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Vol. 14, Issue 7, 3055-3063, July 2003
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* Molecular Membrane Biology Laboratory, RIKEN;
PRESTO, Japan Science and Technology Corporation, 2-1 Hirosawa, Wako, Saitama
351-0198, Japan
Submitted February 26, 2003;
Revised March 23, 2003;
Accepted March 24, 2003
Monitoring Editor: Howard Riezman
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The starting
point of the secretory route is the ER. Transport of newly synthesized
secretory proteins from the ER are sorted from ER-resident proteins and
packaged into COPII-coated vesicles
(Schekman and Orci, 1996). The
formation of the COPII coat on the ER membranes occurs by sequential binding
of the small GTPase Sar1p (Nakano and
Muramatsu, 1989), the Sec23/24p complex, and the Sec13/31p complex
(Barlowe et al.,
1994), which drives vesicle budding. Budding from the ER involves
activation of the Sar1p GTPase by the ER resident protein Sec12p, the
Sar1p-specific guanine-nucleotide exchange factor
(Nakano et al., 1988;
Barlowe and Schekman, 1993).
The molecular events that trigger the activation of Sar1p to the GTP-bound
form to initiate budding are presently unknown. Soon after formation, COPII
vesicles shed their coat and then fuse with the Golgi compartment. The
incorporation of cargo into the COPII vesicle is believed to be selective and
perhaps nonselective to some extent. Certain integral membrane cargos are
concentrated into ER-derived vesicles through direct interactions with COPII
subunits (Springer and Schekman,
1998; Shimoni et al.,
2000). Evidence suggests that several soluble secretory proteins
require specific transmembrane cargo receptors to mediate the interaction of
the cargo proteins with the COPII coats, resulting in selective packaging
(Kuehn et al., 1998;
Appenzeller et al.,
1999; Belden and Barlowe,
2001). The mechanism by which cargo association occurs and the
receptor's mode of action upon exit from the ER are less clear.
The Emp47p and its close homologue Emp46p, type-I membrane proteins that
cycle between the ER and the Golgi, have been proposed as cargo receptors at
the ER exit site (Schroder et
al., 1995; Sato and
Nakano, 2002). Their luminal domains share homology with
leguminous lectins, whereas the C-terminal cytoplasmic regions contain both
COPII and COPI-binding sites. Thus, Emp47p and Emp46p may be required for
selective packaging of specific glycoproteins into ER-derived vesicles.
Indeed, the disruption of both EMP47 and EMP46 leads to a
marked defect in the secretion of a subset of glycoproteins
(Sato and Nakano, 2002).
However, specific cargo proteins for Emp47p and Emp46p have not been
identified thus far. In mammalian cells, the mannose-binding lectin ERGIC-53
appears to be the homolog of Emp46/47p and has been proposed as a soluble
cargo receptor at the ER exit site. A chemical cross-link approach recently
resulted in the coisolation of ERGIC-53 with a soluble
cathepsin-Z–related glycoprotein
(Appenzeller et al.,
1999).
In this report, we demonstrate that Emp47p associates with Emp46p in the ER and acts in selecting Emp46p into COPII vesicles. The Emp46p-Emp47p complex persists in the ER and COPII vesicles but dissociates when it reaches the Golgi. Furthermore, our data indicate that it is the oligomerization state that confers on Emp46/47p the competence for the exit from the ER. Therefore, our findings may have important implications for recruitment of a variety of other secretory cargo into COPII vesicles.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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sec12-4 ura3 leu2 trp1 his3 emp46::3HA-EMP46)
and KSY008 (MAT ura3 lys2 adehis3 leu22 trp1 emp47::LEU2
emp46::HIS3; Sato and Nakano,
2002). To construct pKSY155, pKSY160, pKSY166, and pKSY177
carrying the GFP-myc-EMP47-
383,
GFP-myc-EMP47-333, GFP-myc-EMP47-281, and
myc-EMP47-281-333 deletion mutants, respectively,
under control of the EMP47 promoter, these fragments were generated
by PCR mutagenesis and subcloned into pRS314
(Sikorski and Hieter, 1989).
The fragment encoding HA-EMP46-279-321 was generated
by PCR mutagenesis and subcloned into pRS316
(Sikorski and Hieter, 1989) to
yield the plasmid pKSY187. The plasmids pKSY105, pKSY126, and pKSY113,
encoding myc-EMP47, GFP-EMP46, and HA-EMP46, respectively,
were described previously (Sato and
Nakano, 2002). The overexpression of HA-Emp46p was from the
plasmid pKSY114 (2 µm; Sato and Nakano,
2002). For the overexpression plasmid of myc-Emp47p, the
EcoRI fragment of pKSY105 was inserted into the corresponding site of
the pYO324 (2 µm) to yield the plasmid pKSY169.
Antibodies
The anti-Sec61p and anti-Sec22p antibodies were gifts from R. Schekman.
Monoclonal antimyc and anti-HA antibodies were obtained from Berkeley
Antibody, and the anti-Pho8p antibody was from Molecular Probes (Eugene, OR).
The anti-Sed5p antibody was generated against Ni-NTA purified
Sed5p-his6 lacking the C-terminal transmembrane domains (amino
acids 1–316).
Chemical Cross-linking
For identification of Emp46p binding proteins, 75 mg of microsomes was
incubated on ice with 0.5 mM DSP (Pierce, Rockford, IL) for 30 min in B88 (20
mM HEPES-KOH, pH 6.8, 150 mM KOAc, 5 mM MgOAc, and 250 mM sorbitol). The
reaction was quenched by adding 50 mM Tris-Cl, pH 7.4, followed by 15-min
incubation. Microsomes were lysed in 30 ml of 1% Triton X-100 in B88. After 1
h at 4°C, the detergent extract was centrifuged and supernatant was
incubated with protein G-Sepharose bearing no IgGs at 4°C for 30 min. The
flow through was collected, reapplied to the anti-HA immobilized protein
G-Sepharose prepared as described
(Ungermann et al.,
1998), and incubated overnight at 4°C. The resins were washed,
and bound proteins were eluted with 0.1 M glycine/HCl, 0.2% Triton X-100,
precipitated with TCA, and washed with ice-cold acetone. Samples were analyzed
by SDS-PAGE and Coomassie blue staining. Protein bands were excised from the
gel and rinsed, and the protein samples were digested with trypsin in the gel
matrix. Extracted peptide mixtures were analyzed by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Proteins
were identified by comparing their tryptic peptide mass maps to the S.
cerevisiae sequence database.
Coimmunoprecipitation
For coimmunoprecipitations, membranes were detergent solubilized in B88
containing 0.05% n-dodecyl maltoside. The detergent extract was
placed on ice for 1 h, and the insoluble material was removed by
centrifugation. The supernatant was applied to the protein G–immobilized
antimyc or anti-HA antibody or to the protein A-immobilized anti-GFP antibody
and incubated overnight at 4°C. The beads were washed five times in the
same buffer and then were then eluted with the elution buffer (0.1 M
glycine/HCl, pH 2.5, and 0.05% n-dodecyl maltoside). Samples were
subjected to TCA precipitation, followed by SDS-PAGE and immunoblotting.
Gel Filtration Analyses
Gel filtrations were performed at 4°C on a G3000SWXL column (Tosoh,
Tokyo, Japan) equilibrated in B88 containing 0.05% n-dodecyl
maltoside and run at the flow rate of 0.5 ml/min. Detergent extracts of
membranes were loaded onto the column, 0.2-ml fractions of were collected, and
aliquots from the fractions were analyzed by immunoblotting. The column was
calibrated with gel filtration standard proteins from Amersham Pharmacia
Biotech (Piscataway, NJ).
Miscellaneous
In vitro vesicle budding assays, prebudding formation assays, subcellular
fractionation, and confocal laser microscopy observation were performed as
described (Sato and Nakano,
2002).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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emp47 cells that carried
plasmids encoding HA-Emp46p and myc-Emp47p. Microsomes were solubilized in the
presence of n-dodecyl maltoside and subjected to native
immunoprecipitation with the antimyc antibody. HA-Emp46p was successfully
coimmunopurified when myc-Emp47p was immunoprecipitated with the antimyc
antibody, whereas a control ER protein, Sec61p, was not
(Figure 1B). We conclude that
Emp46p forms a complex with Emp47p in the ER.
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Emp47p Is Required for the ER Exit of Emp46p.
To examine whether the complex formation between Emp46p and Emp47p has a
role in the ER exit of these proteins, we performed the reconstituted in vitro
budding reactions (Figure 2A).
In both wild-type and Emp46p-lacking microsomes, ER-derived COPII vesicles
incorporated Emp46p and Emp47p at a level comparable to another characterized
cargo protein, Sec22p, but not the resident ER protein, Sec61p. Strikingly, no
incorporation of Emp46p into COPII vesicles was detected in the microsomes
lacking Emp47p, whereas the control Sec22p was unaffected. Thus, in the
absence of Emp47p, ER-derived vesicles normally formed but eliminated Emp46p,
suggesting that Emp47p is required for the ER exit of Emp46p.
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As an independent method to evaluate Emp46p exit from the ER in
emp47 cells, we performed cell fractionation experiments to
determine where Emp46p accumulates in the cell
(Figure 2B). Previous sucrose
gradient fractionation of membrane organelles from wild-type cells documented
the Golgi localization pattern for both Emp46p and Emp47p
(Schroder et al.,
1995; Sato and Nakano,
2002). As shown in Figure
2B, the Golgi localization pattern of Emp47p was not affected in
the absence of Emp46p. In contrast, Emp47p deletion caused a strong shift of
residual Emp46p to the ER membrane fractions. These results indicate that
Emp46p fails to exit from the ER and accumulate there in the absence of
Emp47p, which is entirely consistent with the in vitro budding results.
To investigate whether additional factors might be required for the Emp46p-Emp47p interaction, subcellular localizations of overexpressed Emp46p and Emp47p were examined (Figure 2C). Overexpression of Emp46p from a multicopy plasmid with endogenous Emp47p leads to ER accumulation of Emp46p. To examine whether this was because the limited pool of Emp47p in the ER was titrated out, subcellular localization of Emp46p was examined in the cells overproducing both Emp46p and Emp47p. We found that the overproduced Emp46p was efficiently targeted to the Golgi when Emp47p was overexpressed as well. These results suggest that no additional factor is necessary as a bridge between Emp46p and Emp47p in the ER.
Emp46p Binds Emp47p through the Coiled-Coil Region of Emp47p in the
ER and Dissociates in the Golgi
We next sought to identify a region in Emp47p required for Emp46p binding
in the ER. Emp47p has a single transmembrane domain near its C termini, and a
200-residue segment in the N-terminal luminal domain that shares homology with
leguminous lectins. These two regions were connected by a predicted
coiled-coil segment (Figure
3A). For convenience, we divided the Emp47p into three segments,
designated the lectin domain (281), the coiled-coil region (333
and 281–333), and the transmembrane domain with the cytoplasmic
tail (383). Microsomes were prepared from cells expressing HA-Emp46p
and one each of the myc-tagged deletion mutants and applied to native
immunoprecipitation with the anti-HA antibody followed by the antimyc
detections. As shown in Figure
3B, the only deletion proteins found to fail to associate with
Emp46p were the ones lacking the coiled-coil region (281 and
281–333). These results suggest that the molecular escort is
associated with Emp47p through the coiled-coil region in the luminal domain.
We further examined whether the coiled-coil domain of Emp46p is also required
for the Emp46/47p complex formation. Microsomes expressing both the
coiled-coil deleted HA-Emp46p (Emp46p-279–321) and myc-Emp47p
were subjected to native immunoprecipitation with the antimyc antibody as
above (Figure 3C). The result
clearly showed that the Emp46p without coiled-coil region fails to associate
with Emp47p, indicating that the complex formation between Emp47p and Emp46p
is mediated by the coiled-coil region.
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Our results indicating Emp46p and Emp47p as interacting proteins suggest that Emp47p may be a transport receptor for Emp46p targeting. If this were the case, their association might be reversible so that disassembly occurs at some point after export from the ER. To test this possibility, their coimmunoprecipitation was examined in different intracellular compartments. ER and Golgi membranes were prepared from the cells expressing myc-Emp47p and HA-Emp46p by sucrose gradient fractionation. COPII vesicles were also prepared by GTP-driven in vitro budding reactions with this strain. Membranes were solubilized with a detergent and myc-tagged Emp47p was immunoprecipitated. HA-Emp46p associated with these native immune complexes was then examined by immunoblotting (Figure 3D). Strikingly, the Emp46p-Emp47p complex was detected in the ER and COPII vesicles, but not at all in the Golgi fraction. These results demonstrate that the interaction of Emp47p and Emp46p is transient; cargo binding occurs in the ER and release in the Golgi.
Emp47p Forms an Oligomeric Complex in the ER, whereas Emp46p Alone Is
Present as a Monomer
We have shown before that the C-terminal region of Emp46p and Emp47p
contain signals required for COPII binding
(Sato and Nakano, 2002). The
question is raised then why Emp46p requires Emp47p for exit from the ER,
whereas Emp47p alone is efficiently exported out from the ER. We speculated
that the presence of COPII-binding motifs is not enough for incorporation into
COPII vesicles and that some structural difference exists between Emp46p and
Emp47p in the ER. To test this idea, we constructed
emp46emp47 cells expressing differently tagged
Emp47p (myc-GFP-Emp47p and myc-Emp47p) or Emp46p (GFP-HA-Emp46p and HA-Emp46p)
to analyze the oligomeric state of each protein in the ER. myc-GFP-Emp47p
complemented the growth defect of the emp47 strain in the
presence of Ca2+ (Sato and
Nakano, 2002) and showed exactly the same distribution to
myc-Emp47p on sucrose gradients (unpublished data). Microsomes from these
strains were solubilized with a detergent and then a portion of GFP-tagged
protein was immunoprecipitated with the anti-GFP antibody. The amounts of
coprecipitated myc-Emp46/47p were determined by immunoblotting. The results
shown in Figure 4A demonstrate
that myc-GFP-Emp47p was coimmunoprecipitated with myc-Emp47p, suggesting that
Emp47p exists as a homooligomer in the ER at least two Emp47p molecules in a
complex. In contrast, no Emp46p homooligomers were detected at all in the
microsomes (Figure 4B). Our
findings showed that Emp47p alone forms more than a dimer in the ER, whereas
Emp46p alone is present as a monomer.
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The state of Emp47p homooligomerization was further examined. If Emp47p
exists as a homooligomer in the ER, the prediction is that wild-type Emp47p
and Emp47p without the cytoplasmic export signals would be traveling to the
Golgi together. As can be seen in Figure
4C, GFP-Emp47p alone was localized to punctate structures,
representing the yeast Golgi. In contrast, when the mutant that lacks the
C-terminal COPII-binding sites and the transmembrane region,
GFP-Emp47p-383, was expressed alone in
emp46emp47 cells, the Golgi fluorescence was
virtually gone and a continuous pattern typical of the yeast ER emerged. This
indicates the incapability of ER exit of this soluble-form mutant. However,
when GFP-Emp47p-383 was coexpressed with wild-type Emp47p in
emp46emp47 cells, GFP-Emp47p-383 was
now converted to punctate Golgi structures. This shift appears to correlate
with the association of GFP-Emp47p-383 with wild-type Emp47p, again
supporting homooligomerization of Emp47p upon exit from the ER. These results
further indicate that the essential domain for the formation of Emp47p
homooligomer is in the luminal domain of Emp47p. We further examined the
effect of C-terminal deletion of Emp47p on Emp46p localization. When
GFP-Emp46p was expressed with GFP-Emp47p-383, ER accumulation of
GFP-Emp46p was observed (Figure
4C). This result confirms that the ER export of Emp46p is
dependent on Emp47p.
We addressed the mechanism by which Emp47p forms a homooligomer by more
precise identification of the domain of Emp47p required for oligomer
formation. We tested a series of deletion mutants used in
Figure 3A, for intracellular
localization in the presence of wild-type Emp47p as in
Figure 2B. We found that the
C-terminal deletion up to the coiled-coil region had an obvious effect, which
appeared to shift the mutant Emp47p toward ER membrane fractions
(Figure 4D). When the internal
deletion mutant of the coiled-coil region from full-length Emp47p
(myc-Emp47p-281–333) was coexpressed with GFP-tagged wild-type
Emp47p and subjected to native immunoprecipitation as in
Figure 4A, no association was
observed between these proteins in the microsomes
(Figure 4E). This implies that
the coiled-coil region of Emp47p is responsible for the oligomer formation as
well as Emp46p binding. Emp46p has 66% amino acid similarity to Emp47p, and
indeed the alignment of Emp46p protein with Emp47p demonstrates the probable
coiled-coil domain at similar locations in Emp46p and Emp47p
(Sato and Nakano, 2002). It
should be noted, however, that Emp46p has deletions within this region, which
may contribute to homooligomerization. ERGIC-53, a mammalian homologue of
Emp47p, forms disulfide-bond–linked homodimers and homohexamers in the
ER (Lahtinen et al.,
1992). This is not likely the case for Emp47p, because the
coiled-coil region contains no cysteines and Emp47p migrated as its monomer
size in a nonreduced gel (unpublished data).
Emp47p Oligomerization Acts as a Signal for Its Incorporation into
COPII Vesicles but Is Not Required for COPII Binding
We next tested the influence of the myc-Emp47p-281–333
mutation on the incorporation of this protein into COPII vesicles. As shown in
Figure 5A, in the reconstituted
in vitro budding reaction with microsomes expressing both myc-GFP-Emp47p and
myc-Emp47p-281–333, the packaging of
myc-Emp47p-281–333 into ER-derived COPII vesicles was
undetectable, whereas myc-GFP-Emp47p budding was at a level comparable to
Sec22p. This result suggests that the presence of COPII-binding motifs in
Emp47p per se is not sufficient for this cargo to be sorted into COPII
vesicles but the oligomerization is also required. Presumably, the failure of
Emp46p alone to exit the ER is reasoned by its monomeric state in the ER. We
also tested if myc-Emp47p-281–333 affects the packaging of Emp46p
into COPII vesicles using microsomes derived from
emp46emp47 cells expressing HA-Emp46p and
myc-Emp47p-281–333. As expected, the budding efficiency of
HA-Emp46p was very low along with myc-Emp47p-281–333, again
confirming that the coiled-coil region is sufficient for Emp46p binding
(Figure 5A).
|
We next examined whether the coiled-coil deletion of Emp47p and Emp46p
alone failed to be incorporated into COPII vesicles because they could not
bind to COPII subunits or whether they bound but were not packaged into COPII
vesicles. These were investigated by testing whether those monomers were
recovered in detergent-soluble prebudding complexes
(Figure 5B). The prebudding
complexes are detected when microsomes are incubated with a subset of COPII
components, GST-Sar1p, and the Sec23/24p complex in the presence of GMP-PNP
(Kuehn et al., 1998).
GMP-PNP-bound GST-Sar1p stabilizes the assembled prebudding complex for
isolation on glutathione-Sepharose in the presence of detergent. Microsomes
were prepared from cells expressing Emp46p and Emp47p or Emp46p and
Emp47p-281–333 and were incubated in the presence of GST-Sar1p
with GMP-PNP and the Sec23/24p complex. Emp46p and Emp47p from wild-type
microsomes were efficiently recovered in the prebudding complex, whereas the
ER resident protein Sec61p was not. Sec22p was also found in the prebudding
complex, which was shown as a positive control
(Kuehn et al., 1998;
Sato and Nakano, 2002).
We next asked whether the Emp46/47p monomers were able to be incorporated
into the prebudding complex. Surprisingly, when microsomes expressing both
HA-Emp46p and myc-Emp47p-281–333 were tested, both proteins were
present in the prebudding complex. These results indicate that the
oligomerization of Emp46/47p is not required for efficient binding of COPII
subunits. The deletion of the coiled-coil region of the Emp47p may disturb the
protein stability. It should be noted, however, that the
Emp47p-281–333 expressed indistinguishable amounts of protein
from the wild-type Emp47p (our unpublished observation). Moreover, the result
indicating that the coiled-coil deleted Emp47p retains partial function to
form prebudding complexes argues against the possibility that the transport
defect of this protein is caused by its misfolding and interactions with the
ER quality control machinery. Taken together, the results above suggest that
the packaging of Emp46/47p into the COPII vesicles is due to a combined action
of COPII binding and oligomer formation of Emp46/47p.
Emp47p Forms a Large Oligomeric Complex
The results described above strongly suggest that Emp47p is found in
complexes with Emp46p or with Emp47p itself in the ER. To extend our analysis
and to determine the size of Emp47p-containing complex, we performed gel
filtration separation of the detergent solubilized ER and the Golgi isolated
by sucrose gradient, which would be a more direct test of Emp47p
oligomerization. Significant proportions of Emp47p and Emp46p in the ER from
wild-type cells were found to coelute at the molecular mass of 
600 kDa
(Figure 6). When extracts were
prepared from the ER fraction expressing both myc-Emp47p-281–333
and HA-Emp46p, these proteins were eluted at lower molecular mass positions,
most consistent with their monomer sizes. Thus, it seems unlikely that the
high-molecular-mass complexes comprising Emp47p and Emp46p are monomeric forms
of each molecule surrounded by detergent micelles. The figure 600 kDa
corresponds to 10–12 mers if only Emp47p and/or Emp46p are present. In
agreement with the coimmunoprecipitation results in
Figure 3D, 70% of Emp46p
were eluted in fractions corresponding to its monomer size when the Golgi
fractions were analyzed, representing the cargo dissociation from the receptor
in the Golgi. However, no apparent molecular shift was detected for the Emp47p
containing complexes between the ER and the Golgi. We cannot rule out that
this 600-kDa complex may be associated with additional proteins in the
Golgi, and they may contribute to the dissociation of Emp47p-Emp46p protein
complexes. Because efficient transport of the soluble truncated Emp47p is
observed without its cytoplasmic COPII-binding domain when wild-type Emp47p is
present (Figure 4, C and D),
the oligomerization to 600 kDa might not be the minimal requirement for
Emp47p to exit from the ER.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). It remains to be determined if the lectin-like domains of
Emp46/47p bind glycoproteins and indeed act as glycoprotein cargo receptors as
well.
Another saturable transport receptor that has been characterized in yeast,
Erv29p, shows a limited secretion of several soluble cargo molecules when
inactivated (Belden and Barlowe,
2001). Integral membrane cargo proteins have also been thought to
depend on saturable receptors for export from the ER. Candidates for this
function include Emp24p and Erv14p, which have been postulated to be required
for selection of specific GPI anchored and integral membrane cargo,
respectively, into COPII vesicles (Muniz
et al., 2000; Powers
and Barlowe, 2002). Our present study on Emp46/47p provides
another piece of evidence to prove that the integral membrane secretory
protein depends on an adaptor/receptor protein for export from the ER.
We have demonstrated before that both the cytoplasmic regions of Emp46p and
Emp47p have the ability to bind COPII proteins
(Sato and Nakano, 2002). The
decreased rate, but not completely, of ER exit of Emp46p with mutations in
this region was observed. This raised the possibility that the multiple
signals may be required in Emp46p and Emp47p for efficient export. In the
present work, we have demonstrated that Emp46p associates with Emp47p in the
ER and this Emp46/47p hetero-oligomer is competent for export. This finding
readily explains why the disruption of COPII-binding motif cannot completely
block the ER export. The Emp46p with disrupted COPII-binding motif still can
exit the ER through association with Emp47p but at reduced efficiency. The
COPII binding to each subunit of the Emp46/47p complex is likely to be
required for maximal transport efficiency. We also have shown before that the
overexpression of Emp46p suppresses the temperature and calcium sensitivities
of the emp47 cells, whereas Emp47p is required for the ER
export of Emp46p. The explanation for this is that the ER-to-Golgi flow of
Emp46p is achieved by the overexpression presumably by bulk flow export. In
support of this idea, a small amount of Golgi localization of Emp46p is
observed when Emp46p alone is overexpressed (unpublished data), which might be
enough to suppress the phenotypes associated with EMP47 deletion.
The formation of a homooligomeric cargo complex upon exit from the ER is
also reported for the yeast plasma membrane H+-ATPase Pma1p
(Bagnat et al., 2001;
Lee et al., 2002). In
comparison with these examples, Emp47p oligomerization seems to has a
different role upon ER exit, because the ER export of Pma1p is not dependent
on its oligomeric state. Recent studies on Erv41p-Erv46p complex, which cycles
between the ER and Golgi, have identified multiple ER export signals contained
within the C-terminal cytoplasmic tails of both Erv41p and Erv46p. Sequence
information contained in these tails of both the Erv41p and the Erv46p is
required in its proper context for efficient packaging of the Erv41p-Erv46p
complex into COPII vesicles (Otte and
Barlowe, 2002). Requirement of the export signals on separate
subunits of the complex for the ER export is similar to Emp46/47p
transport.
Is cargo oligomerization a general mechanism of protein sorting into COPII
vesicles or specific for the Emp46/47p? It is well known that a number of
proteins need to homo- or heterooligomerize in order to be exported out from
the ER in mammalian cells, such as immune receptors
(Bonifacino et al.,
1990), ATP-dependent K+ channels
(Zerangue et al.,
1999), 
-aminobutyric acid GABAB receptors
(Margeta-Mitrovic et al.,
2000; Pagano et al.,
2001), glutamate NMDA receptors
(Perez-Otano et al.,
2001), and G-protein–coupled receptors
(Bouvier, 2001). However, in
those membrane protein complexes whose trafficking is known to be assembly
dependent, at least one of the subunits contains an ER retrieval signal that
is shielded on subunit assembly, allowing the assembled protein complex to
traffic to the plasma membrane. The unassembled subunits recycle between the
ER and the Golgi until the complexes are properly formed. Therefore, the
oligomerization of those proteins are required for the plasma membrane
targeting of fully assembled complexes, but not essential for each subunit to
exit the ER. In fact, the disruption of those ER retrieval signals allows each
unassembled subunit to localize to the plasma membrane. In comparison with
those documented examples of assembly-dependent ER export, Emp46/47p seems to
act through a different mechanism. Here, Emp46/47p oligomerization itself is
required for the export competence of Emp46/47p complexes. Interestingly, the
oligomeric association of other type-I transmembrane cargos has also been
reported for p24 family members, which also depends on the coiled-coil region
of their luminal domain (Marzioch et
al., 1999; Ciufo and Boyd,
2000). Moreover, the vesicular stomatitis virus (VSV) G protein
trimerizes in the ER and only trimers are transported to the Golgi
(Doms et al., 1987).
Although the precise role of cargo oligomerization in the ER export remains to
be established, these observations strongly support the presence of assembly
dependent cargo selection at the ER exit sites.
In conclusion, although the function of Emp46p and Emp47p proteins remains to be determined, we have demonstrated the Emp47p acts in sorting an integral membrane secretory cargo during export from the ER and this process includes cargo oligomerization. To confirm a direct role of cargo oligomerization in concert with activated Sar1p and Sec23/24p complex, it will be necessary to reconstitute these associations in purified proteoliposomes. Such a system may provide an explanation of the contribution of a cargo oligomerization to its recruitment.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Corresponding author. E-mail address:
kensato{at}postman.riken.go.jp.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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S. Subsequently, digitonin-soluble prebudding complexes were
recovered on glutathione beads. Emp46p, Emp47p, Sec22p, and Sec61p in the
prebudding complex were detected by immunoblotting. Total (T) represents 0.5%
of the total solubilized membranes.