Sec24 Proteins and Sorting at the Endoplasmic Reticulum*
Alessandra
Pagano,
François
Letourneur
,
David
Garcia-Estefania§,
Jean-Louis
Carpentier,
Lelio
Orci, and
Jean-Pierre
Paccaud¶
From the Department of Morphology, University Medical Center,
§ Department of Zoology, Geneva University,
Geneva CH-1211, Switzerland and the
Institute of
Biology and Chemistry of Proteins, CNRS, Lyon, 69367 France
 |
ABSTRACT |
COPII proteins are necessary to generate
secretory vesicles at the endoplasmic reticulum. In yeast, the Sec24p
protein is the only COPII component in which two close orthologues have
been identified. By using gene knock-out in yeast, we found that the absence of one of these Sec24 orthologues resulted in a selective secretion defect for a subset of proteins released into the medium. Data base searches revealed the existence of an entire family of
Sec24-related proteins in humans, worms, flies, and plants. We
identified and cloned two new human cDNAs encoding proteins homologous to yeast Sec24p, in addition to two human cDNAs already present within the data bases. The entire Sec24 family identified to
date is characterized by clusters of highly conserved residues within
the 2/3 carboxyl-terminal domain of all the proteins and a divergent
amino terminus domain. Human (h) Sec24 orthologues co-immunoprecipitate
with hSec23Ap and migrate as a complex by size exclusion
chromatography. Immunofluorescence microscopy confirmed that these
proteins co-localize with hSec23p and hSec13p. Together, our data
suggest that in addition to its role in the shaping up of the vesicle,
the Sec23-24p complex may be implicated in cargo selection and concentration.
 |
INTRODUCTION |
The intracellular transport of secretory proteins from the
endoplasmic reticulum (ER)1
to the cell surface is essentially mediated by vesicles that collect
and concentrate cargo from a donor compartment and deliver it to the
subsequent compartment. Each step of vesicle budding, targeting, and
fusion implies that resident proteins from the donor compartment must
be excluded from the forming vesicle, whereas cargo is selected and
concentrated into it (1-3). The isolation of several yeast mutants
impaired in ER to Golgi transport led to the purification of a set of
five cytosolic proteins sufficient to reconstitute the process of ER
vesicle formation in vitro (4-6). This COPII coat complex
consists of Sar1p, Sec23-Sec24p, and Sec13-Sec31p complexes (7, 8).
Proteins of the secretory machinery appear to be well conserved
throughout evolution, as a set of COPII proteins with significant homologies with the yeast proteins was identified in mammalian cells.
Two mammalian homologues of yeast Sar1p were identified and found to
localize to the transitional zone of the ER (9). The human homologue of
Sec13p was shown to participate in the exit of VSV-G protein from the
ER (10). We recently cloned and characterized two isoforms of human
Sec23p, one of which complements a temperature-sensitive
sec23-1 mutation (11). The in vitro reconstitution of mammalian ER vesicle formation was documented recently and found to be quite analogous to that in yeast (12).
During vesicle formation, cargo proteins are concentrated into the
budding vesicle, and ER resident proteins are selectively excluded (13,
14). Two models can account for this process of cargo selection and
concentration into the nascent vesicle. The first model proposes the
existence of molecular sieves that allow only secretory molecules to
selectively enter the vesicle. The yeast protein Shr3 could be such a
sieve for a particular set of yeast proteins, the amino acid permeases
(15). The second model postulates that vesicle coat components interact
with putative cargo receptors to sort and concentrate cargo molecules.
By interacting either directly with determinants found on secretory
membrane proteins or indirectly with putative "cargo receptors,"
the COPII coat would thus participate in both cargo selection and bud
formation (16). The latter model recently received support from work
carried out in yeast where it was shown that purified COPII components interact with vesicle integral membrane proteins and soluble cargo (17). Moreover, in mammalian cells the transmembrane VSV-G protein was
selectively recruited into the forming vesicle by the pre-budding complex composed of Sar1p and Sec23-24p complex, but no direct interaction between the cytosolic portion of VSV-G with the Sec23-24p complex could be demonstrated (12).
Potential cargo receptors have been tentatively identified recently.
One is the p24 family of transmembrane proteins found both in yeast and
mammals. These proteins, located essentially between the ER and the
Golgi apparatus, recycle between these organelles and are incorporated
into both COPI and COPII vesicles (18-22). However, their role is
still unclear, as p23 in particular appeared to be restricted to a
structural role rather than in sorting (23). In yeast, however, the
deletion of members of this family delays the secretion of a subset of
secretory protein (18, 24, 25). The cytoplasmic tail of mammalian p24
proteins interacts with coatomer (COPI) (26), but we showed recently that it also binds specifically to the mammalian COPII component Sec23Ap via a di-aromatic motif (22). Another potential candidate as
cargo receptor is the mannose-specific lectin-like transmembrane molecule ERGIC53/58. This protein cycles between the ER and the Golgi
and is implicated in the forward transport of glycosylated proteins out
of the ER (27, 28). Its short cytosolic tail contains a KKFF retrieval
motif which mediates its interaction with the COPI complex (29), but
also with the mammalian Sec23-24p complex, via its
di-phenylalanine motif
(30).2
Collectively, these data suggest that COPII components may have a dual
role during vesicle biogenesis; they participate both in the
deformation of the lipid bilayer to shape up the vesicle and in the
sorting of cargo. In this perspective, a likely component to function
as an adaptor during the sorting process is the Sec23-24p complex.
Interestingly, in the yeast genome at least two additional genes highly
related to the essential SEC24 gene can be identified. In
the present report, we investigated the role of these yeast genes in
secretion, and we identify four new human proteins related to yeast
Sec24p. The biochemical and morphological characterization of these
proteins enabled us to demonstrate that they are indeed mammalian forms
of Sec24p, with their corresponding conserved relatives in
Caenorhabditis elegans, Arabidopsis thaliana, and Drosophila melanogaster. Based on our findings, we propose
that the multiple Sec23-24p complexes function as adaptors for
subclasses of secretory cargo.
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EXPERIMENTAL PROCEDURES |
Reagents, Cells Cultures, and General Methods--
Biochemicals
were purchased from Merck unless stated otherwise; T3 RNA polymerase,
rNTPs, and Pfu DNA polymerase were from Promega; restriction
enzymes, ligase, and calf intestinal phosphatase were from New England
Biolabs; pYES2 and pRSETc vectors were obtained from Invitrogen;
pBlue-Script from Stratagene and pCiNeo were obtained from Promega; T7
DNA sequencing kit and Superdex-200 were purchased from Amersham
Pharmacia Biotech; pGEX-KG was kindly provided by K. Guan (31);
anti-rabbit Ig-horseradish peroxidase, anti-mouse Ig-horseradish
peroxidase, ECL reagents kit, [32P]UTP, and
35S-ATP were from Amersham Pharmacia Biotech;
rhodamine-conjugated goat anti-rabbit Ig and FITC-conjugated goat
anti-mouse Ig were from Sigma; monoclonal anti-hemagglutinin 12CA5 was
from Babco; monoclonal anti-FLAG was purchased from Eastman Kodak Co.;
anti-hSec13p antibodies were a kind gift from W. Hong (Singapore).
Mammalian cells were cultured in their appropriate medium supplemented
with 10% fetal calf serum at 5% CO2. Yeast cultures and
manipulations were done according to Guthrie and Fink (32), and unless
otherwise stated, molecular biology procedures were performed as
described by Ausubel et al. (33).
Yeast strains were Saccharomyces cerevisiae RSY607 (MAT
alfa, PEP4::URA3 leu2-3, 112 ura3-52) and PJ69-4A
(Mat a, trp1-901 leu2-3, 112 ura3-52 his3-200 gal4_ gal4_80
LYS::GAL1-HIS3GAL2-ADE2 met2::GAL7-lacZ;
E. coli strains were DH5
and BL21/DE3-LysS.
Cloning Procedures--
Based on the yeast Sec24 cDNA
sequence used to screen ESTs data bases (34, 35), two different probes
were designed. The oligonucleotide 5'-TTAGCCTTGGACTGTTCTGGTCAGC-3'
derived from EST T79533 was used to screen for hSec24A; the primers
5'-TATTCTGCAGGGTGCATC-3' and 3'-GAACGGACAAAGAAGTTAC-5'
(positions 221 and 407, respectively) were constructed based on the EST
Z43853 to obtain a 189-bp PCR product. These two probes were used to
screen a size-selected human B lymphocyte cDNA library as described
previously (11). Sequencing of the cDNA sequences selected was
performed by primer walking in both directions.
KIAA0079 cDNA was kindly provided to us by Dr. Nomura.
The FLAG epitope was introduced at the 5' end of hSec24B cDNA by
PCR from position 159 to position 1349 in the cDNA sequence. The
PCR product was subsequently subcloned into the XhoI and
NheI sites of the pBSKS-hSec24B to obtain Flag-hSec24B. This
epitope-tagged cDNA was subcloned into the mammalian expression
vector pCiNeo. A similar strategy was used to produce Flag-hSec24C. All
the constructs were verified by sequencing.
Gene deletions were carried out according to the method of Baudin
et al. (36), by PCR amplification of the HIS3
gene with oligonucleotides that encode 40 bp of the gene-specific
sequences from each end of the open reading frames to be deleted.
The 5'-rapid amplification of cDNA ends experiment was performed
using the 5'-rapid amplification of cDNA ends kit from Life Technologies, Inc. The antisense gene-specific primer used was 5'-CTGGTTGAAAAGTTGTAGG-3' (positions 364-382 of hSec24A cDNA).
Antibody Production--
Antibodies against hSec24B were
prepared against a glutathione S-transferase fusion protein
containing aa 71-445 of hSec24B. The GST-hSec24B fusion construct was
obtained by PCR from position 212 to position 1349. This PCR product
was cloned into the bacterial expression vector pGEX-KG. Similarly,
anti-Sec24C antibodies were obtained against a glutathione
S-transferase fusion protein containing aa 207-494 of the hSec24C.
The constructions were verified by sequencing the entire PCR product.
Affinity purification of antibodies was performed by coupling the
immunogen to CNBr-activated Sepharose according to the manufacturer's
indications. The affinity matrix was incubated with crude antiserum for
2 h at 4 °C, washed extensively with 20 mM Tris, pH
6.8, NaCl 150 mM; bound antibodies were eluted with 100 mM glycine HCl, pH 2.0, and fractions were immediately neutralized by the addition of 1 M Tris, pH 8.0. The
antibody preparation was dialyzed against PBS, 10% glycerol, 1 mM sodium azide.
Cell Extracts, Fractionation, Immunoprecipitation--
Cell
extracts were either prepared as total Triton X-100 lysate or cytosol.
For Triton X-100 extract, 5 × 106 cells were lysed in
1 ml of lysis buffer (20 mM HEPES, pH 6.8, 125 mM potassium acetate, 5 mM MgCl2, 1 mM EDTA, and the following protease inhibitor, CompleteTM
protease inhibitor mixture tablets, from Boehringer Mannheim). To
obtain cytosol, we resuspended HepG2 cells in lysis buffer (20 mM HEPES, pH 6.8, 100 mM potassium acetate, 5 mM MgCl2, 8% sucrose) and homogenized cells
using 20 strokes of a tight-fitting glass Potter homogenizer in the
presence of 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and protease inhibitors. Postnuclear
supernatant was prepared by centrifuging at 2,000 × g
for 10 min at 4 °C. This postnuclear supernatant was then
centrifuged at 100,000 × g for 60 min at 4 °C,
snap-frozen in liquid nitrogen, and stored at
80 °C.
Aliquots of cytosol were fractionated onto a Superdex 200 column, and
fractions were analyzed as described previously (11).
Immunoprecipitation was performed at 4 °C on 400 µg of Triton
X-100 cell extracts using 5 µl of antiserum in a total volume of 500 µl. The precipitate was collected by adding 25 µl of protein A-agarose beads (1:1 slurry), washed five times with lysis buffer and
once in 20 mM Tris, pH 6.8. The beads were resuspended in SDS sample buffer and then run on 9% polyacrylamide gels before being
transferred to nitrocellulose.
Total protein secretion assay was performed as described previously
(37). Media proteins were precipitated with ConA-Sepharose 4B (Sigma),
and proteins were separated on 8% SDS-polyacrylamide gel electrophoresis.
Rnase Protection Assay--
Total RNA from cultured cells was
purified using the guanidinium acid-phenol method of Chomczynski and
Sacchi (38). Probes for hSec24A, -B, and -C were synthesized with T3
RNA polymerase in the presence of 50 µCi of [32P]UTP
and purified on acrylamide-urea gel. Probes had a length of 365 (position 293-658 of cDNA), 126 (position 2128-2254), and 282 bp
for hSec24A, -B, and -C, respectively. The hybridization, digestion,
and analysis of the protected fragments were done as described
previously (11).
Immunocytochemistry--
Monolayers of cells were fixed in 4%
paraformaldehyde in PBS for 20 min at RT. Cells were either
permeabilized by dehydration and rehydration in ethanol or 0.2%
saponin in PBS for 15 min. Primary antibodies were incubated for 2 h in a moist chamber at RT, followed by washing with PBS. Secondary
antibodies FITC or rhodamine-conjugated goat anti-rabbit or goat
anti-mouse IgG were added for 1 h at RT. When appropriate,
specific antibodies were first adsorbed with 50 µg of the recombinant
immunogen for 30 min at RT prior to incubation with cells.
Yeast Two-hybrid System--
The interaction between the central
region of hSec24Cp and hSec23Ap was tested by the yeast two-hybrid
method, as described by James et al. (39). Different
portions of hSec24C cDNA were cloned into the vector pGBDU-C2 in
frame with the GAL4 binding domain. The constructs prepared for the
assay were the following: hSec24C1 (aa 198-807), hSec24C2 (aa
485-807), hSec24C3 (aa 198-694), hSec24C4 (aa 485-694), and hSec24C5
(aa 485-640). The entire coding sequence of hSec23A and two truncated
forms, hSec23A/1 (aa 1-543) and hSec23A/2 (aa 1-256), were cloned
into the vector pGAD-C1 in frame with the GAL4 activation domain.
 |
RESULTS |
Yeast Homologues and Secretion--
A search through the yeast
genome using the sequence of yeast Sec24 (kindly provided to us by
Randy Schekman) revealed the existence of two additional hypothetical
proteins related to yeast Sec24p (referred to as YNE09 and YHP8,
SWISS-PROT codes p53953 and p38810, respectively). YNE09 and YHP8 share
56 and 23%, respectively, of similarity with the essential yeast gene
SEC24.
The existence of yeast homologues of Sec24 proteins prompted us to
investigate the consequences of their loss on secretion processes. We
knocked out YNE09 and YHP8 alone or in combination, and the secretion
of known secretory proteins such as invertase and CPY was assessed. We
also monitored total protein secretion in the culture medium by
pulse-chase experiments. Although the knock-out of yeast Sec24p is
lethal (40),3 the deletion of
the two other orthologues of Sec24p YNE09 and YHP8 were viable, as well
as the double knock-out.
The pattern of secretion of CPY and invertase of knock-outs were
indistinguishable from that of wild-type cells (not shown). We next
analyzed the general profile of protein secreted in the supernatant
after a pulse-chase with radioactive methionine. In order to potentiate
any secretory defect caused by the absence of a given Sec24p, the cells
were incubated at 37 °C; the viability of cells with a single as
well as a double deletion was not affected by high temperature.
However, when we examined the secretory pattern of such cells,
selective secretory defects became apparent; the deletion of YHP8
prevented almost entirely the secretion of a small subset of proteins
when incubated at 37 °C, a defect already noticed at 30 °C (Fig.
1). The major proteins disappearing from the supernatant had a apparent molecular mass of about 55 and 100 kDa,
whereas smaller species around 30 kDa were also affected but to a
lesser extent (Fig. 1). The identity of the proteins selectively
retained is currently under investigation.

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Fig. 1.
Secretory defects in Sec24p-deleted
strains. Anterograde transport in the indicated strains was tested
by a total secretion assay. Yeast strains were preincubated 20 min at
the indicated temperatures, pulsed with [35S]Met + Cys
for 10 min and chased for 30 min. Supernatants were collected,
incubated with ConA-Sepharose beads, and proteins separated by 8% SDS-
polyacrylamide gel electrophoresis.
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Identification of the Sec24 Isoforms--
When we extended our
data base search, it became apparent that highly related genes existed
in other organisms such as mammals, worms, flies, or plants. We first
identified three different human ESTs sharing a significant degree of
homology with the probe. One EST pointed to an already cloned cDNA
named KIAA0079 (GenBankTM accession number 1723050), which
was kindly provided to us by Dr. Nomura. This cDNA encodes a
protein of 1125 amino acids with a predicted molecular mass of 121 kDa.
We used the remaining two EST sequences to probe a human cDNA
library and isolated two human cDNAs: whereas one cDNA
contained an apparently complete ORF, the other appeared incomplete at
its 5' end, because it lacked the initiator ATG codon. We performed a
rapid amplification of cDNA ends experiment to obtain the 5' end of
the cDNA, and we extended our initial sequence by 360 base pairs,
increasing the previous predicted ORF to a putative protein of about
118 kDa and a length of 1078 amino acids. However, the initiator ATG is still missing, and we are attempting alternative methods to obtain the
complete ORF. This putative protein was named hSec24Ap
(GenBankTM accession number AJ131244). The other cDNA
encoded a protein of 1268 amino acids with a predicted molecular mass
of 137 kDa and was named hSec24Bp (GenBankTM accession
number AJ131245). Recently, an additional mRNA sequence named
KIAA0755 (accession number 3882231) was added to GenBankTM
and encodes a protein of 1032 amino acids related to our hSec24 sequences. We aligned these four human proteins using the multiple alignment analysis software Clustal W (41). We found a strong homology
over the carboxyl-terminal two-thirds of their length (Fig.
2), and although their amino termini are
divergent, the overall similarity between the four proteins is
approximately 35%, but it increases up to 45% in the
carboxyl-terminal region. From the alignment, hSec24A and -B are more
closely related to each other than they are to KIAA0079 and KIAA0755
and vice versa. However, due to the evident similarity of
these sequences with the other two human putative Sec24 proteins, we
propose to rename KIAA0079 and KIAA0755, respectively, hSec24Cp and
hSec24Dp.

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Fig. 2.
Clustal W alignment of the hSec24
family. The four human Sec24 orthologues have the following
length: hSec24Ap, 1078 aa; however, this is an incomplete sequence due
to the missing 5' end of the cDNA; hSec24Bp, 1286 aa; hSec24Cp
(KIAA0079), 1125 aa; and hSec24Dp (KIAA0755), 1032 aa. Shown on the
alignment is the conserved carboxyl-terminal domain of the four human
Sec24 orthologues, which encompasses the last two-thirds of the
proteins. HSec24Ap, from aa 305; hSec24Bp, from aa 489; hSec24Cp, from
aa 375; and hSec24Dp, from aa 313. The order of the proteins reflects
the degree of proximity between the sequences. Shaded areas
indicate conservative amino acid changes, and asterisks
indicate conserved residues. Bars highlight the double
cysteines tandem, characteristic signature of the entire Sec24
family.
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Sec24-related proteins are found in other organisms as well. In
C. elegans, two hypothetical proteins of similar length were identified (GenBankTM accession numbers 1163046 and 137014)
that share more than 45% similarity with hSec24Ap and hSec24Cp,
respectively. An A. thaliana cDNA
(GenBankTM accession number 3063706) encoding
for a protein sharing up to 40% similarity with hSec24Cp was found.
Finally, we identified a genomic DNA sequence
(GenBankTM accession number AC004340) of
D. melanogaster in which putative exons encode a protein
sharing more than 65% similarity over 3/4 of the length of
hSec24Cp. Prosite pattern searching (42) did not retrieve any
significant known protein motif within any member of the family.
However, the alignment of all identified members of the Sec24 family
revealed interesting features as follows: several entirely conserved
positions are found throughout the carboxyl terminal two-thirds of the
proteins, and in particular, a highly conserved region consisting of
two tandems of cysteines separated by 17 or 18 amino acids reminiscent
of a zinc finger-like domain can be considered as the typical signature
of the family (underlined in Fig. 2).
Intracellular Localization of Human Sec24 Isoforms--
From the
above analysis, we hypothesized that our cloned proteins are human
Sec24 homologues. If this assumption is correct, the proteins should be
confined to intracellular locations compatible with their putative role
in ER vesicle formation, namely the transitional elements of the ER and
the intermediate compartment, where other COPII components have been
previously localized (10, 11, 43-44). In order to verify this
hypothesis, we tagged hSec24Bp and hSec24Cp at their amino termini with
the FLAG epitope, and we expressed the constructs transiently in
different human cell lines. We also isolated stable transfected clones
of Chinese hamster ovary cells expressing hSec24Cp. We first verified
biochemically that the tagged proteins would behave as expected. Total
cell extracts were analyzed by Western blotting with the FLAG
monoclonal antibody. For hSec24Bp, a single band migrating at an
apparent molecular mass of 140 kDa was detected using anti-FLAG
antibodies (Fig. 3A). The same
cell extracts were probed with polyclonal anti-hSec24Bp antibodies
generated against a glutathione S-transferase fusion of the
amino-terminal portion of hSec24B. This antibody specifically recognized two bands as follows: the slower migrating form having almost the same apparent molecular mass as the epitope-tagged hSec24Bp,
and the lower band was estimated to have a molecular mass of about 135 kDa (Fig. 3A). Affinity purified antibodies still recognized
both bands, whereas the detection of both bands was abolished when
using immuno-depleted antiserum. This doublet is seen in most human
cell lines tested, although the relative intensity of the each band in
the doublet appears to be cell type-specific (Fig. 3B). We
don't yet know the meaning of this doublet, but it may likely
represent degradation products of the protein. More importantly, this
antibody discriminates hSec24Bp from hSec24Cp (Fig. 3A). The
epitope-tagged hSec24Cp migrated at approximately the same molecular
mass of about 120 kDa (Fig. 3A).

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Fig. 3.
Western blots of cell extracts.
A, 293T cells were transiently transfected with FLAG-tagged
hSec24B construct (lane 1), FLAG-tagged hSec24C (lane
2), or empty vector (lane 3), and cell lysates were
analyzed by Western blotting using either monoclonal anti-FLAG
antibodies or rabbit polyclonal antibodies against hSec24Bp.
B, different human cell extracts were probed with rabbit
anti-hSec24B antibodies: lane 1, 293T cells transfected with
FLAG-hSec24B construct (2 µg); lane 2, untransfected 293T
(25 µg); lane 3, HepG2 (25 µg); lane 4, A431
(25 µg). The right side of the blot is probed with an
antiserum previously adsorbed with the immunogen.
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We then looked at the intracellular distribution of hSec24Bp by
immunofluorescence in various human cell lines. By using antibodies against native hSec24Bp, the protein displayed a punctate pattern scattered throughout the cytoplasm along with a labeling around the
perinuclear region (Fig. 4A).
The same analysis was done with an epitope-tagged construct transiently
expressed in Hela cells; the epitope-tagged hSec24Bp distribution was
indistinguishable from that of the endogenous protein (compare Fig. 4,
A with C). In addition, this distribution is
superimposable to what is seen for COPII components such as hSec23Ap
(not shown) or hSec13p (Fig. 4, C and D).
Treatment with brefeldin A did not modify the distribution of hSec24Bp
or FLAG-tagged hSec24Cp, whereas it significantly affected the
distribution of ManII (not shown). When the intracellular localization
of hSec24Cp was analyzed on Chinese hamster ovary cells transfected
with an epitope-tagged hSec24Cp, it was found to be identical to that
of hSec13p (Fig. 4, E and F). Thus, the intracellular distribution of both proteins as seen by light microscopy is identical to that of other COPII components such as hSec13p and
hSec23Ap, suggesting that they are likely to be involved in COPII
vesicle formation. We were unable to localize by electron microscopy
either hSec24 proteins, as antibodies against hSec24Bp, hSec24Cp, as
well as monoclonal anti-FLAG antibodies did not recognize antigens once
prepared for ultracryo-immunoelectron microscopy.

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Fig. 4.
Intracellular localization of hSec24 proteins
by immunofluorescence. HeLa cells were labeled with the following:
A, affinity purified anti-hSec24B antibodies (1:100
dilution), followed by FITC-conjugated anti-rabbit Ig; B,
anti-hSec24Bp antiserum previously incubated for 30 min at 4 °C with
50 µg of immunogen and used at the same dilution as for A;
C, HeLa cells transfected with FLAG-hSec24B and incubated
with anti-FLAG monoclonal antibodies (1:500) followed by
rhodamine-conjugated anti-mouse Ig; D with anti-hSec13p
antibodies followed by FITC-conjugated anti-rabbit Ig; E,
HeLa cells transfected with FLAG-hSec24C and incubated with anti-FLAG
monoclonal antibodies (1:500) followed by rhodamine-conjugated
anti-mouse Ig; and F, with anti-hSec13p antibodies followed
by FITC-conjugated anti-rabbit Ig. Bar, 20 µm.
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Biochemical Characterization of sec24 Isoforms--
To
substantiate further the assumption that the cloned proteins are indeed
mammalian homologues of yeast Sec24, we submitted to gel filtration
cytosolic extracts of 293T cells and analyzed each fraction by
Western blotting. We found that both hSec24Bp and -Cp eluted in the
same fractions, corresponding to a molecular mass of about 400 kDa,
similarly to what we previously found for hSec23Ap (Fig.
5). The fact that all three proteins
shared the same chromatographic properties is only suggestive of a
direct interaction between them. However, we were able to demonstrate the existence of such interaction by co-immunoprecipitation. Total Triton X-100 extracts of 293T cells were treated with either
anti-hSec24Bp or -Cp antibodies, and the precipitates were analyzed by
Western blotting using anti-hSec23Ap, -hSec24Bp, or -hSec24Cp
antibodies. We found that both hSec24Bp and hSec24Cp would
co-immunoprecipitate with hSec23Ap, whereas hSec24 proteins were
incapable of co-immunoprecipitation with one another (Fig.
6). This demonstrates that each hSec24 orthologue can form a heterodimer with hSec23Ap. Taking into account that there are two homologues of hSec23, there are potentially eight
different hSec23-24p complexes that can be formed if all proteins are
simultaneously expressed within the same cell (see below).

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Fig. 5.
Size exclusion chromatography of cytosol from
293T cells on Superdex 200. Five mg of cytosol was applied to the
column and 1-ml fraction collected. 100 µl of each fraction was
precipitated with acetone, resuspended in SDS-polyacrylamide gel
electrophoresis sample buffer, and applied to a 9% acrylamide gel.
After transfer, the nitrocellulose was probed with anti-hSec23Ap,
anti-hSec24Bp, or anti-hSec24Cp. The column was calibrated with
different molecular weight markers, and the predicted size of the
immunoreactive material is indicated. T, total cell
extract.
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Fig. 6.
Monitoring of interactions between hSec23A
and hSec24 proteins. Triton X-100 extract of 293T cells were
immunoprecipitated with buffer (lane 1), anti-hSec24Bp
antiserum (lane 2), or anti-hSec24Cp antiserum (lane
3). The resulting immunoprecipitates were collected with protein
A-Sepharose beads and analyzed by Western blotting using anti-hSec23Ap,
anti-hSec24Bp, or anti-hSec24Cp. A total cell extract (T) (5 µg) was added as control.
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Interactions of hSec23A-hSec24C by Two-hybrid Assay--
Next we
wished to identify domain(s) of interaction of hSec23Ap with hSec24
proteins. To that end, we used the yeast two-hybrid assay, and we
restricted our analysis to one representative member of the Sec24
family, hSec24Cp, and hSec23Ap. By choosing the more distant member of
the family, we ensured that the domain mapped would represent the least
common denominator necessary to interact with hSec23Ap. As summarized
in Fig. 7, the interacting domain of
hSec24Cp is located within amino acids 485 and 807, whereas the
amino-terminal domain of hSec23Ap is involved up to amino acid 543. Interestingly, the length of the domain necessary for an interaction
varies with respect to the length of each partner: the smallest
portion of hSec23Ap (from its amino terminus up to aa 256) can
interact with hSec24Cp if the later protein fragment extends at least
up to amino acid 809 (see Fig. 2). However, if a longer fragment of
hSec23Ap is provided (i.e. up to aa 543), the required
portion of hSec24Cp can be shortened up to amino acid 694. As expected,
the interacting moiety of hSec24 is found within the conserved domain
of the family.

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Fig. 7.
Characterization of the domain of interaction
between hSec23Ap and hSec24Cp by yeast two-hybrid assay.
Various domains of hSec24Cp were fused to the DNA binding domain
of Gal4 as represented in the figure (fusion proteins 24C1 to
24C5) and introduced into the reporter strain along with three
different hSec23A fragments fused with the activation domain of
GAL4. The interaction was monitored by growth of transformants on
restrictive medium lacking histidine. The strength of the interaction
was estimated by overall growth rate: , no growth: +, slow growth;
++, normal growth. The hatched portion of the bar
representing hSec24Cp delineates the conserved domain of hSec24
proteins.
|
|
We could not detect any interactions between the various hSec24
proteins by co-immunoprecipitation (see above). We tested these
interactions by the two-hybrid assay to confirm these biochemical data,
and we found that indeed hSec24Cp cannot interact directly with itself
(not shown).
Tissue Expression of Isoforms--
The possibility still exists
that the different hSec24 proteins may be functionally identical, and
their expression may be tissue-specific in mammals. We thus analyzed
mRNA levels of three hSec24 members by RNase protection assays in
several human cell lines from different origins. We found that all
three messengers are simultaneously expressed in cell lines originating
from tissue such as fibroblast, hepatocytes, or lymphocytes. The only
major difference was a constant 2-3-fold lower level of expression of the mRNA encoding for hSec24Bp as compared with the A and C
orthologues (data not shown).
 |
DISCUSSION |
Among the three yeast SEC24 genes present in its
genome, only one is essential for growth. However, we showed that the
deletion of a particular Sec24 orthologue resulted in a selective
defect in the secretion rate of a subset of proteins secreted into the culture medium, suggesting that these genes may be implicated at some
stage in sorting cargo molecules out of the ER. This observation prompted us to search for putative Sec24-related proteins in other organisms, particularly in mammals.
We identified and partially characterized four related mammalian
proteins sharing significant homologies with the yeast COPII component
Sec24. Data base searches revealed that this family of proteins is
highly conserved throughout evolution. Prosite pattern searching (46)
did not identify any significant known protein motif within the three
human Sec24 proteins nor within the other members of the family.
However, a closer examination of the alignments revealed a conserved
region containing two groups of two cysteines separated by 17 or 18 amino acids, reminiscent of a GATA zinc finger domain (47). When used
to search data bases, this signature detected only members of the Sec24
family. This conserved domain falls outside of the region that
interacts with hSec23Ap, rendering it unlikely that it plays a role in
the formation of the complex between Sec23 and Sec24. However, it could
be involved in mediating interactions with some docking factor present
on ER membrane, with Sar1p, or with Sec13-31p, whose recruitment to the
forming vesicle is known to require the presence of the Sec23-24p
complex. We are currently testing these possibilities. The other
peculiarity of the family is its lack of amino acid conservation in the
amino-terminal domain. Although no data are yet available on the role
of this domain of the protein, it is tempting to speculate that this
highly variable region enables the various Sec24 proteins to
discriminate functionally different categories of cargo, as discussed below.
All human members of the Sec24 family we identified are likely to be
implicated in the COPII-mediated transport step between ER and the
Golgi. This assumption is supported by several findings. First, both
hSec24 orthologues tested can interact directly with hSec23Ap, forming
a cytosolic complex of approximately 400 kDa as viewed by gel
filtration. Second, hSec23Ap can be co-immunoprecipitated with both
hSec24Bp or -Cp. Finally, the intracellular localization of hSec24Bp
and -Cp as viewed by immunofluorescence is superimposable to the
distribution of other COPII components such as hSec23Ap or hSec13p.
Thus by biochemical and morphological criteria, Sec24 orthologues
cannot be discriminated. Hence from these data one would predict that
all orthologues can be incorporated simultaneously on the coat of the
same vesicle. However, the possibility exists that the various
orthologues are recruited to different sub-domains of the ER by unknown
mechanisms, leading to the generation of vesicles coated preferentially
with one form of Sec24. This point is very difficult to be amenable to
biochemical analysis but could certainly be solved morphologically.
Unfortunately, we cannot yet perform these crucial experiments due to
the lack of antibodies reacting in cryo-immunoelectron microscopy.
The possibility remains that the different hSec24 are redundant
proteins with identical function. However, we do not favor this
hypothesis based on the following observations: first, the striking
conservation of Sec24 orthologues throughout evolution along with the
observation that mRNA of all Sec24 orthologues are simultaneously
expressed in several mammalian cells suggest that each Sec24 protein is
necessary and plays a specific and essential role within the secretory
machinery; second, in yeast only one SEC24 gene is
essential, whereas the loss of two other Sec24 orthologues is not
lethal in the standard condition of growth, indicating that these
additional Sec24-related proteins cannot replace functionally the
knocked out gene, at least within the limits of their endogenous level
of expression. Finally, we demonstrated that when the yeast Sec24C
orthologue is deleted, an impaired secretion of a subset of proteins
becomes evident, reinforcing the assumption that each Sec24 orthologue
is endorsed with a specific secretory function.
What then could be the functional difference between the members of the
Sec24 family? We think that these proteins are involved in sorting and
concentrating subtypes of cargo into the nascent vesicle. It is now
well known that cargo undergoes a dramatic increase in concentration
during its export form the ER (13, 48). The involvement of COPII coat
components in this process has received strong support both in yeast
and in mammalian cells (12, 15, 17). Moreover, a di-phenylalanine motif
in the cytosolic portion of ERGIC53 was identified that interacts with COPII components, defining an anterograde signal for this particular protein (30). In addition, similar motifs found on the cytosolic domain
of the p24 family of proteins were demonstrated to interact efficiently
with hSec23Ap (22). Finally, a mislocalization of ERGIC53 obtained by
mutating this anterograde signal led to an impaired ER exit of
procathepsin C (28). Interestingly, two different human mutations
leading to a complete lack of ERGIC53 protein expression results in a
particular form of hemophilia caused by a deficient secretion of
coagulation factors V and VIII (49). Along with these data, our results
strongly suggest that the Sec24 family of proteins plays the role of
cargo adaptors during the sorting and concentration of secretory
molecules out of the ER, in a similar manner to the adaptin family
found in other limbs of the trafficking pathway.
In summary, we propose that the COPII component Sec24 is likely to be a
key element in the machinery that sorts and packages secretory cargo
proteins from the ER. The region of the molecule implicated in this
process may be the variable amino-terminal portion of the molecule,
whereas the more conserved portion is implicated in coordinating its
interactions with the other components of the COPII coat. Within this
model, these fundamental aspects of ER secretion can now be tested both
functionally and biochemically.
 |
ACKNOWLEDGEMENTS |
We thank Christine Maeder-Garavaglia for
essential technical assistance during the entire project as well as
Marilena Bonaiti; Walter Reith, for providing us with the B lymphocyte
cDNA library; Wanjin Hong for the anti-Sec13p antiserum; Sebastian
Bednarek and Pierre Cosson for helpful discussions and manuscript revision.
 |
FOOTNOTES |
*
This work was supported by the Helmut Horten Stiffung, the
Sandoz Foundation, the de Reuter Foundation, the Julius Thorn Overseas Trust (to J. P. P.), Swiss National Science Foundation Grant
31-43366/2, the Human Frontier Science Program (to L. O.), the
Association pour la Recherche Contre le Cancer, and the Fondation pour
la Recherche Médicale (to F. L.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
41-22-7025238; Fax: 41-22-7025260; E-mail: paccaud{at}cmu.unige.ch.
2
A. Pagano, F. Letourneur, D. Garcia-Estefania,
J.-L. Carpentier, L. Orci, and J.-P. Paccaud, unpublished results.
3
T. Yoshihisa and R. Schekman, personal communication.
 |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic
reticulum;
ORF, open reading frame;
aa, amino acids;
FITC, fluorescein
isothiocyanate;
bp, base pair;
PCR, polymerase chain reaction;
RT, room
temperature;
PBS, phosphate-buffered saline;
h, human.
 |
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