Differential localization of coatomer complex isoforms within the Golgi apparatus

  1. Jörg Moelleken*,
  2. Jörg Malsam*,
  3. Matthew J. Betts†,
  4. Ali Movafeghi‡,
  5. Ingeborg Reckmann*,
  6. Ingrid Meissner*,
  7. Andrea Hellwig§,
  8. Robert B. Russell†,
  9. Thomas Söllner*,
  10. Britta Brügger*, and
  11. Felix T. Wieland*,
  1. *Biochemistry Center and
  2. §Department of Neurobiology, University of Heidelberg, 69120 Heidelberg, Germany;
  3. †Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany; and
  4. ‡Department of Plant Biology, Faculty of Natural Sciences, Tabriz University, Tabriz, Iran

  1. Communicated by James E. Rothman, Columbia University Medical Center, New York, NY, December 20, 2006 (received for review December 8, 2006)

 
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Abstract

Coatomer, the coat protein of coat protein complex (COP)I-vesicles, is a soluble protein complex made up of seven subunits, α-, β-, β′-, γ-, δ-, ε-, and ζ-COP. Higher eukaryotes have two paralogous versions of the γ- and ζ- subunits, termed γ1- and γ2-COP and ζ1- and ζ2-COP. Different combinations of these subunits are known to exist within coatomer complexes, and γ1/ζ1-, γ1/ζ2-, and γ2/ζ1-COP represent the major coatomer populations in mammals. The role of COPI vesicles in the early secretory pathway is the subject of considerable debate. To help to resolve this discussion, we used quantitative immunoelectron microscopy and found that significant localization differences for COPI-isoforms do exist, with a preference for γ1ζ1- and γ1ζ2-coatomer in the early Golgi apparatus and γ2ζ1-coatomer in the late Golgi apparatus. These differences suggest distinct functions for coatomer isoforms in a manner similar to clathrin/adaptor vesicles, where different adaptor proteins serve particular transport routes.

Intracellular vesicular transport is mediated by small protein-coated carriers. Various functions are attributed to coated vesicles, which are defined by their constituent coat proteins. Isoforms with different combinations of coat protein complex (COP)II protein paralogs have been found in the COPII coats on vesicles budding from the endoplasmic reticulum (ER) of mammals and yeast (1–3). Because only one direction of travel from the ER to the Golgi apparatus is known for COPII vesicles, these isoforms might reflect differing cargo specificities (4). In the late Golgi/endocytic pathways, a variety of coat proteins are known, including four different tetrameric adaptor complexes, some of which use the clathrin system as an outer shell of their coat (5). Within these pathways, particular transport routes are served by specific coat complexes (for review, see refs. 6 and 7). Thus, for particular pathways, the selection of cargo as well as the direction might be determined by individual coats.

Just which pathways are mediated by vesicles bearing the COPI coat is the subject of considerable debate. The COPI coat consists of coatomer, which is a stable cytoplasmic complex of seven subunits, α-, β-, β′-, γ-, δ-, ε-, and ζ-COP, and the small GTP-binding protein ADP-ribosylation factor, Arf1. COPI vesicles were found to originate from the Golgi apparatus and are implicated in transport within the Golgi itself in anterograde and retrograde direction, and from the Golgi and the ER–Golgi intermediate compartment (ERGIC) to the ER (8–10). The observation that COPI vesicles participate in several transport directions is difficult to rationalize with just a single isoform of the complex (reviewed in ref. 11). Questions about the function of COPI vesicles are intimately linked to our view of the general mechanisms of transport through the Golgi. Specifically, is transport in both directions, anterograde and retrograde, performed by these carriers, or are COPI vesicles exclusively retrograde carriers within the Golgi and from the Golgi to the ER? Moreover, is anterograde transport across the Golgi exclusively mediated by maturation of Golgi cisternae (12, 13)?

Genome analyses identified a second isoform for each of the two coatomer subunits, γ- and ζ-COP (14), the paralogs γ2- and ζ2-COP. Previously, the corresponding cDNAs were expressed as tagged constructs (15), and the endogenous gene products were shown to be incorporated into coatomer complexes in a stoichiometric manner, which resulted in three major isoforms of the complex with the compositions γ1ζ1-, γ1ζ2-, and γ2ζ1-COP (16). To extend this work, we have prepared polyclonal antibodies specific against γ1-, γ2-, and ζ2-COP and have investigated the localization of coatomer isoforms in NRK cells by using immunoelectron microscopy. We found a striking heterogeneity within the Golgi apparatus: whereas γ1- and ζ2-COP seem restricted to the early Golgi and a pre-Golgi compartment, the majority of γ2-COP-containing isoforms of the complex is localized to the trans side of the organelle. These results strongly suggest the existence of different homogeneously coated isoforms of COPI vesicles that might serve multiple transport routes within the early secretory pathway.

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Results

γ- and ζ-COP Subunits from Other Species.

Sequence searches of γ- and ζ-COP subunits against completely sequenced genomes were used to identify the protein families to which they belong. The resulting peptide sequences were aligned [see supporting information (SI) Table 1], and phylogenetic trees were built from these alignments. The trees strongly support concurrent duplication of γ- and ζ-COP into two paralogs in the vertebrates, sometime after their divergence from Ciona intestinalis (SI Figs. 5 and 6). Ciona, plants, insects, fungi, and the malaria parasite Plasmodium falciparum all have one γ- and ζ-COP isoform each, with very recent duplications and/or alternative transcripts (possibly annotation artifacts) that cluster together, implying that they are duplications specific to a particular species. Absences of isoforms in some vertebrates might be an artifact of unfinished genomes, whereas the fish appear to have really lost γ1-COP after the duplication, because none of the three fish species examined have γ1-COP. The appearance of multiple isoforms of coatomer might be linked to the evolution of complex, multicellular organisms with cells processing a much wider range of cargo proteins.

Generation and Characterization of Antibodies Directed Against COP Subunit Isoforms.

γ-COP resembles γ-adaptin, which is made of an N-terminal “trunk” domain and a C-terminal “appendage” domain (17–20). The appendage domains are generally the least similar in sequence between γ1- and γ2-COP; for example, in mouse, the sequence identity is 75% in the appendage domains, compared with 81% in the trunk. We therefore expressed constructs in Escherichia coli that contained the appendage domains of γ1- and γ2-COP linked to GST. The purified products were used to raise antibodies in rabbits, and the resulting antisera (anti-γ1-app and anti-γ2-app) were characterized by using immunoprecipitation experiments, Western blotting, and immunofluorescence microscopy. Both antisera are able to precipitate coatomer from cytosol (Fig. 1 A). Antibodies that are directed against β′-COP (lane 1), a subunit of coatomer that is shared by all complex isoforms, or CM1 antibodies (lane 3), which are directed against native coatomer, precipitate all isoforms of coatomer (containing γ1-, γ2-, ζ1-, and ζ2-COP). However, anti-γ1-app antibodies and anti-γ2-app antibodies specifically precipitate coatomer containing γ1-COP (lane 5) or γ2-COP (lane 7), respectively. Likewise, an antibody directed against an N-terminal sequence of ζ2-COP (16) showed a remarkable preference for coatomer containing ζ2- and γ1-COP (lane 11). As a control, an unrelated antibody (lane 2 and 8) and the corresponding preimmune sera (lanes 4, 6, and 10) do not significantly precipitate coatomer.

Fig. 1.
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Fig. 1.

Specificity of the antibodies used. (A) Immunoprecipitations (IP) with cytosol were performed with anti-β′-COP (lane 1), CM1 (lanes 3 and 9), anti-γ1-app (lane 5), anti-γ2-app (lane 7), and anti-ζ2-COP (lane 11) antibodies. The eluates were separated by using SDS/PAGE and analyzed by Western blotting, using the indicated antibodies. Okt8 (lanes 2 and 8) is a CD8-specific antibody. Lanes 4, 6, and 10 reflect corresponding γ1-, γ2-, and ζ2-preimmune sera, respectively. (B) Indirect immunofluorescence analysis using anti-γ1-app (b), anti-γ2-app (f), and anti-ζ2-COP (k) antibodies shows a typical perinuclear staining. The γ1-COP signal can be eliminated by coincubation of the antibody with GST-γ1-app (c) but not GST-γ2-app protein (d). The γ2-COP signal is unaffected by a coincubation of GST-γ1-app (g) but is decreased drastically in the presence of GST-γ2-app protein (h). That the ζ2-COP signal is significantly reduced in the presence of the corresponding peptide (l) indicates the specificity of this antibody. Micrographs a, e, and i show the signal background of the corresponding Alexa-fluor-conjugated secondary antibodies in the absence of primary antibodies.


We investigated the specificity of the antibodies further by using immunofluorescence microscopy (Fig. 1 B). For this investigation, fixed and permeabilized 3T3/NIH fibroblasts were incubated with the various antisera, and bound antibodies were visualized by using fluorescence-labeled secondary antibodies. The antisera against γ1-COP (Fig. 1 Bb), γ2-COP (Fig. 1 Bf), and ζ2-COP (Fig. 1 Bk) gave rise to a pattern typical of the Golgi apparatus, which colocalizes with coatomer markers (data not shown). The staining could be blocked by competition from the respective protein constructs (Fig. 1 Bc and Bh) and by a peptide representing the N-terminal domain of ζ2-COP (Fig. 1 Bl). Coincubation of the anti-γ1-app antiserum with GST-γ2-app protein (Fig. 1 Bd) and the anti-γ2-app antiserum with GST-γ1-app (Fig. 1 Bg) did not eliminate specific binding. Therefore, these antibodies can be used for immunocytochemistry and immunoprecipitation with high specificity for γ1-COP-, γ2-COP-, and ζ2-COP-containing coatomer. No antibody with specificity for ζ1-COP was available for these applications. Because coatomer is a stable complex that is recruited to the donor membrane en bloc (21), these antibodies can be used to analyze the stoichiometry and localization of coatomer isoforms.

Stoichiometry of Coatomer Isoforms.

The antibodies were used to reassess the stoichiometry of the various coatomer isoforms in a mammalian cytosol. Immunoprecipitations were performed on 3T3/NIH cytosol, and the individual subunits of precipitated coatomer were quantified by using the recombinant GST constructs that contained the γ1- or γ2-appendage domain and recombinant His-tagged versions of ζ1- and ζ2-COP as mass standards (Fig. 2). We found a 70:30 ratio of γ1- to γ2-COP in total coatomer after immunoprecipitation with the CM1 antibody (directed against all coatomer isoforms) (Fig. 2 Aa and Ab). Similarly, immunoprecipitations with anti-γ1-app or anti-γ2-app antibodies gave a ζ1- and ζ2-COP ratio of 77:23 in γ1-coatomer isoforms (Fig. 2 Ac and Ad) and a 86:14 ratio in γ2-COP that contained immunoprecipitates (Fig. 2 Ae and Af). These numbers allowed the calculation of the relative proportion of coatomer isoforms, as shown in Fig. 2 B. Isoforms of coatomer containing γ1/ζ1-, γ1/ζ2-, and γ2/ζ1-COP were found at a ratio of ≈10:3:5. The γ2ζ2-coatomer is found in a much smaller quantity, which is probably close to the sensitivity limit of the analytical method used.

Fig. 2.
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Fig. 2.

Quantification of coatomer isotypes. (A) Immunoprecipitations (IPs) using CM1 (a and b), anti-γ1-app (c and d), or anti-γ2-app (e and f) antibodies were performed with freshly prepared cytosol. Calibration curves obtained from Western blot analyses with GST-γ1-app, GST-γ2-app, His6-ζ1-COP, or His6-ζ2-COP proteins allowed the determination of the absolute amounts of protein in the samples. To the right of the quantification blots are the molar ratios, taking molar masses into account. (B) Diagram showing the relative proportion of coatomer isotypes. Error bars represent the SD about the mean.


Intra-Golgi Localization of the Coatomer Isoforms Containing γ1-, γ2-, and ζ2-COP.

The antibodies were used for immunogold labeling on ultrathin cryosections of NRK cells. Primary antibodies were visualized with protein A that was labeled with 15-nm gold. To validate the polarity of the Golgi sections investigated, antibodies that were directed against the well established cis-Golgi marker protein GM130 were used (22). This marker antibody was visualized with 10-nm gold bound to protein A. Representative labeling patterns are shown in Fig. 3. Whereas preferential gold staining is observed at the cis half of the Golgi with antibodies directed against γ1- (Fig. 3 A and B) and ζ2-COP (Fig. 3 E), the γ2-coatomer (Fig. 3 C and D) is restricted more to the trans half of the organelle. As expected, labels of antibodies directed against the common subunit β′-COP are observed throughout the Golgi apparatus (Fig. 3 F). Images that were obtained with the various antibodies were evaluated quantitatively by separating Golgi areas along an arbitrary middle line, defining a cis half and a trans half of the organelle, and counting the gold dots in each section. As shown in Fig. 4, ≈70% of γ1-COP-containing coatomer was found in the cis half. The ζ2-COP coatomer showed an even stronger preference, with ≈80% localized to the early Golgi. The γ2-coatomer, in contrast, showed a distribution of >60% trans and <40% cis. As a control, only a slight preference, <55% cis and >45% trans, was observed when overall coatomer localization was studied with the anti-β′-COP antibody (Fig. 3 F). This is in agreement with a previous study in endocrinic pancreatic cells (23). The stringent localization of the cis-Golgi marker protein GM130 (22) to only one half of the Golgi corroborates the validity of the analytical method used. The marked localization of ζ2-coatomer to the early Golgi has led us to investigate this finding in further detail, by comparison with the KDEL receptor, a resident of the ERGIC and the cis-Golgi (24, 25). A pattern was obtained as depicted in Fig. 3 G, where the antibodies against the KDEL receptor were labeled with 10-nm gold, and antibodies against ζ2-COP with 15-nm gold. Again, a significant preference of ζ2-coatomer for the ERGIC and the cis-Golgi was observed. Taken together, the significant differential localization of coatomer isoforms suggests that they serve different functions within the Golgi apparatus.

Fig. 3.
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Fig. 3.

Localization of isotypic coatomer subunits within the Golgi. Immunogold labeling was performed on ultrathin slices of 2% paraformaldehyde/0.2% glutaraldehyde-fixed NRK cells. Shown are representative pictures of Golgi stacks labeled with anti-γ1-app (A and B), anti-γ2-app (C and D), anti-ζ2-COP (E), anti-β′-COP (F), and anti-ζ2-COP antibodies (G), each bound to 15-nm protein A-gold. Double labeling was performed by using anti-GM130 (A–F) or anti-KDEL receptor antibodies (G), and 10-nm protein A-gold. Average labeling densities per μm2 of total Golgi were 25.0 ± 2.2 for γ1-COP, 27.7 ± 2.8 for γ2-COP, 13.3 ± 1.4 for ζ2-COP, 38.2 ± 5.5 for β′-COP, and 40.3 ± 3.8 for GM130.


Fig. 4.
View larger version:
Fig. 4.

Quantification of coatomer isoforms in the cis and trans halves of the Golgi. The relative labeling densities within the cis half and the trans half of the Golgi with the indicated antibodies are shown. The labeling densities were normalized to allow comparison of independent experiments. The amount of Golgi stacks analyzed is indicated for each antibody. The cis-Golgi marker GM130 was used as an internal standard to validate polarity of the Golgi; therefore, the number of Golgi stacks analyzed for GM130 reflects the sum of all Golgi stacks that were taken into account. Error bars represent the SD about the mean.


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Discussion

We have identified clear coatomer isoforms with different combinations of the γ- and ζ-COP subunits in the vertebrates. These isoforms, like the other five COPs shared by all coatomers, are present stoichiometrically in their constituent complexes (16). Phylogeny strongly suggests that this diversification of γ- and ζ-COP subunits is a vertebrate innovation. Such gene family expansions are common in vertebrate-specific processes, such as the immune system, olfaction, and particular developmental processes (26).

Using new antibodies available against the two γ-COP subunits, we have reassessed the relative abundance of coatomer in mammalian cytosol and have found coatomer isoforms that contain the subunits γ1/ζ1-, γ1/ζ2-, and γ2/ζ1-COP in a ratio of ≈10:3:5. Coatomer with γ2ζ2-COP, if it exits, represents <5% of the total.

Strikingly, the coatomer isoforms are differentially distributed within the Golgi apparatus, with γ1-COP found preferentially in the early Golgi and γ2-coatomer found preferentially in the late Golgi. A double labeling experiment with the KDEL receptor, an ERGIC/cis-Golgi marker (27, 28), underscores the preference of ζ2-coatomer for these compartments.

The distinct localization of individual coatomer isoforms strongly suggests the presence of various types of COPI vesicles in the cell, each of which is homogeneously coated with a defined isotypic coatomer complex. Given the pattern emerging from immunoelectron microscopy, gradients are highly likely to exist in the relative abundance of such vesicles, with more γ1- and ζ2-COPI vesicles in the early Golgi and more γ2 vesicles in the late Golgi. Alternatively, or in addition, COPI vesicles that are enveloped by various mixtures of coatomer isoforms might exist (although this possibility is less likely given, e.g., the strong preference of ζ2-coatomer for the early Golgi).

At present, it is unknown how the specific coatomer isoforms are recruited to a certain Golgi cisterna. Interestingly, the difference of the isoforms lies in their γζ-subcomplexes, and it is γ-COP that has two binding sites for oligomeric forms of the members of the p24 family of membrane proteins (29) that constitutively cycle between ER and Golgi (30–33). Differential γζ-COP combinations might well have different affinities to the cytoplasmic tails of these type 1 transmembrane proteins or to different combinations thereof. Thus, if these proteins or their heteromeric complexes were unequally distributed across the Golgi, their differential interaction with coatomer isoforms could explain differential recruitment of the complex. Indeed, a preferential localization of p23, p24, and p27 at the cis-Golgi has been shown (32–34). Additionally, individual coatomer isoforms might differ in their affinity for the cytoplasmic tails of various Golgi-resident membrane proteins, causing differential recruitment to individual cisternae. Indeed, COPI vesicles that contain either p24 proteins or the Golgi-resident enzymes mannosidase I and II have been described (35). Likewise, strong evidence for functionally different COPI vesicles comes from an immunoelectron microscopy study in which two distinct populations of COPI vesicles were defined by inclusion of either the KDEL receptor or proinsulin (23). Similarly, GOS28, a Golgi v-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor), was found in COPI vesicles devoid of the KDEL receptor and vice versa (36). The existence of different populations of COPI vesicles had been deduced previously, based on the analysis of carriers immuno-isolated with antibodies directed against p27 (37). However, because these carriers were uncoated, it was not completely clear whether they were derived from COPI-coated vesicles.

A variety of functions have been attributed to COPI vesicles. These include the transport of proteins cycling between the Golgi and the ER and the retrieval of proteins that have escaped the ER (10, 38). Originally, COPI vesicles were defined and characterized as carriers for biosynthetic cargo across the Golgi apparatus, and various anterograde cargo proteins were found to be passengers (23, 39–42). Later, in the context of explaining anterograde transport mediated by the maturation process of cisternae of the organelle, a role was attributed to COPI vesicles as carriers for residential proteins that need to be sorted back to allow the individual cisternae to achieve and maintain their identity (11, 13, 43).

Sequence similarities exist between the clathrin/adaptor proteins and coatomer. The tetrameric adaptor complex resembles a coatomer “core” made up of β-, δ-, γ-, and ζ-COP (17, 42, 44, 45), and there is a distant similarity of the trimeric subcomplex α-, β′-, and ε-COP with the clathrin proteins (18). Furthermore, the analogy to the clathrin system became even more apparent when the appendage domain of γ1-COP revealed a striking structural similarity to the appendage domains of α- and β-adaptin (19, 20).

Our observations of different distributions strongly suggests that coatomer complex isoforms serve different functions, within the early secretory pathways, that are similar to the various functions individual adaptor proteins exert in the late secretory pathways (5). Thus, it becomes unlikely that COPI vesicles exclusively mediate back transport of Golgi-resident proteins during maturation of Golgi cisternae.

Further work will be needed to focus on the isolation and proteomics of individual COPI vesicles to elucidate their functions. With a preference for the cis-Golgi and ERGIC, for example, γ1ζ2-coatomer is a likely candidate to form vesicles for recycling and retrieval of proteins from these organelles to the ER. Determining the particular functions of the complex isoforms will be crucial for a better understanding of the Golgi within the vertebrates.

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Materials and Methods

Antibodies.

The following polyclonal antibodies were used: anti-α-COP (CT peptide) (F.T.W.); anti-β-COP (46); anti-β′-COP (47); anti-ζ1-COP (peptide, human, amino acids 63–74) (F.T.W.); anti-ζ2-COP (16); and anti-KDEL receptor (Erd2p, kindly provided by the late H.-D. Söling, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany). The monoclonal antibodies that were used are as follows: anti-GM130 (BD Transduction Laboratories, San Jose, CA); Okt8 (48); CM1 (49). HRP-conjugated secondary antibodies: goat anti-mouse and donkey anti-guinea pig (Dianova, Hamburg, Germany) and mouse anti-rabbitnative (RG16) (Sigma–Aldrich, Munich, Germany). True Blot was obtained from eBiosciences (San Diego, CA). Alexa-fluor-conjugated secondary antibodies were obtained from Molecular Probes (Leiden, The Netherlands). Preimmune serum was obtained from the rabbits that were used to raise antibodies.

Preparation of Cytosol from Cultured Cells.

3T3/NIH cells grown to confluency on 15-cm dishes were washed three times with PBS and scraped off in the presence of 0.25 ml of immunoprecipitation buffer (25 mM Tris·HCl, pH 7.4/150 mM NaCl/1 mM EDTA) that was complemented with EDTA-free protease inhibitor. All further steps were performed at 4°C. The cells were passed 20 times each through 21- and 27-gauge needles. The homogenate was centrifuged at 800 × g for 5 min, and the resulting supernatant was subjected to a further centrifugation step at 100,000 × g for 60 min. Typically, the high-speed supernatant (cytosol) had a protein concentration of 2 mg/ml.

Immunoprecipitations and Western Blotting.

Antibodies were incubated with protein A-Sepharose (CL-4B) (Amersham, Little Chalfont, U.K.) on a rotating wheel in immunoprecipitation buffer for 60 min at room temperature. After washing three times with immunoprecipitation buffer, freshly prepared cytosol (0.5 mg/10 μl of beads) was added, and incubation continued for 60 min at room temperature. Beads were collected by centrifugation, extensively washed, and eluted in reducing SDS sample buffer at 70°C. Samples were subjected to SDS/PAGE and analyzed by Western blotting, using the indicated antibodies. To reduce background when rabbit antibodies were used for immunoprecipitation and subsequent Western blot analysis, monoclonal mouse anti-rabbitnative secondary antibodies (RG16) (Sigma–Aldrich) were applied. True Blot was obtained from eBiosciences.

Cloning and Expression of γ1- and γ2-Appendage Domains.

mRNA was isolated from 3T3/NIH mouse fibroblasts with oligo(dT)25 Dynabeads (Dynal Biotech, Hamburg, Germany) according to the manufacturer's protocol. Reverse transcription was performed by using the 5′ RACE System V 2.0 kit (Life Technologies, Paisley, Scotland). The regions corresponding to the γ1- and γ2-COP appendage domains (nucleotides 1663–2625 and 1663–2616, respectively) were amplified by using PCR with the following primer pairs: 5′-TCT AGA ATC CCT GGT CTG GAG AAA GCC CTG-3′ (forward) and 5′-GGA TTC CTA GCC CAC GGA TGC CAA GAT GAT-3′ (reverse) for γ1 and 5′-GCT AGC ATA CCA GGG ATG GAA AAG GCC TTA-3′ (forward) and 5′-GGA TTC TTA TCC CAC AGA AGC CAA GAT AAC ATC-3′ (reverse) for γ2. γ1-COP was cloned as an XbaI/BamHI- and γ2-COP as an NheI/BamHI fragment in the expression vector pPro-GST-TEV (kindly provided by M. Lutzmann, Heidelberg University Biochemistry Center). GST-γ1-app and GST-γ2-app were over-expressed in E. coli BL21(DE3) (Promega, Madison, WI) within 4 h at 37°C after induction with 1 mM isopropyl β-d-thiogalactoside. All further steps were performed at 4°C. Cells were washed once with PBS, lysed in lysis buffer (PBS/1 mM DTT) complemented with EDTA-free protease inhibitor by using an Emulsiflex (Avestin, Ottawa, ON, Canada) at 15,000–20,000 psi (1 psi = 6.89 kPa). The lysate was centrifuged at 8,000 × g for 10 min, and the resulting supernatant was subjected to a further centrifugation step at 100,000 × g for 60 min. Glutathione-Sepharose beads (Glutathione-Sepharose 4 Fast Flow; Amersham) were added to the high-speed supernatant, and binding proceeded overnight. After extensive washing in lysis buffer, the recombinant proteins were eluted in 50 mM Tris·HCl, pH 8.0/150 mM KCl/1 mM DTT/20 mM glutathione. The proteins were concentrated by using ultrafiltration (Biomax, 30-kDa cut-off; Millipore, Billerica, MA) to 10 mg/ml. Exclusion-size chromatography (Superdex 75, XK16/30; GE Healthcare, Little Chalfont, U.K.) allowed purification to near homogeneity. Typically, the yield was 10 mg of GST-γ1-app and 5 mg of GST-γ2-app per 6 liters of E. coli culture, each.

Generation of Anti-γ1- and Anti-γ2-COP Specific Antibodies.

Recombinant GST-appendage (app) domains fusion proteins (γ1-COP: amino acids 555–874; γ2-COP: amino acids 555–871; both mouse) were used to raise antibodies in rabbits. GST-γ1-app or GST-γ2-app protein (2.5 mg of each) were bound to glutathione-Sepharose beads overnight at 4°C. The beads were washed with PBS. GST-γ1-app or GST-γ2-app antiserum (7.5 ml of each) was adjusted to 1× PBS and was added to the GST-γ2-app- or GST-γ1-app-loaded glutathione-Sepharose beads, respectively (negative affinity purification). After 2 h of incubation at room temperature, the resulting flow-through (equal to anti-γ1-app or anti-γ2-app antibody) was collected, adjusted to 50% glycerol, and stored at −20°C.

Quantification of Coatomer Isotypes.

Total coatomer immunoprecipitations using the CM1 antibody were performed with cytosol freshly prepared from 3T3/NIH cells. A given sample was analyzed by Western blotting, using anti-γ1-app or anti-γ2-app antibody. Comparison of γ1- or γ2-COP Western blot signals with GST-γ1-app or GST-γ2-app protein calibration curves, respectively, allowed the determination of the mass. The molar ratio of γ1- and γ2-COP was calculated by taking molar masses into account. γ1- or γ2-coatomer immunoprecipitations were performed by using anti-γ1-app or anti-γ2-app antibodies, respectively. Each sample was analyzed by Western blotting, using anti-ζ1-COP- or anti-ζ2-COP-specific antibodies. His-tagged ζ1-COP or ζ2-COP proteins (16) were used as protein standards. The molar ratio of ζ1- and ζ2-COP within γ1- or γ2-coatomer was calculated by taking molar masses into account. The relative proportion of coatomer isotypes matches the product of the corresponding molar ratios; e.g., γ1ζ1-coatomer/total coatomer = γ1-COP/total coatomer (0.7) × ζ1-COP/γ1-coatomer (0.77) = 0.54. Data represent the mean ± SEM of five independent experiments.

Electron Microscopy and Immunogold Labeling.

NRK cells grown to 50% confluency on 10-cm dishes were fixed with 2% paraformaldehyde/0.2% glutaraldehyde in EM buffer (250 mM Hepes/KOH, pH 7.4/100 mM NaCl) for 1 h. Incubation was extended overnight at 4°C. Cells were embedded gradually in 2%, 5%, and 10% gelatin in EM buffer, infused with 2.3 M sucrose, frozen in liquid nitrogen, and sectioned with a cryo-ultramicrotome (50-nm slices). Ultrathin sections were picked up in a 1:1 mixture of 2.3 M sucrose in PBS and 2% methylcellulose in distilled water, collected on Formvar- and carbon-coated grids, and stored at 4°C. Sections were processed and labeled with the indicated primary antibodies and 15-nm protein A-gold (Department of Cell Biology, Utrecht University, The Netherlands), as described in ref. 50. After fixation with 1% glutaraldehyde for 10 min at room temperature to destroy protein A binding sites of bound antibodies (51), sections were incubated with anti-GM130 antibody and 10-nm protein A-gold. Contrasting was done with 0.5% uranyl acetate/1.8% methylcellulose in distilled water, and then the sections were air-dried and examined in an electron microscope (EM10 or LEO 906E; Zeiss, Oberkochen, Germany). Antibodies were diluted as follows: anti-γ1-app 1:100, anti-γ2-app 1:300, anti-ζ2-COP 1:20, anti-β′-COP 1:4,000, anti-KDEL receptor 1:500, and anti-GM130 1:300. Gold conjugates were diluted according to the manufacturer's protocol. Quantification was performed as described in ref. 23. Briefly, the cis side of the Golgi was identified by the presence of GM130 labeling. The stack was split into two halves by an arbitrary line drawn in the middle of the stack. The labeling density per μm2 within each half was evaluated and normalized to allow comparison of different stacks. Data represent the mean ± SEM of the relative labeling densities.

Cell Culture.

NRK cells and 3T3/NIH cells were grown in DMEM containing 10% heat-inactivated FCS or 10% heat-inactivated calf serum, respectively, and supplemented with 100 units of penicillin per ml, 100 μg of streptomycin per ml, 2 mM l-glutamine, and 0.5 mg of G418 per ml. Cells were incubated at 37°C under 5% CO2/95% air.

Bioinformatics.

For bioinformatics procedures, see SI Text.

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Acknowledgments

We thank Sigrid Berger-Seidel and Karin Gorgas for their help in the early stages of this work; Hilmar Bading and Joachim Kirsch for generously providing their EM facilities; George Posthuma for invaluable advice for EM; and Emily Stoops, Julian Langer, and Julien Béthune for reading the manuscript. This work was supported by German Research Council Grant SFB 638, A10 (to B.B. and F.T.W.) and The European Union Grant 3D repertoire (M.J.B.), Contract LSHG-CT-2005-512028.

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Footnotes

  • To whom correspondence should be addressed. E-mail: felix.wieland{at}bzh.uni-heidelberg.de

  • Author contributions: J. Moelleken, T.S., and F.T.W. designed research; J. Moelleken, J. Malsam, M.J.B., A.M., I.R., I.M., and A.H. performed research; J. Malsam, M.J.B., A.M., and A.H. contributed new reagents/analytic tools; J. Moelleken, J. Malsam, M.J.B., R.B.R., T.S., B.B., and F.T.W. analyzed data; and J. Moelleken, R.B.R., B.B., and F.T.W. wrote the paper.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0611360104/DC1.

  • Abbreviations:
    COP,
    coat protein complex;
    ER,
    endoplasmic reticulum;
    ERGIC,
    endoplasmic reticulum–Golgi intermediate compartment.
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