mutagenesis, and the fragment containing the mutated sequence
was fully sequenced before being reintroduced into phaseolin cloned
in the plasmid pDHA (Pedrazzini et al., 1994; Tabe et al., 1995) for
constitutive expression in plant cells. Phaseolin constructs in the
pDHA vector were used for transient expression in protoplasts. For
constitutive expression in transgenic tobacco, the pDHA vector without inserts or containing wild-type, T343F, or A363 phaseolin was inserted into the Hindlll site of the binary vector pGA470 (An et al.,
1985),which was then used to transform strain LBA4404 of Agrobacterium tumefaciens by triparental mating.
Accuracy of the Recognition Mechanism
How accurate is quality control? The introduction of a peptide enriched in methionine residues in the structure of
phaseolin does not make the protein incompetent for trans-
Transformation of Tobacco
Luria-Bertani medium (5 mL) containing 20 pg/mL rifampicin (Sigma)
and 12.5 pg/mL tetracycline (Sigma) was inoculated with a single
1878
The Plant Cell
colony of the different transformed agrobacteria and incubated at
29°C for 2 days. The cultures were diluted 1:100, incubated overnight
of l), and diluted 150 in shootto obtain 108 cells per mL (OD,
inducing medium (SIM; Murashige and Skoog salts containing 0.5
mg/L 6-benzylaminopurine; Sigma). This suspension was used to
transform leaf discs from Nicotiana tabacum cv SR1 by cocultivation
in 10-cm Petri dishes (20 leaf discs per dish containing 10 mL of suspension) for 48 hr at 28°C in the dark (An et al., 1986).After cocultivation, bacterial cells were washed off with SIM containing 250 mg/L
cefotaxime, and the leaf discs were grown at 25°C under full light on
SIM containing agar, 250 mg/L cefotaxime, and 1O0 mg/L kanamycin
(10 leaf discs per 10-cm Petri dish). After 5 weeks, shoots were
plated on half-strengthMurashige and Skoog salts, 100 mg/L kanamycin, and 250 mg/L cefotaxime until the new plants developed.
Transformed plants were grown at 25°C in 16 hr of light in axenic culture in Magenta GA-7 vessels (Sigma)without antibiotics and propagated every 5 to 6 weeks.
Production of the Anti-BiP Antiserum
The sequence encoding amino acids 551 to 667 of tobacco BiP
clone NTBLP4 (Denecke et al., 1991) was amplified by polymerase
chain reaction. The amplified sequence was fused in frame to the 3’
end of the mal€ gene in the expression vector pMAL-c2 (Riggs,
1992), generating plasmid pMALBiP. The fscherichia c06 XL1-Blue
transformed with pMALBiP plasmid was cultured in 400 mL of LuriaBertani medium, and expression of the fusion protein was induced
by isopropylp-D-thiogalactopyranoside treatment for 2 hr. Total bacteria1 proteins were extracted by sonication and fractionated on an
amylose resin column (1.5 X 10 cm), as described by Riggs (1992).
The fusion protein was retarded but not retained by the column; fractions eluting from the amylosecolumn and containing the fusion protein were then pooled, dialyzed against 100 mM NaCl and 20 mM
Tris, pH 7.5, and loaded onto a DEAE-Sephacel (Pharmacia, Uppsala, Sweden) column (1.5 x 7 cm) at 4°C. The resin was extensively
washed with the same buffer and then with 30 mL of 500 mM NaCl
and 20 mM Tris, pH 7.5, collecting fractions of 2 mL. An aliquot of
each fraction was analyzed by SDS-PAGE, and fractions containing
the purifiedfusion protein were pooled and concentrated to 0.75 mg/
mL in PBS using Centriplus 10 (Amicon, Beverly, MA) concentrators.
The concentrated protein was used to immunize rabbits.
Total Leaf Protein Extraction, Subcellular Fractionation,
and Protein Gel Blot Analysis
For analysis of total proteins, leaves from transformed plants were
homogenized in an ice-cold mortar with 0.2 M NaCI, 1 mM EDTA, 2%
p-mercaptoethanol, 0.2% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, and 0.1 M Tris-CI, pH 7.8, supplemented with a Complete
(Boehringer Mannheim) protease inhibitor cocktail. The buffer-to-tissue ratio was 6:l. The homogenate was centrifuged at 15009 at 4°C
for 10 min, and the supernatant was analyzed by SDS-PAGE on 15%
acrylamidegels. lmmunodetectionof phaseolin and BiP in protein gel
blots was performed using the enhanced chemiluminescence(ECL)
system (Amersham)following the manufacturer’s protocol, using rabbit polyclonal antisera raised against phaseolin purified from bean cotyledons (D’Amico et al., 1992), against plant complex N-linked
glycans (Lainé et al., 1991), or against a recombinant MAL-BiP fusion
protein (see above). Subcellular fractionation on isopycnic sucrose
gradients in the presence of EDTA or magnesium was performed as
described by Ceriotti et al. (1995).
Protoplast Preparation, Pulse-Chase Labeling,
Immunoprecipitation, and Endoglycosidase H Digestion
Protoplasts were prepared from leaves (5 to 7 cm long), as described
by Pedrazzini et al. (1994). Radioactivelabeling was performed by incubation at 25°C in the dark in K3 medium (Gamborg’sB5 basal medium with minimal organics [Sigma], supplemented with 750 mg/L
CaC1,~2H,O, 250 mg/L NH,NO,
136.2 g/L sucrose, 250 mg/L xylose, 1 mg/L 6-benzylaminopurine, and 1 mg/L a-naphthalenacetic
acid, pH 5.5) supplemented with 150 pg/mL of BSA and 1O0 pCi/mL
of PRO-MIX (a mixture of 35S-methionineand cysteine; Amersham).
The protoplast concentration was 106cells per mL. Chase was performed by adding unlabeled methionine and cysteine to 10 mM and
5 mM, respectively, from a x10 concentrated stock. In some experiments, before radioactive labeling, protoplasts were incubated for
45 min in K3 medium supplemented with 10 pg/mL brefeldin A
(Boehringer Mannheim; stock solution of 2 mg/mL in ethanol stored
at -20°C) or 50 pg/mL tunicamycin (Boehringer Mannheim; stock
solution of 5 mg/mL in 10 mM NaOH stored at 4°C) and maintained
under the same conditions for the entire experiment. At the desired
time points, 3 volumes of ice-cold W5 medium (154 pM NaCI, 5 pM
KCI, 125 pM CaCI2.2H,O, 5 pM glucose) was added, and samples
were centrifuged for 5 min at 509. The supernatant, containing secreted proteins, was removed, leaving 100 pL to cover the protoplasts. The supernatant and protoplasts were frozen separately in
liquid nitrogen and stored at -80°C.
Homogenizationwas performed by adding to the frozen samples 2
volumes of ice-cold protoplast homogenizationbuffer (150 mM TrisCI, 150 mM NaCI, 1.5 mM EDTA, and 1.5% Triton X-100, pH 7.5)
supplemented, immediately before use, with 1.5 mM phenylmethylsulfonyl fluoride and the complete protease inhibitor cocktail. After
brief vortexing, the samples were used for immunoprecipitation, as
described by D’Amico et al. (1992), using the antisera mentioned
above. In some cases, the immunoprecipitatewas treated with ATP,
as described by Vitale et al. (1995). Digestion of immunoprecipitated
proteins with endoglycosidase H (recombinant; Boehringer Mannheim) was performed as described by Ceriotti et al. (1991).
Radioactive samples were analyzed by SDS-PAGE on 15% acrylamide gels. Rainbow i4C-methylated proteins (Amersham) were
used as molecularweight markers. Gels were treated with 2,5-diphenyloxazole dissolved in DMSO, and radioactive polypeptides were
revealed by autoradiography.
Transient Transformation of Protoplasts
For transient expression of phaseolin constructs, protoplasts prepared from leaves of untransformed plants were subjected to polyethylene glycol-mediated transfection, as described by Pedrazzini et
al. (1994),with the following modifications.The concentration of protoplasts before the addition of polyethylene glycol was 106cells per
mL; no carrier herring sperm DNA was added. After overnight incubation at ZYC, transformed protoplasts were then subjected to radioactive labeling, as described above.
Quantification of Radioactive Proteins and lmmunoblots
After fluorography (SDS-PAGE of radioactive proteins) or ECL immunodetection of protein blots, quantificationof the relative intensities
of the bands on the films was performed by microdensitometry, using a TLC Scanner II (Camag, Muttenz, Switzerland).Care was taken
to use film exposures that were in the linear range of film darkening.
Quality Control in the Secretory Pathway
ACKNOWLEDGMENTS
We thank Jürgen Denecke for providing clone NTBLP4 and Lo’ic
Faye for providing the antiserum against the plant complex N-linked
glycans. This work was supported in part by the European Union
(Grant No. CHRX-CT94-0590) and by the Progetto Speciale Biologia
e produzioni agrarie per un’agricoltura sostenibile of the Consiglio
Nazionale delle Ricerche.
ReceivedApril 30, 1997; accepted August 8, 1997.
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Plant Cell 1997;9;1869-1880
DOI 10.1105/tpc.9.10.1869
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