Sec24 Proteins and Sorting at the Endoplasmic Reticulum*

Alessandra Pagano, François LetourneurDagger , 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 Dagger  Institute of Biology and Chemistry of Proteins, CNRS, Lyon, 69367 France

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
REFERENCES

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.

    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 DH5alpha 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.

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.

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.

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.

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.

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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