Differential localization of coatomer complex isoforms within the

Jo¨ rg Moelleken*, Jo¨ rg Malsam*, Matthew J. Betts†, Ali Movafeghi‡, Ingeborg Reckmann*, Ingrid Meissner*, Andrea Hellwig§, Robert B. Russell†, Thomas So¨ llner*, Britta Bru¨ gger*, and Felix T. Wieland*¶

*Biochemistry Center and §Department of Neurobiology, University of Heidelberg, 69120 Heidelberg, Germany; †Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany; and ‡Department of Plant Biology, Faculty of Natural Sciences, Tabriz University, Tabriz, Iran

Communicated by James E. Rothman, Columbia University Medical Center, New York, NY, December 20, 2006 (received for review December 8, 2006) Coatomer, the coat of coat protein complex (COP)I-vesicles, Genome analyses identified a second isoform for each of the is a soluble protein complex made up of seven subunits, ␣-, ␤-, ␤؅-, two coatomer subunits, ␥- and ␨-COP (14), the paralogs ␥2- and ␥-, ␦-, ␧-, and ␨-COP. Higher eukaryotes have two paralogous ␨2-COP. Previously, the corresponding cDNAs were expressed versions of the ␥- and ␨- subunits, termed ␥1- and ␥2-COP and ␨1- as tagged constructs (15), and the endogenous products and ␨2-COP. Different combinations of these subunits are known to were shown to be incorporated into coatomer complexes in a exist within coatomer complexes, and ␥1/␨1-, ␥1/␨2-, and ␥2/␨1- stoichiometric manner, which resulted in three major isoforms of COP represent the major coatomer populations in mammals. The the complex with the compositions ␥1␨1-, ␥1␨2-, and ␥2␨1-COP role of COPI vesicles in the early secretory pathway is the subject (16). To extend this work, we have prepared polyclonal antibod- ␥ ␥ ␨ of considerable debate. To help to resolve this discussion, we used ies specific against 1-, 2-, and 2-COP and have investigated quantitative immunoelectron microscopy and found that signifi- the localization of coatomer isoforms in NRK cells by using immunoelectron microscopy. We found a striking heterogeneity cant localization differences for COPI-isoforms do exist, with a ␥ ␨ preference for ␥1␨1- and ␥1␨2-coatomer in the early Golgi appa- within the Golgi apparatus: whereas 1- and 2-COP seem restricted to the early Golgi and a pre-Golgi compartment, the ratus and ␥2␨1-coatomer in the late Golgi apparatus. These differ- majority of ␥2-COP-containing isoforms of the complex is ences suggest distinct functions for coatomer isoforms in a manner localized to the trans side of the organelle. These results strongly similar to /adaptor vesicles, where different adaptor pro- suggest the existence of different homogeneously coated iso- teins serve particular transport routes. forms of COPI vesicles that might serve multiple transport routes within the early secretory pathway. coat protein complex Results ntracellular vesicular transport is mediated by small protein- ␥- and ␨-COP Subunits from Other Species. Sequence searches of ␥- Icoated carriers. Various functions are attributed to coated vesi- and ␨-COP subunits against completely sequenced genomes were cles, which are defined by their constituent coat . Isoforms used to identify the protein families to which they belong. The with different combinations of coat protein complex (COP)II resulting peptide sequences were aligned [see supporting informa- protein paralogs have been found in the COPII coats on vesicles tion (SI) Table 1], and phylogenetic trees were built from these ␥ budding from the (ER) of mammals and alignments. The trees strongly support concurrent duplication of - ␨ yeast (1–3). Because only one direction of travel from the ER to the and -COP into two paralogs in the vertebrates, sometime after Golgi apparatus is known for COPII vesicles, these isoforms might their divergence from Ciona intestinalis (SI Figs. 5 and 6). Ciona, plants, insects, fungi, and the malaria parasite Plasmodium falcipa- reflect differing cargo specificities (4). In the late Golgi/endocytic ␥ ␨ pathways, a variety of coat proteins are known, including four rum all have one - and -COP isoform each, with very recent different tetrameric adaptor complexes, some of which use the duplications and/or alternative transcripts (possibly annotation artifacts) that cluster together, implying that they are duplications clathrin system as an outer shell of their coat (5). Within these specific to a particular species. Absences of isoforms in some pathways, particular transport routes are served by specific coat vertebrates might be an artifact of unfinished genomes, whereas the complexes (for review, see refs. 6 and 7). Thus, for particular fish appear to have really lost ␥1-COP after the duplication, because pathways, the selection of cargo as well as the direction might be none of the three fish species examined have ␥1-COP. The appear- determined by individual coats. ance of multiple isoforms of coatomer might be linked to the Just which pathways are mediated by vesicles bearing the evolution of complex, multicellular organisms with cells processing COPI coat is the subject of considerable debate. The COPI coat a much wider range of cargo proteins. consists of coatomer, which is a stable cytoplasmic complex of seven subunits, ␣-, ␤-, ␤Ј-, ␥-, ␦-, ␧-, and ␨-COP, and the small Generation and Characterization of Antibodies Directed Against COP GTP-binding protein ADP-ribosylation factor, Arf1. COPI ves- Subunit Isoforms. ␥-COP resembles ␥-adaptin, which is made of icles were found to originate from the Golgi apparatus and are an N-terminal ‘‘trunk’’ domain and a C-terminal ‘‘appendage’’ 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 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

observation that COPI vesicles participate in several transport CELL BIOLOGY A.H. contributed new reagents/analytic tools; J. Moelleken, J. Malsam, M.J.B., R.B.R., T.S., directions is difficult to rationalize with just a single isoform of B.B., and F.T.W. analyzed data; and J. Moelleken, R.B.R., B.B., and F.T.W. wrote the paper. the complex (reviewed in ref. 11). Questions about the function The authors declare no conflict of interest. of COPI vesicles are intimately linked to our view of the general Abbreviations: COP, coat protein complex; ER, endoplasmic reticulum; ERGIC, endoplasmic mechanisms of transport through the Golgi. Specifically, is reticulum–Golgi intermediate compartment. transport in both directions, anterograde and retrograde, per- ¶To whom correspondence should be addressed. E-mail: [email protected] formed by these carriers, or are COPI vesicles exclusively heidelberg.de. retrograde carriers within the Golgi and from the Golgi to the This article contains supporting information online at www.pnas.org/cgi/content/full/ ER? Moreover, is anterograde transport across the Golgi exclu- 0611360104/DC1. sively mediated by maturation of Golgi cisternae (12, 13)? © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611360104 PNAS ͉ March 13, 2007 ͉ vol. 104 ͉ no. 11 ͉ 4425–4430 Downloaded by guest on September 25, 2021 against an N-terminal sequence of ␨2-COP (16) showed a remarkable preference for coatomer containing ␨2- and ␥1-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. We investigated the specificity of the antibodies further by using immunofluorescence microscopy (Fig. 1B). 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 ␥1-COP (Fig. 1Bb), ␥2-COP (Fig. 1Bf), and ␨2-COP (Fig. 1Bk) 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 ␨2-COP (Fig. 1Bl). Coincubation of the anti-␥1-app antiserum with GST-␥2-app protein (Fig. 1Bd) and the anti-␥2-app antiserum with GST-␥1-app (Fig. 1Bg) did not eliminate specific binding. Therefore, these antibodies can be used for immunocytochemistry and immunoprecipitation with high specificity for ␥1-COP-, ␥2- COP-, and ␨2-COP-containing coatomer. No antibody with spec- ificity for ␨1-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 ␥1- or ␥2-appendage domain and recombinant His-tagged versions of ␨1- and ␨2-COP as mass standards (Fig. 2). We found a 70:30 ratio of ␥1- to ␥2-COP in total coatomer after immunoprecipitation with the CM1 antibody (directed against all Fig. 1. Specificity of the antibodies used. (A) Immunoprecipitations (IP) with ␤Ј ␥ coatomer isoforms) (Fig. 2 Aa and Ab). Similarly, immunoprecipi- cytosol were performed with anti- -COP (lane 1), CM1 (lanes 3 and 9), anti- 1- ␥ ␥ ␨ app (lane 5), anti-␥2-app (lane 7), and anti-␨2-COP (lane 11) antibodies. The tations with anti- 1-app or anti- 2-app antibodies gave a 1- and eluates were separated by using SDS/PAGE and analyzed by Western blotting, ␨2-COP ratio of 77:23 in ␥1-coatomer isoforms (Fig. 2 Ac and Ad) using the indicated antibodies. Okt8 (lanes 2 and 8) is a CD8-specific antibody. and a 86:14 ratio in ␥2-COP that contained immunoprecipitates Lanes 4, 6, and 10 reflect corresponding ␥1-, ␥2-, and ␨2-preimmune sera, respec- (Fig. 2 Ae and Af). These numbers allowed the calculation of the tively. (B) Indirect immunofluorescence analysis using anti-␥1-app (b), anti-␥2- relative proportion of coatomer isoforms, as shown in Fig. 2B. app (f), and anti-␨2-COP (k) antibodies shows a typical perinuclear staining. The Isoforms of coatomer containing ␥1/␨1-, ␥1/␨2-, and ␥2/␨1-COP ␥1-COP signal can be eliminated by coincubation of the antibody with GST-␥1-app were found at a ratio of Ϸ10:3:5. The ␥2␨2-coatomer is found in a ␥ ␥ (c) but not GST- 2-app protein (d). The 2-COP signal is unaffected by a coincu- much smaller quantity, which is probably close to the sensitivity bation of GST-␥1-app (g) but is decreased drastically in the presence of GST-␥2- app protein (h). That the ␨2-COP signal is significantly reduced in the presence of limit of the analytical method used. the corresponding peptide (l) indicates the specificity of this antibody. Micro- graphs a, e, and i show the signal background of the corresponding Alexa-fluor- Intra-Golgi Localization of the Coatomer Isoforms Containing ␥1-, ␥2-, conjugated secondary antibodies in the absence of primary antibodies. and ␨2-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 domain (17–20). The appendage domains are generally the least validate the polarity of the Golgi sections investigated, antibodies similar in sequence between ␥1- and ␥2-COP; for example, in that were directed against the well established cis-Golgi marker mouse, the sequence identity is 75% in the appendage domains, protein GM130 were used (22). This marker antibody was visual- compared with 81% in the trunk. We therefore expressed ized with 10-nm gold bound to protein A. Representative labeling constructs in Escherichia coli that contained the appendage patterns are shown in Fig. 3. Whereas preferential gold staining is domains of ␥1- and ␥2-COP linked to GST. The purified observed at the cis half of the Golgi with antibodies directed against products were used to raise antibodies in rabbits, and the ␥1- (Fig. 3 A and B) and ␨2-COP (Fig. 3E), the ␥2-coatomer (Fig. resulting antisera (anti-␥1-app and anti-␥2-app) were character- 3 C and D) is restricted more to the trans half of the organelle. As ized by using immunoprecipitation experiments, Western blot- expected, labels of antibodies directed against the common subunit ting, and immunofluorescence microscopy. Both antisera are ␤Ј-COP are observed throughout the Golgi apparatus (Fig. 3F). able to precipitate coatomer from cytosol (Fig. 1A). Antibodies Images that were obtained with the various antibodies were eval- that are directed against ␤Ј-COP (lane 1), a subunit of coatomer uated quantitatively by separating Golgi areas along an arbitrary that is shared by all complex isoforms, or CM1 antibodies (lane middle line, defining a cis half and a trans half of the organelle, and 3), which are directed against native coatomer, precipitate all counting the gold dots in each section. As shown in Fig. 4, Ϸ70% isoforms of coatomer (containing ␥1-, ␥2-, ␨1-, and ␨2-COP). of ␥1-COP-containing coatomer was found in the cis half. The However, anti-␥1-app antibodies and anti-␥2-app antibodies ␨2-COP coatomer showed an even stronger preference, with Ϸ80% specifically precipitate coatomer containing ␥1-COP (lane 5) or localized to the early Golgi. The ␥2-coatomer, in contrast, showed ␥2-COP (lane 7), respectively. Likewise, an antibody directed a distribution of Ͼ60% trans and Ͻ40% cis. As a control, only a

4426 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611360104 Moelleken et al. Downloaded by guest on September 25, 2021 Fig. 2. Quantification of coatomer isotypes. (A) Immunoprecipitations (IPs) using CM1 (a and b), anti-␥1-app (c and d), or anti-␥2-app (e and f) antibodies were performed with freshly prepared cytosol. Calibration curves obtained from Western blot analyses with GST-␥1-app, GST-␥2-app, His6-␨1-COP, or His6-␨2-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.

slight preference, Ͻ55% cis and Ͼ45% trans, was observed when Strikingly, the coatomer isoforms are differentially distributed overall coatomer localization was studied with the anti-␤Ј-COP within the Golgi apparatus, with ␥1-COP found preferentially in antibody (Fig. 3F). This is in agreement with a previous study in the early Golgi and ␥2-coatomer found preferentially in the late endocrinic pancreatic cells (23). The stringent localization of the Golgi. A double labeling experiment with the KDEL receptor, an cis-Golgi marker protein GM130 (22) to only one half of the Golgi ERGIC/cis-Golgi marker (27, 28), underscores the preference of corroborates the validity of the analytical method used. The marked ␨2-coatomer for these compartments. localization of ␨2-coatomer to the early Golgi has led us to The distinct localization of individual coatomer isoforms investigate this finding in further detail, by comparison with the strongly suggests the presence of various types of COPI vesicles KDEL receptor, a resident of the ERGIC and the cis-Golgi (24, 25). in the cell, each of which is homogeneously coated with a defined A pattern was obtained as depicted in Fig. 3G, where the antibodies isotypic coatomer complex. Given the pattern emerging from against the KDEL receptor were labeled with 10-nm gold, and immunoelectron microscopy, gradients are highly likely to exist antibodies against ␨2-COP with 15-nm gold. Again, a significant in the relative abundance of such vesicles, with more ␥1- and preference of ␨2-coatomer for the ERGIC and the cis-Golgi was ␨2-COPI vesicles in the early Golgi and more ␥2 vesicles in the observed. Taken together, the significant differential localization of late Golgi. Alternatively, or in addition, COPI vesicles that are coatomer isoforms suggests that they serve different functions enveloped by various mixtures of coatomer isoforms might exist within the Golgi apparatus. (although this possibility is less likely given, e.g., the strong preference of ␨2-coatomer for the early Golgi). Discussion At present, it is unknown how the specific coatomer isoforms are We have identified clear coatomer isoforms with different combi- recruited to a certain Golgi cisterna. Interestingly, the difference of nations of the ␥- and ␨-COP subunits in the vertebrates. These the isoforms lies in their ␥␨-subcomplexes, and it is ␥-COP that has isoforms, like the other five COPs shared by all coatomers, are two binding sites for oligomeric forms of the members of the p24

present stoichiometrically in their constituent complexes (16). family of membrane proteins (29) that constitutively cycle between CELL BIOLOGY Phylogeny strongly suggests that this diversification of ␥- and ␨-COP ER and Golgi (30–33). Differential ␥␨-COP combinations might subunits is a vertebrate innovation. Such gene family expansions are well have different affinities to the cytoplasmic tails of these type common in vertebrate-specific processes, such as the immune 1 transmembrane proteins or to different combinations thereof. system, olfaction, and particular developmental processes (26). Thus, if these proteins or their heteromeric complexes were un- Using new antibodies available against the two ␥-COP subunits, equally distributed across the Golgi, their differential interaction we have reassessed the relative abundance of coatomer in mam- with coatomer isoforms could explain differential recruitment of malian cytosol and have found coatomer isoforms that contain the the complex. Indeed, a preferential localization of p23, p24, and p27 subunits ␥1/␨1-, ␥1/␨2-, and ␥2/␨1-COP in a ratio of Ϸ10:3:5. at the cis-Golgi has been shown (32–34). Additionally, individual Coatomer with ␥2␨2-COP, if it exits, represents Ͻ5% of the total. coatomer isoforms might differ in their affinity for the cytoplasmic

Moelleken et al. PNAS ͉ March 13, 2007 ͉ vol. 104 ͉ no. 11 ͉ 4427 Downloaded by guest on September 25, 2021 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-␥1-app (A and B), anti-␥2-app (C and D), anti-␨2-COP (E), anti-␤Ј-COP (F), and anti-␨2-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 ␮m2 of total Golgi were 25.0 Ϯ 2.2 for ␥1-COP, 27.7 Ϯ 2.8 for ␥2-COP, 13.3 Ϯ 1.4 for ␨2-COP, 38.2 Ϯ 5.5 for ␤Ј-COP, and 40.3 Ϯ 3.8 for GM130.

tails of various Golgi-resident membrane proteins, causing differ- Sequence similarities exist between the clathrin/adaptor proteins ential recruitment to individual cisternae. Indeed, COPI vesicles and coatomer. The tetrameric adaptor complex resembles a that contain either p24 proteins or the Golgi-resident enzymes coatomer ‘‘core’’ made up of ␤-, ␦-, ␥-, and ␨-COP (17, 42, 44, 45), mannosidase I and II have been described (35). Likewise, strong and there is a distant similarity of the trimeric subcomplex ␣-, ␤Ј-, evidence for functionally different COPI vesicles comes from an and ␧-COP with the clathrin proteins (18). Furthermore, the immunoelectron microscopy study in which two distinct popula- analogy to the clathrin system became even more apparent when tions of COPI vesicles were defined by inclusion of either the KDEL the appendage domain of ␥1-COP revealed a striking structural ␣ ␤ receptor or proinsulin (23). Similarly, GOS28, a Golgi v-SNARE similarity to the appendage domains of - and -adaptin (19, 20). (soluble N-ethylmaleimide sensitive factor attachment protein re- Our observations of different distributions strongly suggests ceptor), was found in COPI vesicles devoid of the KDEL receptor that coatomer complex isoforms serve different functions, within and vice versa (36). The existence of different populations of COPI the early secretory pathways, that are similar to the various vesicles had been deduced previously, based on the analysis of functions individual adaptor proteins exert in the late secretory pathways (5). Thus, it becomes unlikely that COPI vesicles carriers immuno-isolated with antibodies directed against p27 (37). exclusively mediate back transport of Golgi-resident proteins However, because these carriers were uncoated, it was not com- during maturation of Golgi cisternae. pletely clear whether they were derived from COPI-coated vesicles. Further work will be needed to focus on the isolation and A variety of functions have been attributed to COPI vesicles. proteomics of individual COPI vesicles to elucidate their func- These include the transport of proteins cycling between the tions. With a preference for the cis-Golgi and ERGIC, for Golgi and the ER and the retrieval of proteins that have escaped example, ␥1␨2-coatomer is a likely candidate to form vesicles for the ER (10, 38). Originally, COPI vesicles were defined and recycling and retrieval of proteins from these organelles to the characterized as carriers for biosynthetic cargo across the Golgi ER. Determining the particular functions of the complex iso- apparatus, and various anterograde cargo proteins were found to forms will be crucial for a better understanding of the Golgi be passengers (23, 39–42). Later, in the context of explaining within the vertebrates. anterograde transport mediated by the maturation process of cisternae of the organelle, a role was attributed to COPI vesicles Materials and Methods as carriers for residential proteins that need to be sorted back to Antibodies. The following polyclonal antibodies were used: anti-␣- allow the individual cisternae to achieve and maintain their COP (CT peptide) (F.T.W.); anti-␤-COP (46); anti-␤Ј-COP (47); identity (11, 13, 43). anti-␨1-COP (peptide, human, amino acids 63–74) (F.T.W.); anti-

4428 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611360104 Moelleken et al. Downloaded by guest on September 25, 2021 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.

␨2-COP (16); and anti-KDEL receptor (Erd2p, kindly provided by ary antibodies (RG16) (Sigma–Aldrich) were applied. True Blot the late H.-D. So¨ling, Max Planck Institute for Biophysical Chem- was obtained from eBiosciences. istry, Go¨ttingen, Germany). The monoclonal antibodies that were used are as follows: anti-GM130 (BD Transduction Laboratories, Cloning and Expression of ␥1- and ␥2-Appendage Domains. mRNA San Jose, CA); Okt8 (48); CM1 (49). HRP-conjugated secondary was isolated from 3T3/NIH mouse fibroblasts with oligo(dT)25 antibodies: goat anti-mouse and donkey anti-guinea pig (Dianova, Dynabeads (Dynal Biotech, Hamburg, Germany) according to the Hamburg, Germany) and mouse anti-rabbitnative (RG16) (Sigma– manufacturer’s protocol. Reverse transcription was performed by Aldrich, Munich, Germany). True Blot was obtained from eBio- using the 5Ј RACE System V 2.0 kit (Life Technologies, Paisley, sciences (San Diego, CA). Alexa-fluor-conjugated secondary an- Scotland). The regions corresponding to the ␥1- and ␥2-COP tibodies were obtained from Molecular Probes (Leiden, The appendage domains (nucleotides 1663–2625 and 1663–2616, re- Netherlands). Preimmune serum was obtained from the rabbits spectively) were amplified by using PCR with the following primer that were used to raise antibodies. pairs: 5Ј-TCT AGA ATC CCT GGT CTG GAG AAA GCC CTG-3Ј (forward) and 5Ј-GGA TTC CTA GCC CAC GGA TGC Preparation of Cytosol from Cultured Cells. 3T3/NIH cells grown to CAA GAT GAT-3Ј (reverse) for ␥1 and 5Ј-GCT AGC ATA CCA confluency on 15-cm dishes were washed three times with PBS GGG ATG GAA AAG GCC TTA-3Ј (forward) and 5Ј-GGA TTC and scraped off in the presence of 0.25 ml of immunoprecipi- TTA TCC CAC AGA AGC CAA GAT AAC ATC-3Ј (reverse) for tation buffer (25 mM Tris⅐HCl, pH 7.4/150 mM NaCl/1 mM ␥2. ␥1-COP was cloned as an XbaI/BamHI- and ␥2-COP as an EDTA) that was complemented with EDTA-free protease in- NheI/BamHI fragment in the expression vector pPro-GST-TEV hibitor. All further steps were performed at 4°C. The cells were (kindly provided by M. Lutzmann, Heidelberg University Biochem- passed 20 times each through 21- and 27-gauge needles. The istry Center). GST-␥1-app and GST-␥2-app were over-expressed in homogenate was centrifuged at 800 ϫ g for 5 min, and the E. coli BL21(DE3) (Promega, Madison, WI) within4hat37°C after resulting supernatant was subjected to a further centrifugation induction with 1 mM isopropyl ␤-D-thiogalactoside. All further step at 100,000 ϫ g for 60 min. Typically, the high-speed steps were performed at 4°C. Cells were washed once with PBS, supernatant (cytosol) had a protein concentration of 2 mg/ml. lysed in lysis buffer (PBS/1 mM DTT) complemented with EDTA- free protease inhibitor by using an Emulsiflex (Avestin, Ottawa, Immunoprecipitations and Western Blotting. Antibodies were incu- ON, Canada) at 15,000–20,000 psi (1 psi ϭ 6.89 kPa). The lysate was bated with protein A-Sepharose (CL-4B) (Amersham, Little Chal- centrifuged at 8,000 ϫ g for 10 min, and the resulting supernatant font, U.K.) on a rotating wheel in immunoprecipitation buffer for was subjected to a further centrifugation step at 100,000 ϫ g for 60 CELL BIOLOGY 60 min at room temperature. After washing three times with min. Glutathione-Sepharose beads (Glutathione-Sepharose 4 Fast immunoprecipitation buffer, freshly prepared cytosol (0.5 mg/10 ␮l Flow; Amersham) were added to the high-speed supernatant, and of beads) was added, and incubation continued for 60 min at room binding proceeded overnight. After extensive washing in lysis temperature. Beads were collected by centrifugation, extensively buffer, the recombinant proteins were eluted in 50 mM Tris⅐HCl, washed, and eluted in reducing SDS sample buffer at 70°C. Samples pH 8.0/150 mM KCl/1 mM DTT/20 mM glutathione. The proteins were subjected to SDS/PAGE and analyzed by Western blotting, were concentrated by using ultrafiltration (Biomax, 30-kDa cut-off; using the indicated antibodies. To reduce background when rabbit Millipore, Billerica, MA) to 10 mg/ml. Exclusion-size chromatog- antibodies were used for immunoprecipitation and subsequent raphy (Superdex 75, XK16/30; GE Healthcare, Little Chalfont, Western blot analysis, monoclonal mouse anti-rabbitnative second- U.K.) allowed purification to near homogeneity. Typically, the yield

Moelleken et al. PNAS ͉ March 13, 2007 ͉ vol. 104 ͉ no. 11 ͉ 4429 Downloaded by guest on September 25, 2021 was 10 mg of GST-␥1-app and 5 mg of GST-␥2-app per 6 liters of and sectioned with a cryo-ultramicrotome (50-nm slices). Ultrathin E. coli culture, each. 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- Generation of Anti-␥1- and Anti-␥2-COP Specific Antibodies. Recom- and carbon-coated grids, and stored at 4°C. Sections were processed binant GST-appendage (app) domains fusion proteins (␥1-COP: and labeled with the indicated primary antibodies and 15-nm amino acids 555–874; ␥2-COP: amino acids 555–871; both mouse) protein A-gold (Department of Cell Biology, Utrecht University, ␥ ␥ were used to raise antibodies in rabbits. GST- 1-app or GST- 2- The Netherlands), as described in ref. 50. After fixation with 1% app protein (2.5 mg of each) were bound to glutathione-Sepharose glutaraldehyde for 10 min at room temperature to destroy protein beads overnight at 4°C. The beads were washed with PBS. GST- ␥ ␥ A binding sites of bound antibodies (51), sections were incubated 1-app or GST- 2-app antiserum (7.5 ml of each) was adjusted to with anti-GM130 antibody and 10-nm protein A-gold. Contrasting 1ϫ PBS and was added to the GST-␥2-app- or GST-␥1-app-loaded was done with 0.5% uranyl acetate/1.8% methylcellulose in distilled glutathione-Sepharose beads, respectively (negative affinity purifi- water, and then the sections were air-dried and examined in an cation).After2hofincubation at room temperature, the resulting ␥ ␥ electron microscope (EM10 or LEO 906E; Zeiss, Oberkochen, flow-through (equal to anti- 1-app or anti- 2-app antibody) was ␥ collected, adjusted to 50% glycerol, and stored at Ϫ20°C. Germany). Antibodies were diluted as follows: anti- 1-app 1:100, anti-␥2-app 1:300, anti-␨2-COP 1:20, anti-␤Ј-COP 1:4,000, anti- Quantification of Coatomer Isotypes. Total coatomer immunopre- KDEL receptor 1:500, and anti-GM130 1:300. Gold conjugates cipitations using the CM1 antibody were performed with cytosol were diluted according to the manufacturer’s protocol. Quantifi- freshly prepared from 3T3/NIH cells. A given sample was analyzed cation was performed as described in ref. 23. Briefly, the cis side of by Western blotting, using anti-␥1-app or anti-␥2-app antibody. the Golgi was identified by the presence of GM130 labeling. Comparison of ␥1- or ␥2-COP Western blot signals with GST-␥1- The stack was split into two halves by an arbitrary line drawn in the app or GST-␥2-app protein calibration curves, respectively, allowed middle of the stack. The labeling density per ␮m2 within each half the determination of the mass. The molar ratio of ␥1- and ␥2-COP was evaluated and normalized to allow comparison of different was calculated by taking molar masses into account. ␥1- or ␥2- stacks. Data represent the mean Ϯ SEM of the relative labeling coatomer immunoprecipitations were performed by using anti-␥1- densities. app or anti-␥2-app antibodies, respectively. Each sample was analyzed by Western blotting, using anti-␨1-COP- or anti-␨2-COP- Cell Culture. NRK cells and 3T3/NIH cells were grown in DMEM specific antibodies. His-tagged ␨1-COP or ␨2-COP proteins (16) containing 10% heat-inactivated FCS or 10% heat-inactivated calf were used as protein standards. The molar ratio of ␨1- and ␨2-COP serum, respectively, and supplemented with 100 units of penicillin ␥ ␥ within 1- or 2-coatomer was calculated by taking molar masses per ml, 100 ␮g of streptomycin per ml, 2 mM L-glutamine, and 0.5 into account. The relative proportion of coatomer isotypes matches mg of G418 per ml. Cells were incubated at 37°C under 5% the product of the corresponding molar ratios; e.g., ␥1␨1-coatomer/ CO2/95% air. total coatomer ϭ ␥1-COP/total coatomer (0.7) ϫ ␨1-COP/␥1- ϭ Ϯ coatomer (0.77) 0.54. Data represent the mean SEM of five Bioinformatics. For bioinformatics procedures, see SI Text. independent experiments. We thank Sigrid Berger-Seidel and Karin Gorgas for their help in the early Electron Microscopy and Immunogold Labeling. NRK cells grown to stages of this work; Hilmar Bading and Joachim Kirsch for generously 50% confluency on 10-cm dishes were fixed with 2% paraformal- providing their EM facilities; George Posthuma for invaluable advice for dehyde/0.2% glutaraldehyde in EM buffer (250 mM Hepes/KOH, EM; and Emily Stoops, Julian Langer, and Julien Be´thune for reading the pH 7.4/100 mM NaCl) for 1 h. Incubation was extended overnight manuscript. This work was supported by German Research Council Grant at 4°C. Cells were embedded gradually in 2%, 5%, and 10% gelatin SFB 638, A10 (to B.B. and F.T.W.) and The European Union Grant 3D in EM buffer, infused with 2.3 M sucrose, frozen in liquid nitrogen, repertoire (M.J.B.), Contract LSHG-CT-2005-512028.

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