Correspondence to: J. Paiement, Département de Pathologie et Biologie Cellulaire, Faculté de Médecine, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Québec, Canada, H3C 3J7., paiemej{at}patho.umontreal.ca (E-mail), (514) 343-7259 (phone), (514) 343-2459 (fax)
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Abstract |
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A two-step reconstitution system for the generation of ER cargo exit sites from starting ER-derived low density microsomes (LDMs; 1.17 g/cc) is described. The first step is mediated by the hydrolysis of Mg2+ATP and Mg2+GTP, leading to the formation of a transitional ER (tER) with the soluble cargo albumin, transferrin, and the ER-to-Golgi recycling membrane proteins 2p24 and p58 (ERGIC-53, ER-Golgi intermediate compartment protein) enriched therein. Upon further incubation (step two) with cytosol and mixed nucleotides, interconnecting smooth ER tubules within tER transforms into vesicular tubular clusters (VTCs). The cytosolic domain of
2p24 and cytosolic COPI coatomer affect VTC formation. This is deduced from the effect of antibodies to the COOH-terminal tail of
2p24, but not of antibodies to the COOH-terminal tail of calnexin on this reconstitution, as well as the demonstrated recruitment of COPI coatomer to VTCs, its augmentation by GTP
S, inhibition by Brefeldin A (BFA), or depletion of ß-COP from cytosol. Therefore, the p24 family member,
2p24, and its cytosolic coat ligand, COPI coatomer, play a role in the de novo formation of VTCs and the generation of ER cargo exit sites.
Key Words:
cell-free assembly, transitional endoplasmic reticulum, endoplasmic reticulum cargo exit sites, 2p24, COPI
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Introduction |
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CURRENT views on the transport of newly synthesized secretory cargo in eukaryotic cells have been guided by two opposing theories. According to the membrane flow model (
Recently, however, the visualization of large (1 micron diam) structures generated from the ER coincident with cargo transport has given a strong impetus towards establishing the validity of the membrane flow model (
To resolve the controversy, one approach is to identify the proteins that regulate the formation of the early secretory pathway and assign these functions to either the generation of discrete vesicular intermediates or larger membranes undergoing gradual maturation. Identification of such regulatory proteins has relied on three approaches: cell-free intra-Golgi and ER-to-Golgi transport assays to identify and purify molecules regulating protein transport (
Recently, these three approaches have focused on cytosolic coat proteins, as well as their potential integral membrane protein receptors. Thus, the elucidation of the COPI coatomer coat was deduced from Golgi cell-free transport assays (
In the case of the COPI coatomer coat, uncertainty exists as to the direction (direct anterograde versus indirect retrograde) that COPI primarily affects the secretory apparatus (2p24 and its cytosolic COPI ligand is defined in a characterized cell-free assay that reconstitutes the generation of a nascent secretory apparatus from ER-derived low density microsomes (LDMs).
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Materials and Methods |
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Preparation and Characterization of Microsomes
Total microsomes were obtained by differential centrifugation of rat liver homogenates (
Preparation of Rat Liver Cytosol
After centrifugation of the total microsomes at 100,000 gav, 1 mM PMSF, 1 mM DTT, and 0.9 µg/ml leupeptin was added to the supernatant and centrifuged at 200,000 gav for 2 h at 4°C. Ammonium sulfate was added to the resulting supernatant (60% saturation, added from solid). After 1 h of stirring at 4°C, the precipitate was recovered by centrifugation at 10,000 rpm for 30 min at 4°C. Pellets were resuspended in buffer containing 25 mM Tris-HCl, pH 7.4, 50 mM KCl, and 1 mM DTT, desalted in the same buffer by chromatography on a Sephadex G-25 column, and concentrated to 4050 mg protein/ml using an ultrafiltration stirred cell and Diaflo ultrafilter PM10 (Amicon; W.R. Grace and Co.). The precipitate formed at this stage was removed by centrifugation at 200,000 gav for 60 min. The supernatant obtained was aliquoted and stored at -80°C.
Preparation of COPI-depleted Cytosol
Protein ASepharose beads were first incubated in 500 µl KOAc buffer (25 mM Hepes, pH 7.4, 115 mM KOAc, 100 mM NaCl, 2.5 mM MgCl2) containing 15 µg of rabbit antimouse IgG for 30 min at 4°C. Beads were washed three times with KOAc buffer and then incubated with or without 10 µl of anti ß-COP monoclonal IgG in 500 µl of KOAc buffer for 45 min with agitation at 4°C. Beads were finally washed three times with KOAc buffer devoid of NaCl and incubated with 40 µl of rat liver cytosol (37.5 µg/µl), 160 µl KOAc buffer (without NaCl), and protease inhibitor cocktail (0.25 mM benzamidine, 2.5 µg/ml leupeptin, 1 µg/ml soybean trypsin inhibitor) for 45 min at 4°C. The mixture was centrifuged and the supernatant collected, concentrated with Centricon 10 (Amicon, W.R. Grace and Co.), and frozen in aliquots at -80°C.
Cell-free Incubation Conditions
Unless otherwise indicated, the medium consisted of 0.25 ml of buffer containing 150 µg microsomal protein, 100 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM GTP, 2 mM ATP, an energy regenerating system (7.3 IU/ml creatine kinase, 2 mM creatine phosphate), 0.1 mM DTT, 0.02 mM PMSF, 1 µg/ml leupeptin, and 50 mM sucrose. Incubations were carried out at 37°C for 180 min. For studies on the effect of cytosol, 750 µg of rat liver cytosolic protein (in the presence or absence of 1 mM GTPS) was added to the medium described above after 180 min and the incubation was continued for 560 min. To assess the effect of Brefeldin A (BFA; 200 µM final concentration), this reagent was added to the medium 10 min before addition of cytosol. For fusion of classical rough ER, high density rough microsomes were incubated at 37°C in the presence of Mg2+GTP (
2p24 or anticalnexin was added where indicated.
ß-COP Binding Reaction
LDMs (300 µg) were incubated for 180 min to form transitional ER (tER). 200 µM BFA was added or methanol (BFA solvent) and incubation continued for 10 min at 37°C. 3 mg of rat liver cytosol in the presence or absence of 1 mM GTPS were added and reactions were incubated for an additional 15 min at 37°C. The samples were then placed on ice and received 1 ml each of ice-cold washing buffer containing 100 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2, and KOAc (final concentration of 250 mM). The samples were then centrifuged at 16,000 gav for 15 min at 4°C. The supernatant was removed and the pellet subjected to immunoblot analysis using ß-COP specific antibodies (Sigma Chemical Co).
SDS-PAGE and Immunoblotting
Proteins were separated by SDS-PAGE using 715% polyacrylamide gradients. After electrophoresis, the separated proteins were transferred to nitrocellulose membranes. Electrophoretic blotting procedure and immunodetection were carried out as described in
Antibodies
Rabbit polyclonal antibodies against calnexin (2p24 (
Endoglycosidase H and N-Glycosidase F Treatment and Galactosyl Transferase Assay
Deglycosylation experiments were performed according to the recommendation of the supplier (Boehringer Mannheim GmbH). Galactosyl transferase activity using ovomucoid as acceptor was assayed as described by
Thin-Section EM
After incubation, membranes were fixed 12 h using 2.5% glutaraldehyde in cacodylate buffer (100 mM, pH 7.4), recovered onto Millipore membranes (0.45-µm pores) by the random filtration technique of
Morphometry of ER Membranes and Microsomes
Estimates of the lengths of embedded and sectioned rough and smooth membranes in the membrane networks were obtained by morphometry using the membrane intersection counting procedure (
Immunogold Labeling
Immunolocalization of ß-COP was modified from that used by S for 10 min for the generation of vesicular tubular clusters (VTCs). After incubation, the membrane fraction was recovered by centrifugation (1500 rpm for 25 min). Sedimented membranes were resuspended in 750 µl containing 100 mM Tris-HCl, pH 7.4, 60 mM sucrose, and 60 µl antiß-COP. After an incubation of 60 min at room temperature, membranes were fixed using 0.05% glutaraldehyde/cacodylate 0.1 M, pH 7.4, at 4°C for 30 min. After fixation, the membranes were filtered onto Millipore membranes as previously outlined (
2p24 immunolocalization was carried out as follows. LDMs (300 µg protein) were incubated with Mg2+GTP and Mg2+ATP for 180 min, and for an additional 10 min in the presence of cytosol at 37°C. Anti-
2p24 antibodies were added and membranes were incubated an additional 30 min at room temperature. Membranes were then fixed using 0.05% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 4°C and treated for analysis by EM as described above. Unincubated microsomes were incubated with anti-
2p24 for 30 min at 10°C and treated for analysis by EM as described above. For the protein Agold complexes, 10-nm colloidal gold particles were prepared according to
Immunogold Labeling of Cryosections.
After incubation, membranes were fixed using 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 4°C. Cryoprotection, freezing, sectioning, immunolabeling, and contrasting were carried out as previously described by
Analysis of Gold Label Distribution.
For quantification of COPI labeling after incubation of reconstituted membranes with cytosol gold particles associated with rough membranes, SER and VTCs comprising the ER networks were counted. Rough membrane cisternae were defined as large cisternal profiles limited by ribosome-studded membranes. SER were defined as branching and anastomosing tubules limited by membrane devoid of associated ribosomes. In the presence of cytosol, these tubules are transformed into clusters of closely apposed vesicles and convoluted tubules designated VTCs. The number of gold particles over rough ER membranes, as well as those over the combined SER and VTCs that made up the reconstituted networks, were expressed as average number of gold particles per ER membrane network. Counts were compared for different membrane incubation conditions.
For quantification of 2p24, p58, albumin, transferrin, calnexin, and ribophorin labeling on cryosections, gold particles associated with rough membranes and SER comprising the ER networks were counted. Gold particles were counted over parallel juxtaposed ER cisternae (representing rough ER cisternae) and over the adjacent continuous mass of interconnecting membranes (corresponding to interconnecting smooth ER tubules; see Figure 2). Surface area measurements of each compartment comprising the reconstituted ER networks were measured as previously described for ER membranes in situ (
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Cryoimmune EM of Liver Parenchyma.
Rat liver was prepared for ultracryotomy as described previously (2p24 were diluted 1:5 and those to ß-COP were diluted 1:20 in PBS containing 2% BSA/2% casein/0.5% ovalbumin (PBS-BCO). Sections were washed six times for 5 min in PBS followed by blocking in PBS-BCO (5 min) and incubation in appropriate secondary antibodies conjugated to gold particles for 30 min. Sections again were washed six times for 5 min in PBS, six times for 5 min in distilled water, stained for 5 min with uranyl acetate-oxalate solution (pH 7.0), washed twice for 1.5 min in distilled water, and finally transferred to drops of methyl cellulose containing 0.4% aqueous uranyl acetate for 10 min on ice. Grids were picked up with copper loops and excess methyl cellulose was removed with filter paper. Sections were viewed in a Phillips 400 T electron microscope operating at 80 kV. E5A3 mAb to ß-COP was kindly provided by the late Dr. Thomas Kreis, (University of Geneva, Geneva).
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Results |
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Cytosol-dependent Formation of ER Cargo Exit Sites
Previously, we have demonstrated that incubation of LDMs with Mg2+GTP and Mg2+ATP leads to the formation of a partially rough, partially smooth transitional region corresponding to the tER (
In the first step, incubation of LDMs in the presence of Mg2+ATP alone had no effect on the appearance of these microsomes (not shown). However, incubations with Mg2+GTP led to membrane fusion and the formation of large rough ER cisternae (not shown). The majority (75%) of the vesicle profiles closely apposed to the reconstituted cisternae were devoid of associated ribosomes and had an average diameter of 83 ± 29 nm. This vesicle size is similar to the vesicle size of unincubated smooth microsomes (
When a mixture of Mg2+GTP and Mg2+ATP was added, membrane differentiation consequent to membrane fusion was observed (Figure 1 A). As a consequence of mixed nucleotide hydrolysis (
In the second step (Figure 1), further incubation of such networks with cytosol and the same mixture of nucleotides generated VTCs of identical morphology to ER export sites characterized in situ and in detergent permeabilized cells (
Rough ER cisternae within the membrane networks were least affected by incubation in the presence of cytosol and were often observed as parallel rough cisternae, even after 60 min of incubation (not shown). Minitubules (~30 nm in diameter) were often observed in association with ER networks, particularly after treatment with cytosol and mixed nucleotides (Figure 1B, Figure I, and Figure J, mt). These represented membranous structures and not microtubules, since unit membranes were seen encompassing the circumference of the minitubules. Hence, LDMs led to the generation of morphological compartments of the early secretory pathway with membrane fusion and transformation (network formation) dependent on both GTP and ATP hydrolysis (
Distribution of Soluble Cargo and Integral Membrane Proteins
To study cargo and membrane protein distribution in reconstituted ER, gold immunolabeling was carried out after the first step generation of tER. The secretory cargo albumin and transferrin revealed a higher density within SER as compared with the rough ER cisternae (Figure 2A and Figure B) as verified by quantitation (Table 1). A higher concentration in albumin density has also been described in the smooth ER in situ (Dahan, S., M. Dominguez, J. Gushue, P. Melançon, and J.J.M. Bergeron, manuscript submitted for publication).
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The distribution of four integral membrane proteins was also assessed, i.e., ribophorin, calnexin, 2p24, and p58.
2p24, a type I integral membrane protein implicated in cargo sorting and membrane biogenesis, is localized in the cis-Golgi network with ~1/3 found in the ER (
2p24 was found concentrated over buds (Figure 2 C, inset). Quantitation revealed a 2.1-fold concentration for
2p24 in the SER compartment, as compared with the surrounding rough membranes (Table 1). As for
2p24, p58 immunolabeling was observed in the SER (Figure 2 D) at a higher concentration to that of the immunolabeled protein in the fused parallel cisternae (Table 1). By contrast, ribophorin was more concentrated in the parallel rough membranes juxtaposed to the SER of the networks (Figure 2 E; Table 1). The ribophorin distribution along parallel membranes mimics that of the distribution of the ribosomes (not shown), as would be expected since this rough ER marker has been shown to be in equimolar amounts with ribosomes (
2p24, p58, and calnexin, were observed in higher concentrations in reconstituted smooth ER tubules. These opposing protein gradients were maintained, despite the fact that the two membrane subcompartments of the ER were continuous. Because the SER containing albumin and transferrin, as well as
2p24 and p58, transforms into VTCs by the addition of cytosol and mixed nucleotides, it was concluded that ER cargo exit site formation was reconstituted.
To address whether protein concentrations were different in the rough and smooth microsomes before incubation, calnexin and 2p24 protein concentrations were studied in the starting membrane preparation. Direct labeling with antibodies to the cytosolic domain of calnexin and
2p24 using preembedding gold immunolabeling was used to determine the concentrations of the proteins in the membranes. Cryosections were ineffective for our studies, since it was not possible to distinguish rough from smooth vesicles in the starting material. The results revealed gold particles over rough and smooth components (Figure 3A and Figure B). Labeling density was calculated along the surface of rough and smooth microsomes. For
2p24 labeling, gold particle labeling over smooth microsomes (151 vesicles measured, 4.0 gold particles/µm of membrane) was slightly higher, 1.2 times, compared with that over rough microsomes (136 vesicles measured, 3.3 gold particles/µm of membrane). For calnexin labeling, gold particle labeling over smooth microsomes (175 vesicles measured, 5.5 gold particles/µm of membrane) was also slightly higher, 1.2 times, compared with that over rough microsomes (279 vesicles measured, 4.5 gold particles/µm of membrane). Thus, labeling densities for
2p24 and calnexin were slightly higher in smooth microsomes of the starting membrane preparation. Because of the nature of the preembedding immunogold labeling method, we cannot exclude the possibility that ribosomes associated with the surface of rough microsomes may have partially inhibited labeling by steric hindrance. Thus, the labeling ratios may have been closer to unity for both proteins. In any case, a progressive increase in concentration was found following incubation in the presence of Mg2+GTP and Mg2+ATP in which
2p24 increased to 2.1 times and calnexin increased to 1.6 times (Table 1) in density over the SER of the tER, as compared with the surrounding rough ER.
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Organization of Cargo Exit Sites by the Cytosolic Domain of 2p24
The effect of antibodies to the cytosolic domain of 2p24 was tested under different conditions of ER assembly and compared with the effects of antibodies to the cytosolic domain of the ER resident membrane protein calnexin. Anticalnexin did not affect Mg2+GTP-dependent membrane fusion or mixed nucleotide-dependent formation of the tER (Figure 4, ac, control, df, anticalnexin). In contrast, based on the reduced size of the membrane networks, anti-
2p24 inhibited GTP-dependent membrane fusion (Figure 4 h), as well as mixed nucleotide dependent formation of the tER (Figure 4i and Figure j). Quantitation confirmed the effect of anti-
2p24 on the formation of networks with little effect noted by anticalnexin (Figure 4 k).
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As a further control, neutralization experiments were attempted. The effect of anti-2p24 was neutralized by its antigenic peptide, but not by a peptide corresponding to the cytosolic domain of the ER membrane protein calnexin (Figure 4 l). Evidence for the efficacy of binding of anticalnexin antibodies to reconstituted ER membranes was observed by immunogold labeling of cryosections (Table 1) and reconstituted ER membranes using preembedding immunolabeling (Figure 3 B and 4 o), and confirmed by immunoblot analysis of unincubated membranes (data not shown). Hence, the cytosolic domain of
2p24, but not of calnexin, modulated step one of the cell-free assembly system that led to the generation of a tER as caused by mixed nucleotide hydrolysis.
Because quantitation also confirmed the effect of the anti-2p24 antibody on membrane fusion effected by GTP hydrolysis alone (Figure 4 m), this was compared with GTP-mediated membrane fusion of classical rough ER (high density rough microsomes). Membrane fusion of such rough microsomes required the prior removal of the associated ribosomes (
2p24 (or calnexin; Figure 4 n). These results further attest to the distinct microdomains that can be distinguished by these membrane fusion assays. Furthermore, when high density microsomes stripped of associated ribosomes were incubated with LDMs, the former were unable to participate in tER formation (data not shown). Therefore, there are two populations of rough ER that can be separated by subcellular fractionation. One is involved in the assembly of tER and is isolated as LDMs, the other is involved in the formation of large rough ER cisternae and is isolated as high density microsomes, and the two exhibit different fusion properties.
Finally, the effect of anti-2p24 antibodies on cytosol-dependent loss of SER in reconstituted ER networks was tested. Incubation of reconstituted ER networks with anti-
2p24 antibodies before incubation with cytosol led to inhibition of loss of SER within ER networks. In three separate experiments, a total of 106 reconstituted networks were analyzed, and of these networks, 42 ± 4% had recognizable smooth tubules. In contrast, membrane networks incubated with anticalnexin antibodies before treatment with cytosol lost most of their associated smooth tubules. In three separate experiments, a total of 71 reconstituted networks were analyzed, and of these networks, 20 ± 14% had recognizable smooth tubules. Thus, antibodies to the cytosolic tail of
2p24 led to partial inhibition of cytosol-dependent loss of SER in reconstituted ER networks.
Recruitment of COPI Coatomer
The sites of location of 2p24 antigenicity using preembedding gold immunolabeling was studied after induction of VTC formation in the presence of cytosol. Gold immunolabeling revealed
2p24 antigen in vesicular-tubular structures associated with fused rough ER (Figure 5). The reduced gold immunolabeling is thought to be due to the masking of determinants of the cytosolic domain of
2p24 caused by binding of proteins to the membranes during preincubation in the presence of cytosol. The cytosolic domain of
2p24 binds COPI and COPII coatomer with high affinity and a specificity attributed to the KKXX motif at its COOH-terminal domain for COPI and a diphenylalanine-based motif affecting COPII binding (
2p24 cytosolic domain on the cell-free system by studying the role of COPI coatomer.
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The predicted binding of COPI to the cell-free system at the ER cargo exit sites (VTCs) was tested following incubations at step two of the reconstitution system with cytosol, Mg2+ATP, and the nonhydrolyzable GTP analogue, GTPS. Biochemical studies (Figure 6 A) revealed an augmented association of ß-COP to membranes. Visualization of COPI coatomer during these incubations (Figure 7i) revealed an association of ß-COP with the VTCs generated after the tER was incubated with cytosol and Mg2+GTP/ATP (Figure 7i B) or with Mg2+ATP and Mg2+GTP
S (Figure 7i, Figure C and Figure D). Little labeling with antiß-COP was found in the absence of cytosol (Figure 7i A). Quantitation of gold particle distribution confirmed the cytosol and GTP
S-dependent association of ß-COP with the VTCs (Figure 7ii A).
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Quantitation was also carried out to determine the effects of cytosol on the formation of VTCs. The amount of the SER remaining in the reconstituted membrane networks after treatment with cytosol was used as a measure of the amount of transformation of the tER into VTCs. Percent number of networks with VTCs was also calculated. Thus, a diminution of the amount of SER and a coincident increase in amount of associated VTCs was observed after incubation of reconstituted ER networks in the presence of cytosol plus Mg2+ATP/GTP or Mg2+ATP/ GTPS (Figure 7ii B). A prediction of these results is that VTC formation should be sensitive to the fungal metabolite BFA via its action on inhibiting an ARF1-GEF activity (
2p24 and ß-COP Are Localized to ER and Golgi Elements in Rat Liver Hepatocytes
As determined by cryoimmune EM using well characterized antibodies specific to 2p24 (
2p24 (Figure 8 A). Tubulo-vesicular smooth ER networks in the Golgi region of hepatocytes, as well as the cis-Golgi intermediate compartment, were also labeled by anti-
2p24 (Figure 8B and Figure C). The COPI coatomer subunit ß-COP reveals a distribution that overlaps that of
2p24 in liver parenchyma (Figure 8D and Figure E). Thus,
2p24 and ß-COP are associated with similar structures, including tubular-vesicular elements of the ER often found next to the Golgi apparatus in situ within the rat hepatocyte.
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2p24 in LDMs Is Golgi Apparatus-derived
The cargo molecules albumin and transferrin represent newly synthesized protein cargo of the ER. However, this was unlikely to be the case for 2p24 (and p58) observed in reconstituted ER or for
2p24 observed in hepatocyte ER in situ by immunolabeling. Whether in preparations of LDMs, or even in highly purified stripped rough microsomes (SRM),
2p24 is terminally glycosylated as evident from its lack of sensitivity to endoglycosidase H (endoH), but complete sensitivity to PNGase F (Figure 9). Equal amounts of protein from each fraction was applied to each lane (100 µg).
2p24 is also highly enriched in Golgi fractions, due to its abundance in the cis-Golgi network that coisolates with hepatic Golgi fractions (
2p24 found in the liver ER fraction employed in the in vitro ER reconstitution assay is terminally glycosylated, and thus a molecular derivative of the Golgi apparatus.
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Discussion |
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A Cell-free System to Study Assembly of tER and the Formation of ER Exit Sites
A two-step in vitro reconstitution system starting from well-characterized ER-derived LDMs purified from rat liver homogenates (
The Role of Membrane Fusion in Assembly of tER
The nucleotide-dependent fusion of LDMs to yield tER is thought to involve both fusion of like (homotypic) and unlike (heterotypic) ER membrane derivatives. Mg2+GTP hydrolysis is required to stimulate fusion of partially rough ER membrane derivatives and Mg2+ATP hydrolysis is required to stimulate fusion of smooth ER membrane derivatives. Hence, these nucleotides contribute to homotypic membrane fusion. At some stage during tER formation, continuity is established between these two ER subcompartments and this would be expected to occur by heterotypic membrane fusion.
However, if partially rough microsomes containing microdomains of smooth ER initially fused in the presence of Mg2+GTP, this would permit subsequent Mg2+ATP-dependent fusion with additional smooth microsomes and obviate a necessity for heterotypic membrane fusion. This possibility has not been ruled out yet. The assays developed here using quantitative morphology and quantitative immunolabeling may now be used to screen for antibodies to proteins that can distinguish between the homotypic and heterotypic membrane fusion events.
Rough ER Membranes within tER Exhibit Unique Fusion Properties
The rough ER subcompartment comprising the tER assembled when LDMs are incubated in the presence of Mg2+GTP and Mg2+ATP is very different from classical rough ER, which is recovered from tissue homogenates as high density rough microsomes. Although high density rough microsomes undergo GTP-dependent fusion, as do low density rough microsomes, the fusion events are different. For example, antibodies to 2p24 inhibit fusion of the partially rough ER comprising tER, but not that of classical rough ER (Figure 4 n). Fusion of classical rough ER requires prior removal of associated ribosomes (
2p24 may affect the generation of tER by influencing the heterooligomerization of p24 family members (
2p24 and p58 in tER
The membrane proteins 2p24 and p58 were found in microdomains of the tER. These membrane proteins are found at steady state to be enriched in the cis-Golgi network and ERGIC compartments (
2p24 was terminally glycosylated (endoH resistant) in LDMs, and even in highly purified rough ER membranes, is consistent with a constantly recycling scenario for
2p24.
In the starting preparation of LDMs, 2p24 was found in slightly higher concentration in smooth microsomes. After incubation in step one conditions, a higher concentration of the protein was observed in the SER. The evidence suggests that segregation of
2p24 into SER occurred coincident with tER formation.
The membrane proteins p58 and 2p24 share KKXX motifs at their COOH termini, and in vitro binding assays show that the cytosolic domains of these membrane proteins bind COPI coatomer, and unexpectedly, COPII coatomer as well (
2p24 in VTCs formed in the presence of cytosol and the in vitro reconstitution assay revealed inhibition of tER formation by antibodies to the cytosolic domain of
2p24. Antibodies to the cytosolic domain of p58 inhibit ER-to-Golgi transport, as well as COPI coatomer binding (
2p24, possible confounding effects of steric hindrance by antibodies to an abundant protein cannot be excluded. Remarkably, in our studies, antibodies to the cytosolic domain of
2p24 affected the surrounding parallel rough ER cisternae themselves. With Mg2+GTP as sole nucleotide, this membrane fusion step could be studied in isolation. This fusion step was specifically inhibited by antibodies to the cytosolic domain of
2p24, but not calnexin. Because this step was shown to be an early event in the formation of tER (
2p24 in affecting the formation of ER cargo exit sites extends to the rough ER portion of the rough/smooth ER boundary of the tER. This boundary is a predicted consequence of incoming retrograde smooth membranes derived from the Golgi apparatus and outgoing anterograde rough membranes transforming into intermediate compartment elements.
Mistargeting of a mutated form of ERGIC-53 to the ER of HeLa cells was shown to impair secretion of a lysosomal enzyme while apparently not affecting gross (light microscope) morphological changes of the early secretory pathway (
Role of 2p24 and its COPI Coatomer Ligand in ER Cargo Exit Site Formation
An effect of COPI coatomer on VTC formation was found. This was concluded from the visualization of ß-COP in VTCs after the addition of cytosol, the enhancement of VTC formation and ß-COP association with VTCs by cytosol with GTPS, the BFA sensitivity of VTC formation, and the inhibition of VTC formation when ß-COP was depleted from cytosol. These coincidental observations are consistent with, but do not prove a direct link between COPI coatomer and the cytosolic domain of
2p24 in the formation of ER cargo exit sites. Indeed, we cannot completely rule out the possibility that binding of antibodies to
2p24, but not calnexin, affects the ability of other abundant membrane proteins in microsomes to access coat proteins. These observations do, however, provide a structural explanation for the observations that ARF1 dominant negative mutants (
Roles for 2p24 and its COPI coatomer ligand in ER cargo exit site formation have been suggested by results obtained using the novel two-step reconstitution system described. Based on available data, the simplest explanation for the possible involvement of these two proteins is that
2p24 promotes assembly of tER and COPI coatomer is required for subsequent formation of VTCs. The structural modifications implicating
2p24 and COPI involvement are summarized in diagrammatic form (Figure 10). Because
2p24 is known to bind COPI coatomer, and since anti-
2p24 antibodies were observed to inhibit cytosol-dependent transformation of tER, we cannot exclude a role for
2p24 in the last step of formation of ER exit sites.
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A Possible Structural Role for the p24 Family of Proteins
A structural role for 2p24 may explain all properties thus far documented for the p24 family of proteins. This would include their enrichment in vesicles derived from ER cargo exit sites and the physical association of p24 family members as heterooligomers in yeast (
COPI and Anterograde Transport
The role of COPI coatomer (and consequently the ARF1 GTPase) as a defining feature required for the generation of membranes of the early secretory apparatus has been argued by
The identification of motifs (FF) involved in COPII binding (
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Acknowledgements |
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We thank Dr. Peter McPherson (McGill University) for criticism of the manuscript and Dr. Tommy Nilsson for support and advice throughout these experiments. We thank Dr. Jacopo Saraste for kindly supplying antibodies to p53. We thank Anne Guénette and Ali Fazel for expert assistance, and Jean Léveillé for photographic assistance.
This work was supported by grants from the Medical Research Council of Canada to J. Paiement and J.J.M. Bergeron. C. Lavoie was a recipient of a studentship from the Medical Research Council of Canada.
Submitted: September 2, 1998; Revised: May 17, 1999; Accepted: May 19, 1999.
1.used in this paper: BFA, Brefeldin A; endoH, endoglycosidase H; ERGIC-53, ER-Golgi intermediate compartment protein; SER, interconnecting smooth ER tubules; LDMs, low density microsomes; tER, transitional endoplasmic reticulum; VTCs, vesicular tubular clusters
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