Download Protein Quality Control Mechanisms and Protein

Update on Protein Bodies and Quality Control
Protein Quality Control Mechanisms and Protein
Storage in the Endoplasmic Reticulum.
A Conflict of Interests?1
Alessandro Vitale* and Aldo Ceriotti
Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy
More than 30 years ago it was observed that the
storage proteins of maize (Zea mays) seeds accumulate
as ‘‘bulges or localized dilatations along the endoplasmic reticulum cisternae’’ of developing endosperm
cells (Khoo and Wolf, 1970). It later became evident
that, whereas the widespread seed storage proteins of
the 7S and 11S classes travel from the endoplasmic
reticulum (ER) to the Golgi complex and are then
deposited in vacuoles, a number of cereal storage
proteins instead form electron-dense, round-shaped
structures with diameters of 0.5 to 2.0 mm, termed
protein bodies, within the ER lumen (Herman and
Larkins, 1999). These large aggregates are then either
permanently stored in the ER or delivered to storage
vacuoles by unconventional protein traffic pathways.
Today, the mechanisms by which some of the most
important proteins for human nutrition form protein
bodies within the ER remain a fascinating but still
puzzling issue in cell biology. This developmentally
programmed use of the ER to store vast amounts of
specific proteins in highly condensed forms has been
found only in plants. On the other hand, aggregation
of newly synthesized proteins in the ER, due to stress
or genetic defects, is usually treated by the cell as
a pathology that must be avoided by disposing of the
misfolded proteins (Sitia and Braakman, 2003). Many
ER resident proteins indeed have the role of preventing protein aggregation, maintaining nascent and
newly synthesized polypeptides in a state that is
compatible with further structural maturation or, for
defective proteins, with degradation. Thus, proteins
destined for storage in the ER must have evolved to
condense in a controlled fashion and thus to avoid
both export from the ER and degradation. How this
might be achieved is the topic of this update.
The ER is part of the endomembrane system, which
also includes the Golgi complex, vacuoles, and the
plasma membrane as major components. The system
1
This work was supported by the Ministero dell’Istruzione,
dell’Università e della Ricerca – Fondo per gli Investimenti della
Ricerca di Base (project nos. RBNE018BHE and RBNE01TYZF) and
by the European Union Research Training Networks Contract
HPRN–CT–2002–00262 (BioInteractions).
* Corresponding author; e-mail [email protected]; fax 39–02–
23699–411.
www.plantphysiol.org/cgi/doi/10.1104/pp.104.050351.
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hosts the secretory pathway, which synthesizes and
delivers to the correct location most of the proteins of
the above-mentioned compartments and of the cell
wall. These proteins are collectively termed secretory
proteins. The ER plays the role of a protein nursery,
assisting in the folding and assembly of newly synthesized secretory proteins before they traffic to the
Golgi complex and then the vacuoles or the cell surface
(Vitale and Denecke, 1999). Many residents of the ER
have signals that promote their localization in this
compartment (Vitale and Denecke, 1999), but storage
proteins that accumulate within the ER do not carry
any of these known signals. How do they form stable
structures in the ER? To try answering, we need to take
into consideration many aspects of ER functions and
their regulation.
PROTEIN QUALITY CONTROL
Sequencing of plant genomes shows that thousands
of genes encode proteins with signals for insertion into
the secretory pathway; consistently, the ER is very well
equipped to assist in the folding and assembly of
newly synthesized secretory polypeptides (Vitale and
Denecke, 1999). Indeed, it seems that, directly or
indirectly, most ER residents are helpers of protein
folding. Proteins that fail to undergo correct conformational maturation are dislocated from the ER into
the cytosol or delivered to vacuoles and then degraded. The promotion of protein folding and the
disposal of defective proteins are strictly related and
are collectively termed quality control (Vitale and
Denecke, 1999; Sitia and Braakman, 2003).
The difference between a polypeptide in the process
of folding and a defective one that cannot fold or
assemble may, however, be very subtle; thus, the ER is
subject to an apparent paradox: it must degrade defective proteins but is continuously loaded with proteins that are in the process of folding and assembly
and therefore could be considered transiently defective (Fig. 1). How does the ER quality control machinery discriminate between the two? A timing
mechanism within the ER seems to be the answer in
the case of glycoprotein disposal (Sitia and Braakman,
2003). In addition, the interactions of structurally
defective proteins with chaperones and other helpers
are more persistent than those of wild-type proteins,
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Protein Bodies and Quality Control
Figure 1. A schematic view of quality
control. Secretory proteins emerge cotranslationally into the ER lumen. Nascent
chains undergo intermediate steps of
structural maturation. During this process
they are retained in the ER. Once a protein
has folded and, in the case of multimeric
proteins, assembled, it is available for
traffic to its final destination. Proteins with
permanent defects are retained in the ER
for prolonged periods of time (or rapidly
retrieved from the Golgi complex) and
often eventually dislocated into the cytosol for degradation; alternatively, they
accumulate as aggregates in the ER. In
plant cells, a number of proteins are
naturally retained and stored permanently
or transiently in the ER as large assembled/
aggregated structures.
and this may be crucial in maintaining unfolded
proteins in a state that is compatible with degradation.
However, some native or artificial storage proteins
accumulated as protein bodies in the ER also associate
for a long time after synthesis with the plant homologue
of the immunoglobulin heavy chain binding protein
(BiP), which is the ER member of the heat shock 70
chaperone family (Li et al., 1993; and in this issue
Mainieri et al., 2004). Persistent interactions with the ER
helpers are therefore not sufficient per se for degradation, and this seems to constitute a second paradox, at
least in the cells that accumulate storage proteins in
the ER.
Storage proteins accumulating in the ER must
therefore be able to take advantage of the folding
machinery of this compartment to reach a conformational state that is not compatible with export and that,
at the same time, does not direct the protein to the
degradative phase of quality control. To understand
how this can be achieved we first need to examine the
mechanisms of protein export from the ER.
LEAVING THE ER
Export from the ER requires the recruitment of
a specific coat protein complex, termed COPII, on the
cytosolic face of the ER membrane, and it is thought
to occur at the level of a subcompartment (transitional
ER) defined by the presence of COPII components
(Barlowe, 2003). This forward movement is balanced
by backwards recycling traffic that retrieves membrane
lipids and specific proteins and uses a different coat,
termed COPI. Two nonexclusive models have been
proposed for the export of soluble proteins (Vitale and
Denecke, 1999; Barlowe, 2003). According to the first,
the bulk-flow model, proteins are not concentrated at
ER exit sites and diffuse in a passive fashion into COPII
vesicles. Concentration of secretory cargo would not
occur at the exit of the ER, but rather by exclusion from
the COPI vesicles originating from the Golgi complex
that recycle ER proteins. According to the second
model, receptor-mediated mechanisms allow cargo
concentration into COPII vesicles, and evidence has
been collected indicating that membrane proteins ERGIC53, Emp24p, and Erv29p are implicated in the
recognition of certain soluble proteins and in their
export from the ER (Barlowe, 2003). These receptors
must be able to bind sorting signals present in soluble
secretory proteins and to actively recruit COPII. These
signals may be concealed in misfolded proteins, and
this could contribute to their retention in the ER.
Retention of misfolded proteins may also be due to
their interaction with ER folding factors. ER chaperones
and folding helpers often contain signals (such as the
KDEL/HDEL motif in soluble ER residents and
the dilysine/diarginine motif in membrane residents;
Vitale and Denecke, 1999) that are recognized in the
Golgi complex and promote retrieval into the ER by
COPI vesicles. Certain misfolded proteins can thus be
retrieved from the Golgi complex by virtue of their
association with a recycling chaperone. Chaperones
may also be retained because they form an insoluble
protein matrix that effectively reduces their inclusion in
budding COPII vesicles. It would seem reasonable to
assume that binding to such a resident protein matrix
could effectively hamper the export of unfolded proteins from the ER (Sitia and Braakman, 2003).
In conclusion, depending on the protein and on the
mechanism of export, the lack of (exposed) export
signals in misfolded proteins, together with the binding to molecular chaperones in the ER and to retrieval
systems, can contribute to quality control.
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Vitale and Ceriotti
PROTEIN AGGREGATES
One additional mechanism of protein retention in
the ER is mediated by the formation of protein aggregates. In this case, the intrinsic biophysical properties
of the protein lead to the formation of complexes that
are unable to be inserted in the lumen of COPII
vesicles. The accumulation of protein aggregates in
the ER is normally induced by stress conditions or by
the expression of defective proteins. In these situations, the degradative capacity of the ER may be
insufficient to take care of the bulk of misfolded
proteins, which may then be segregated into subregions of the ER (Sitia and Braakman, 2003).
It should be stressed that the accumulation of
aggregated proteins in the ER is clearly not sufficient
to drive the formation of protein body-like structures,
as illustrated in the following examples. Tunicamycin
is a bacterial antibiotic that inhibits N-glycosylation,
a process that occurs cotranslationally in the ER lumen
and is required for proper folding of many glycoproteins. In developing lima bean (Phaseolus lunatus)
cotyledons, tunicamycin treatment leads to gross misfolding of a set of storage glycoproteins, and BiP is
found in association with the aggregated proteins
(Sparvoli et al., 2000). The aggregates are detected by
centrifugation in velocity gradients, but they do not
form specific protein body-like structures within the
ER, which enlarges in an apparently uniform process
(Fig. 2A). Similarly, in Phaseolus vulgaris developing
cotyledons, heat shock blocks traffic of the vacuolar
lectin phytohemagglutinin and causes uniform dilatation of ER cisternae (Chrispeels and Greenwood,
1987). These results imply that specific features of the
aggregating proteins are required for the formation of
discrete protein body-like accretions within the ER
lumen.
Depending on the protein, individual chaperones
can be included or excluded from the aggregates, but
sequestration of excessive amounts of folding helpers
in these aggregates is likely to be detrimental to the cell.
Consistent with this view, it has been proposed that BiP
sequestration into aggregates can lead to a reduction of
free BiP to a level that cannot be tolerated by a yeast
(Saccharomyces cerevisiae) strain unable to respond to ER
stress (Umebayashi et al., 1999).
PROTEIN BODIES
Figure 2. Artificial overaccumulation of protein in the ER does not
always result in the same alteration of ER morphology. When developing lima bean cotyledons are treated with tunicamycin (A), the
storage proteins destined for storage vacuoles aggregate and the ER
cisternae (arrows) enlarge, maintaining an electron-transparent aspect.
When zeolin, a chimeric protein composed of phaseolin and g-zein
domains, is expressed in tobacco leaves (B), it forms round-shaped,
electron-dense protein bodies within the ER. Immuno-gold labeling in
A, performed with anti-7S storage protein antiserum, decorates the
storage protein already accumulated in storage vacuoles before the
tunicamycin treatment, as well as some aggregates within the ER.
Storage proteins accumulating in the ER may take
advantage of any of the above-mentioned retention or
retrieval mechanisms and use controlled aggregation
as a way to escape the final degradative phase of
quality control and accumulate in the ER lumen
Immuno-gold labeling in B, performed with antiphaseolin antiserum,
decorates the protein body. PB, Protein body; PSV, protein storage
vacuole; V, vegetative vacuole. Bars 5 1 mM. For more details on these
experiments see Sparvoli et al. (2000) and Mainieri et al. (2004).
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Protein Bodies and Quality Control
without exposing this compartment to an excessive
level of stress.
Protein body formation has been extensively studied
in maize. The protein bodies that form in the ER of
maize endosperm cells contain a set of polypeptides
that are classified as a-, b-, g-, and d-zeins. Individual
b- or g- zeins can form stable protein body-like
accretions in the ER of transgenic plants in the absence
of other subunits (for review, see Herman and Larkins,
1999), but it is clear that formation of protein bodies in
maize normally involves both homotypic and heterotypic interactions (Kim et al., 2002). The latter have
been shown to be important for the stability of a- and
d-zeins in transgenic tobacco (Nicotiana tabacum) plants.
The accumulation of these two proteins is highly
increased when they are coexpressed with g-zein
or b-zein, respectively (Herman and Larkins, 1999).
Interestingly, g- and b-zeins are synthesized before the
a- and d- polypeptides during maize seed development, supporting the hypothesis of their fundamental
role in the formation of a stable protein body.
The mechanisms that determine the retention of zein
polypeptides in the ER are still unclear. According to
the models of protein export outlined above, either
binding to ER chaperones or lack of export signals
could raise the concentration of all these proteins into
the ER to a level compatible with the formation of
protein aggregates. Alternatively, aggregation of these
proteins could be so fast as to avoid altogether their
diffusion to ER export sites. Direct interactions with
membrane lipids may also occur, due to the particular
secondary structure of certain domains of g-zein
(Kogan et al., 2004). Which of these models is correct
is at present unknown (Fig. 3).
A region necessary for retention within the ER has
been identified in a g-zein polypeptide by mutagenesis and expression in transgenic Arabidopsis (Arabidopsis thaliana) plants. This polypeptide consists of
a Pro-rich tandem repeat domain, followed by a linker
region and by a C-terminal Cys-rich domain. While
the wild-type protein is retained by Arabidopsis leaf
protoplasts and localizes to protein bodies from which
BiP is largely excluded, a deletion mutant lacking the
repeat and linker domains is secreted (Geli et al., 1994).
These results strongly suggest that retention in the ER
is not simply due to the absence of an active export
signal and implicate the repeat domain in retention.
Interestingly, a truncated version of the protein consisting of the repeat and linker domains accumulates
in enlarged regions of the ER of Arabidopsis cells that
can also be stained with an anti-BiP antiserum (Geli
et al., 1994). Whether these sequences contain a BiPbinding site remains to be determined, but these data
would be compatible with the view that the N-terminal
region of the g-zein protein may mediate retention
within the ER by first binding to BiP and then driving
protein body formation. Consistently, when the repeat
and linker regions of g-zein are appended to the
trimeric vacuolar protein phaseolin, the fusion protein,
termed zeolin, is retained in the ER and stably de-
posited in protein body-like structures (Fig. 2B; see
Mainieri et al., 2004). These protein bodies sequester
a proportion of BiP, which can be released by ATP in
vitro treatment, indicating that BiP is not trapped but
is acting as a chaperone (heat shock-70 chaperones are
ATPases and use ATP hydrolysis to perform their
function). Zeolin protein bodies are insoluble and can
be solubilized and disassembled by reducing agents
(Mainieri et al., 2004). These results indicate that
disulfide bonds are a key factor in zeolin protein body
assembly and that the process is extensively assisted
by BiP. Further work directly addressing the question
whether BiP or other chaperones bind the N-terminal
domain of g-zeins within the context of the native
protein will be necessary to clarify the mechanism by
which the repetitive domain drives retention in the ER.
As mentioned above, a role can also be played by
direct interactions with lipids because a synthetic
version of the repetitive domain of g-zein strongly
interacts with liposomes in vitro (Kogan et al., 2004).
g-Zeins belong to the prolamin superfamily of
storage proteins, which has representatives in a range
of cereals such as wheat (Triticum aestivum), oats (Avena
sativa), and rice (Oryza sativa). The rice prolamins do
not contain a repeated sequence, but their deposition
has been shown to be assisted by the chaperone BiP (Li
et al., 1993). BiP is associated to the surface of rice
prolamin protein bodies (Muench et al., 1997) and has
been proposed to be necessary to maintain the prolamins in a competent state for subsequent assembly in
the ER. In wheat endosperm, BiP is found within the
prolamine protein bodies rather than bound to their
surface, possibly suggesting that this chaperone plays
different functions in the biogenesis of protein bodies
in different species (Levanony et al., 1992).
It is also worth noting that proteins with similar
primary structure may be differently recognized by
the ER quality control system and that the level of
export competency of a given protein may be dependent on the environment encountered within
the ER lumen. In wheat, the sulfur-rich prolamins
include both low Mr (LMW) glutenin subunits and
g-gliadins. The two classes of proteins share the same
domain architecture, with an N-terminal repetitive
domain rich in Gln and Pro, and a C-terminal domain
stabilized by a set of conserved disulfide bonds.
However, while gliadins are mainly monomeric,
LMW glutenin subunits assemble into disulfide-linked
polymers. Individual members of these two classes of
proteins have different intracellular fates when expressed in Xenopus oocytes. While a g-gliadin polypeptide is in large part export competent, an LMW
glutenin subunit is instead fully retained, possibly at
the level of the ER (Altschuler and Galili, 1994). The
mechanism(s) that mediate the complete retention
of the LMW glutenin subunit remain unclear, but
structural features within the C-terminal nonrepetitive
domain may be involved (Altschuler and Galili, 1994).
It is interesting to note that the g-gliadin polypeptide
was found to be transport competent also when
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Vitale and Ceriotti
Figure 3. Possible mechanisms of protein
body biogenesis. Newly synthesized polypeptides destined for storage in the ER
interact with chaperones and folding helpers, similarly to the other secretory proteins.
These interactions are, however, persistent.
Specific domains may also interact directly
with the lipids of the ER membrane. The
possible relationships between the two
events are unknown, and both may contribute to retention in the ER until protein
bodies are formed. The involvement of
each of these processes is likely to vary
between different protein body proteins.
The mature protein bodies are too large to
enter COPII vesicles and are permanently
retained in the ER, or transported to the
vacuole by alternative pathways. Chaperones like BiP may remain present on the
surface of the protein body, to assist the late
steps of assembly and avoid uncontrolled
exposure of hydrophobic domains that
could negatively affect ER functions.
expressed in transgenic tobacco plants (Napier et al.,
1997), but it was found to form protein body-like
structures in the yeast ER (Rosenberg et al., 1993).
The cell type-specific destiny of a g-gliadin when expressed in heterologous systems may reflect differences
in the ER machinery or in the concentration reached by
the newly synthesized protein in the ER lumen. Indeed,
protein assembly is a concentration-dependent process
and can be dramatically affected by changes in the rate of
protein synthesis (Ceriotti et al., 1991). In the native
tissue, high biosynthetic levels, interactions with other
polypeptides, or other seed-specific factors might reduce the ability of g-gliadins to leave the ER, as reflected
by the scarce proliferation of Golgi complex in wheat
endosperm cells during the onset of storage protein
synthesis and deposition (Levanony et al., 1992).
The role of tissue-specific modifications or interactions with partner prolamins in determining the final
destiny of a polypeptide after translocation into the ER
is also highlighted by the observation that a modified
g-zein is correctly incorporated into native protein bodies when expressed in maize endosperm but, unlike its
wild-type counterpart (see above), in part secreted in
transgenic Arabidopsis plants (Alvarez et al., 1998).
THE UNFOLDED PROTEIN RESPONSE
The abundance and workload of the ER are certainly
not uniform in different tissues, as for example testified by the high ER proliferation during seed development, and are also influenced by stress situations or
pathogen attacks (most pathogen defense proteins are
secretory proteins). An interesting issue of cell biology
is how the ER regulates its functions to accommodate
increased production of secretory proteins or to alleviate the stress imposed by sudden perturbation of
protein folding. This process has been termed un-
folded protein response (UPR) and has been more
deeply studied in yeast and in mammalian cells
(Rutkowski and Kaufman, 2004).
UPR has obvious relationships with quality control
(Sitia and Braakman, 2003; Rutkowski and Kaufman,
2004). The major link seems to be constituted by BiP.
Besides its interactions with newly synthesized secretory proteins, in mammalian cells BiP also binds more
permanently three transmembrane proteins that reside
in the ER: PERK, IRE1, and ATF6 (Rutkowski and
Kaufman, 2004). When, for any reason, the amount of
BiP ligands in the ER increases, the chaperone is
displaced from these three proteins to become involved
as a folding helper. As a result, PERK and IRE1 dimerize, and ATF6 is transported to the Golgi complex.
These three events start a cascade of reactions resulting
in the specific transcription of genes that encode folding
helpers of the ER, coupled to a general inhibition of
protein synthesis. If these UPR reactions do not succeed
in alleviating the stress, apoptosis can eventually be
induced. Therefore, the cell senses the amount of BiP
that is not working as a chaperone. Structurally defective proteins introduced into the ER undergo abnormally persistent interactions with BiP before being
targeted for degradation, and therefore they displace
a high proportion of BiP from the three sensors
(Rutkowski and Kaufman, 2004). A rapid increase
of workload of the ER, as a result of programmed or
stress-induced increase in the expression of secretory
protein genes, can also have the same effect.
WHO COMES FIRST?
Because many secretory proteins are N-glycosylated
and because glycosylation is often important for protein folding and solubility, tunicamycin is a potent and
widely used inducer of UPR but, obviously, tunica-
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Protein Bodies and Quality Control
mycin is not a frequent inducer of UPR during the life
of a plant. It is therefore interesting to analyze other
situations in which increased levels of ER folding
helpers have been detected. BiP and protein disulfide isomerase (an ER-located oxidoreductase) are
up-regulated in storage cells of developing seeds, compared to vegetative green tissues (Kalinski et al., 1995;
Shimoni et al., 1995; Muench et al., 1997). In developing seeds, UPR induction could be due to displacement of BiP from the transmembrane ER effectors
because of its action as a chaperone on the newly
synthesized storage proteins. However, in wheat endosperm the levels of protein disulfide isomerase are
already up-regulated days before storage protein accumulation can be detected by protein blot (Shimoni
et al., 1995). This suggests that, at least initially, induction is independent of the known UPR signaling.
Similarly, during pathogen response, up-regulation of
the mRNA of BiP occurs before that of pathogeninduced proteins (Jelitto-Van Dooren et al., 1999). A
recent proteomic study on the changes occurring when
mammalian B cells differentiate into immunoglobulinsecreting cells confirms the plant biology results:
induction of the ER machinery precedes immunoglobulin production (van Anken et al., 2003). It is reasonable to hypothesize that once the high synthesis of
storage or pathogen-induced proteins has started, ER
loading induces further UPR, but the first stage of ER
preparation must be triggered by something else than
the entry of these new proteins into the ER.
In this respect, the observation that UPR is induced
by a number of opaque endosperm maize mutations is
important (Herman and Larkins, 1999; Hunter et al.,
2002). The mutations resulting in stronger induction directly affect zein structural genes. The fl2 and De*B-30
mutants have been extensively characterized: a single
amino acid change inhibits removal of the signal
peptide in the two affected zein proteins (Herman
and Larkins, 1999; Kim et al., 2004). Therefore, in spite
of the high level of zein synthesis, wild-type maize still
has spare inducibility of UPR, indicating that protein
body formation in not an unspecific aggregation process and that zein storage proteins are not treated as
defective proteins. This situation recalls the one observed in cells expressing proteins engineered to form
conditional aggregates in the ER (Rivera et al., 2000).
Abnormal protein body formation is observed in
the above-described fl2 and De*B-30 maize mutants
(Zhang and Boston, 1992). Although the altered morphology of protein bodies could be directly ascribed to
the presence of the mutated zein polypeptides, an
alternative and intriguing possibility is that chaperone
overexpression due to UPR interferes with the process
of protein body formation. The influence of altered
chaperone levels in a protein assembly process is
testified by the observation that the artificial overexpression of BiP transiently inhibits protein assembly
in the plant ER (Foresti et al., 2003).
CONCLUSIONS
The synthesis of large amounts of secretory proteins
requires substantial remodeling at the subcellular level
and a coordinated effort between various cellular
pathways. Work performed during the last decades
has started shedding light on the way these processes
are integrated and elucidating the mechanisms that
allow the cell to cope with the heavy biosynthetic load
encountered during the period of storage protein
deposition.
In some instances, plant cells have exploited the
flexibility of the ER to directly accumulate the synthesized polypeptides without the need for further transport along the secretory pathway. Although this
process could be seen as the mere aggregation of
essentially insoluble polypeptides, it is clear that
specific characteristics of the accumulated proteins
and specific interactions with cellular factors must
be important to allow the successful deposition of the
storage proteins without subjecting the ER to an intolerable level of stress. The details of these processes
have started being investigated in some species, but
many questions still remain unanswered. The role of
the UPR in the developmental process leading to ER
proliferation during seed development needs to be
analyzed in more detail. The requirement of high rates
of storage protein synthesis for the formation of protein bodies is an issue that should be investigated. The
widespread occurrence of interchain disulfide bonds
in cereal storage proteins calls for a more detailed
analysis of their role in the aggregation/assembly
process. The search for more mutants of protein body
formation can also lead to the discovery of novel
protein maturation helpers. The question whether
there are specialized regions of the ER where protein
body formation can begin remains open. Addressing
these issues should help us understand not only the
mechanism of storage protein deposition but also how
eukaryotic cells in general manage to deal with aggregation-prone proteins translocated into the ER.
ACKNOWLEDGMENTS
We thank Franco Faoro and Michele Bellucci for the pictures in Figure 2, A
and B, respectively. We thank the many colleagues with whom we have
discussed during the last years several of the issues detailed in this update, and
we apologize to those whose work could not be cited due to space constraints.
Received July 22, 2004; returned for revision September 4, 2004; accepted
September 7, 2004.
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