GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system
1999; Springer Nature; Volume: 18; Issue: 18 Linguagem: Inglês
10.1093/emboj/18.18.4949
ISSN1460-2075
Autores Tópico(s)Autophagy in Disease and Therapy
ResumoArticle15 September 1999free access GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system James Shorter James Shorter Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Rose Watson Rose Watson Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Maria-Eleni Giannakou Maria-Eleni Giannakou IBLS, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ Scotland, UK Search for more papers by this author Mairi Clarke Mairi Clarke IBLS, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ Scotland, UK Search for more papers by this author Graham Warren Graham Warren Present address: Department of Cell Biology, SHM, C441, Yale University School of Medicine, 33 Cedar Street, PO Box 208002, New Haven, CT, 06520-8002 USA Search for more papers by this author Francis A. Barr Corresponding Author Francis A. Barr IBLS, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ Scotland, UK Search for more papers by this author James Shorter James Shorter Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Rose Watson Rose Watson Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Maria-Eleni Giannakou Maria-Eleni Giannakou IBLS, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ Scotland, UK Search for more papers by this author Mairi Clarke Mairi Clarke IBLS, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ Scotland, UK Search for more papers by this author Graham Warren Graham Warren Present address: Department of Cell Biology, SHM, C441, Yale University School of Medicine, 33 Cedar Street, PO Box 208002, New Haven, CT, 06520-8002 USA Search for more papers by this author Francis A. Barr Corresponding Author Francis A. Barr IBLS, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ Scotland, UK Search for more papers by this author Author Information James Shorter1, Rose Watson1, Maria-Eleni Giannakou2, Mairi Clarke2, Graham Warren3 and Francis A. Barr 2 1Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK 2IBLS, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ Scotland, UK 3Present address: Department of Cell Biology, SHM, C441, Yale University School of Medicine, 33 Cedar Street, PO Box 208002, New Haven, CT, 06520-8002 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:4949-4960https://doi.org/10.1093/emboj/18.18.4949 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have identified a 55 kDa protein, named GRASP55 (Golgi reassembly stacking protein of 55 kDa), as a component of the Golgi stacking machinery. GRASP55 is homologous to GRASP65, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system. GRASP65 exists in a complex with the vesicle docking protein receptor GM130 to which it binds directly, and the membrane tethering protein p115, which also functions in the stacking of Golgi cisternae. GRASP55 binding to GM130, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system. Cryo-electron microscopy revealed that GRASP65, like GM130, is present on the cis-Golgi, while GRASP55 is on the medial-Golgi. Recombinant GRASP55 and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for GRASP65. These results demonstrate that GRASP55 and GRASP65 function in the stacking of Golgi cisternae. Introduction In mammalian cells, the Golgi apparatus is composed of a highly ordered parallel array of cisternae which form a stacked structure typically found in the perinuclear region of the cell (Rambourg and Clermont, 1997). The major functions of the Golgi apparatus are thought to be in the synthesis of the complex carbohydrate structures that are attached to many cellular and secretory proteins and lipids, and in the sorting of these proteins and lipids to their correct subcellular destinations (Mellman and Simons, 1992). The ordered structure of the Golgi apparatus is thought to reflect the requirement for these enzymes and of the protein sorting machinery to be compartmentalized to allow a specific series of post-translational modifications and sorting reactions to be carried out (Farquhar, 1985). In recent years a number of studies have identified components that act in the fusion of Golgi membranes to give rise to cisternae, and in the subsequent stacking of these cisternae to form stacks. These studies have used in vitro assays in which the Golgi apparatus can be disassembled and reassembled under defined conditions, thus allowing the identification of components important for different aspects of Golgi structure (Acharya et al., 1995; Rabouille et al., 1995a). One cell-free system (Rabouille et al., 1995a) has exploited the disassembly of the Golgi apparatus into many small vesicles and membrane fragments during cell division (Lucocq et al., 1987, 1989). In this system, isolated Golgi membranes are treated with mitotic cell cytosol to generate a population of mitotic Golgi fragments (MGFs) that can reassemble into stacked Golgi membranes when incubated under the correct conditions. The N-ethylmaleimide (NEM)-sensitive factor (NSF), its cofactors the soluble NSF attachment proteins (SNAPs) and the vesicle tethering protein p115 act in conjunction with p97 and its cofactor p47 to rebuild cisternae from MGFs (Rabouille et al., 1995b, 1998). Electron microscopic studies on cells and isolated Golgi membranes have described filamentous structures, bridging the gaps between adjacent cisternae, that may be components of a structural exoskeleton helping to hold them together (Franke et al., 1972; Cluett and Brown, 1992). There are many proteins that are candidates for components of these filamentous structures. One group of proteins is the golgins, a large family of coiled-coil proteins including GM130 and giantin originally described as autoantigens, either peripherally or integrally associated with the Golgi apparatus (Chan and Fritzler, 1998). Another group of proteins are more often thought of as components of the actin cytoskeleton; these are the recently identified Golgi-localized isoforms of ankyrin and spectrin (Beck et al., 1994; Devarajan et al., 1996; Stankewich et al., 1998). Studies using the system for the reassembly of stacked Golgi apparatus membranes from MGFs have begun to provide some clues as to the nature of the filamentous bridge structures. When MGFs are pre-treated with the alkylating agent NEM they can reassemble in the presence of either interphase cytosols or the purified membrane fusion components NSF, SNAPs, p115 and p97 to give single cisternae, but not stacks of cisternae (Rabouille et al., 1995a,b). This observation was used to identify GRASP65 (Golgi reassembly stacking protein of 65 kDa), an NEM-sensitive membrane component required for the stacking of cisternae (Barr et al., 1997). Two other membrane components are known to be required for stacking, GM130 and giantin, something that is distinct from their role in the NSF pathway of membrane fusion (Shorter and Warren, 1999). GRASP65 exists in Golgi membranes as part of a complex with GM130 (Barr et al., 1998), which acts as the receptor for p115 during the docking of vesicles with Golgi membranes (Nakamura et al., 1997; Sönnichsen et al., 1998). During vesicle docking, p115 is thought to bind to giantin on the vesicle, and GM130 on the target Golgi membrane (Sönnichsen et al., 1998), thus tethering the two membranes together and allowing specific membrane fusion to occur. A recent study has shown that p115 also functions upstream of GRASP65 in the stacking of Golgi cisternae (Shorter and Warren, 1999). The p115–GM130–GRASP65 complex together with giantin might therefore act in a specialized docking pathway bringing cisternae together at the cis-face of the Golgi apparatus, then handing them over to other protein complexes that would hold later Golgi cisternae together. Results Cloning of a mammalian GRASP65-related protein During the course of previous studies on GRASP65 a number of antibodies have been raised against either the full-length protein expressed in bacteria, or synthetic peptides. One of these antibodies, FBA19, raised against the sequence YLHRIPTQPSSQYK (underlined in Figure 1), which is conserved amongst the known forms of GRASP65 from various yeasts and Caenorhabditis elegans, recognizes 65 and 55 kDa proteins in rat liver Golgi membranes, as shown below. Other antibodies to GRASP65 that recognize epitopes in the less well conserved C-terminal domain of the protein recognize only a 65 kDa protein in Golgi membranes (Barr et al., 1997). This led us to believe that there might be a second form of GRASP65, a view supported by the existence of expressed sequence tags (ESTs) with only partial homology to the known rat GRASP65 sequence. A RACE cloning strategy was adopted in order to obtain a clone for the putative GRASP65 homologue, using nested primers designed from a mouse testis EST (DDBJ/EMBL/GenBank accession number AA061790). To obtain 5′ and 3′ clones corresponding to this GRASP65-related protein, nested primer pairs TR1 to TR4 and adaptor primers AP-1 and AP-2 were used. These clones were sequenced and a new pair of primers designed to amplify the full open reading frame. Analysis of the sequence and predicted open reading frame of this clone revealed that it had a high level of homology, but not identity with GRASP65 in the first 212 amino acids, and after this point became highly divergent (Figure 1). The cDNA encodes a 454 amino acid protein with a predicted molecular weight of 55 kDa, confirmed by in vitro translation (Figure 6A, lane 9) and Western blotting with specific antibodies (Figure 3, lane 1), and it was therefore named GRASP55. Like GRASP65, it has a consensus site for myristoylation at the N-terminus and could be anchored to membranes by means of this modification (Barr et al., 1997). The sequence in this N-terminal first 21 amino acids is slightly divergent in the two proteins, and GRASP55 has an insertion at position 14, the only point in the first 212 amino acids at which the two proteins are not co-linear. This raises the possibility that they are not targeted to the same regions of the Golgi apparatus, or that their interaction with membranes is differentially regulated. Comparison of the first 212 amino acids of GRASP65 and 213 amino acids of GRASP55 reveals that 66% of residues are identical and 14% conserved in this region, with the residues currently known to be important for GM130 binding being identical (asterisks in Figure 1; Barr et al., 1998). The region against which the FBA19 antibody was raised is partially conserved between the two proteins, YLHRIPTRPFEEGK in GRASP55 and YLHRIPTQPSSQYK in GRASP65 (underlined in Figure 1), thereby explaining why it gives rise to two bands in purified Golgi membranes. Figure 1.Comparison of the GRASP55 and GRASP65 sequences. Alignment of the GRASP55 (af110267) and GRASP65 (AF015264) sequences; shading indicates identity and boxed residues are conserved. Residues in GRASP65 important for GM130 binding are indicated by asterisks. Underlined residues indicate the peptide used to raise the FBA19 antiserum. Download figure Download PowerPoint Figure 2.GRASP55 and GRASP65 are both ubiquitously expressed. A Northern blot of the rat tissues indicated in the figure was hybridized with probes specific for (A) GRASP65, (B) GRASP55 and (C) a probe common to both GRASP65 and GRASP55. As a control the blot was also hybridized with an actin-specific probe (D). Download figure Download PowerPoint To determine the expression patterns of GRASP65 and GRASP55, Northern blots on a panel of rat tissues were performed using a combination of probes specific or common to the two mRNAs (Figure 2). A probe specific for GRASP65 recognized a message at the correct size in all tissues, although only very faintly in spleen (Figure 2A). As previously reported, a second message of ∼1.5 kb was observed in testis. The GRASP55-specific probe detected two messages of slightly smaller size than seen by the GRASP65 probe, again present in all tissues (Figure 2B). The lower band of this doublet was especially noticeable in liver, but was visible in other tissues upon longer exposure of the blot. In testis, the lower band of the doublet was more prominent than the upper band, in contrast to other tissues, and a second, smaller message of ∼1.4 kb was also observed. A probe common to both GRASP65 and GRASP55 gave a complex pattern of bands corresponding to the addition of the signals seen with the GRASP65- and GRASP55-specific probes (Figure 2C). An actin probe used as a control for the loading of the Northern blot gave the expected pattern of hybridization (Figure 2D). Therefore, messages for GRASP65 and GRASP55 would appear to be present in all tissues tested, with multiple possible splice variants being present in testis. Figure 3.Tissue Western blots for GRASP55 and GRASP65. (A) Purified Golgi membranes were Western blotted for GRASP55, polyclonal FBA34, GRASP65, monoclonal 7E10, GRASP55 + GRASP65, polyclonal FBA19 or GM130, polyclonal MLO-7). Western blots of the tissues indicated in the figure were probed with these antibodies specific for either GRASP65 (B), GRASP55 (C) or GM130 (D). Download figure Download PowerPoint To confirm that the proteins are actually expressed, Western blots were performed using protein extracts from these tissues. These Western blots were probed with antibodies specific for GRASP65, GRASP55 and GM130 (Figure 3). To demonstrate that these antibodies are in fact specific for the GRASP65 and GRASP55, purified rat liver Golgi membranes were blotted with these and with an antibody that sees both proteins. In Golgi membranes, the sheep polyclonal FBA34 recognizes a 55 kDa protein, the 7E10 monoclonal to GRASP65 sees a 65 kDa protein and the FBA19 antibody recognizes both GRASP65 and GRASP55 (Figure 3A). The antibody to GRASP65 detected a protein of 65 kDa in all tissues, with a fainter second band in testis at ∼60 kDa possibly corresponding to the second messenger RNA seen in this tissue (Figure 3B). Similar results were obtained with an antibody to GRASP55; this detected a 55 kDa band in all tissues (Figure 3C). Blotting for the GRASP65 partner protein GM130 revealed that this protein is also present in all the tissues examined (Figure 3D). Together, these data demonstrate that GRASP55 is ubiquitously expressed in mammalian tissues, and therefore, like GRASP65, could act in the stacking of Golgi cisternae. Localization of GRASP55 to the Golgi apparatus The N-terminal domain of GRASP65 when fused to green fluorescent protein (GFP) can target to the Golgi apparatus, something that is abolished by mutations in its GM130 binding site (Barr et al., 1998). Given the similarity between the N-terminal domains of GRASP65 and GRASP55, it was likely that GRASP55 would also target the Golgi apparatus. To test this, full-length GRASP65 and full-length GRASP55 fused to GFP were transfected into HeLa cells either singly or together (Figure 4). When HeLa cells were transfected with constructs for either rat GRASP65 (Figure 4A) or GRASP55–GFP (Figure 4B), a pattern typical of the perinuclear ribbon-like structure of the Golgi apparatus was observed. To confirm that this was indeed the Golgi apparatus, equivalent samples were stained with antibodies to the Golgi marker protein GM130, and GRASP65 (Figure 4C), or GRASP55–GFP fluorescence visualized (Figure 4D). Comparison of the individual staining patterns reveals that GRASP55, like GRASP65 and GM130, is localized to the Golgi apparatus. Cells were also transfected with both GRASP65 and GRASP55–GFP to allow a direct comparison of the distributions of these two proteins (Figure 4E). Comparison of the two images shows that GRASP65 and GRASP55 have similar distributions within the Golgi apparatus. The targeting of GRASP65 to the Golgi apparatus requires its N-terminal myristoylation site, and a series of residues in the domain which is involved in binding to GM130 (Barr et al., 1998). To find out if the targeting of GRASP55 was similar to that of GRASP65, point mutants were constructed analogous to those known to abolish GRASP65 targeting. When the glycine at position 2 was mutated to alanine, GRASP55 G2A, the protein was found to accumulate in the cytoplasm with only a very weak signal for the Golgi apparatus (Figure 4F). Mutations of two residues that cause a complete loss of binding to GM130 and of Golgi targeting in GRASP65, the G196A and H199A mutations (Barr et al., 1998), caused only a partial defect in Golgi targeting when introduced into GRASP55 G197A (Figure 4G and H200A (Figure 4H), seen as an increased diffuse cytoplasmic fluorescence relative to the wild-type protein (Figure 4B). These data show that GRASP55 localizes to the Golgi apparatus, and that this requires the N-terminal myristoylation site. Unlike GRASP65, mutations in the putative GM130 binding region have only a small effect on the Golgi localization of GRASP55, implying that this does not require interaction with a target protein at this site, or that the mutations have no effect on binding of GRASP55 to its target protein. Figure 4.Localization of GRASP55 to the Golgi apparatus by immunofluorescence. HeLa cells were transfected with rat GRASP65 (A and C), rat GRASP55–GFP (B and D), rat GRASP65 and GRASP55–GFP (E) or the GRASP55–GFP mutants G2A (F), G197A (G) and H200A (H). Cells were processed for immunofluorescence with antibodies to rat GRASP65 only (A and E), rat GRASP65 and GM130 (C) or GM130 only (D, F–H). GFP fluorescence was used to visualize GRASP55 (B, D–H). Download figure Download PowerPoint To determine where GRASP55 and GRASP65 were localized within the Golgi apparatus, antibody labelling on cryo-sections of HeLa cells transfected with rat GRASP65 and GRASP55–GFP was performed (Figure 5). Under the electron microscope it could be seen that the labelling for both proteins was over the Golgi apparatus, consistent with the localization at the light microscope level. GRASP65 labelling was typically seen over the cis-face of the stack (Figure 5A), although it cannot be ruled out that it is present further into the stack but is simply not accessible to antibodies. The observation that GRASP65 only becomes accessible to the small alkylating agent NEM after treatment of stacked Golgi membranes with mitotic cytosol indicates that it is sequestered in some protein complex in these structures, and indicates that it may not be easily accessible to some antibodies under native conditions. GRASP55 labelling was found to be more over the stack with some labelling over the cis-face of the Golgi (Figure 5B), and was at a level similar to GRASP65. Double-labelling experiments confirm that GRASP65 labelling (Figure 5C and D, large gold particles), marked by the arrows, is over the cis-face of the Golgi stack, while GRASP55 labelling (Figure 5C and D, small gold particles) is seen over the stack and at the cis-Golgi cisternae. The labelling efficiency for GRASP65 in the double-labelling experiments was lower than that seen for GRASP55–GFP, and also lower than for GRASP65 detected by a polyclonal antibody in single-labelling experiments. This might be explained by the use of a monoclonal antibody to detect GRASP65, which by definition sees only a single epitope, in the double-labelling experiments, as opposed to the use of a polyclonal antibody, likely to recognize multiple epitopes, to detect GRASP55–GFP. In order to be able to compare the localizations of GRASP65 and GRASP55 within the Golgi apparatus, the distributions of gold particles corresponding to antibody labelling of the proteins were quantitated. Due to the differences in labelling efficiencies discussed above this was carried out for both the single- and double-labelling experiments, and the results compared. For the single labellings, the distribution of gold particles over 22 (GRASP65; 133 gold particles) and 20 (GRASP55; 147 gold particles), Golgi regions with a defined cis to trans polarity were quantitated. Plotting these distributions as a function of cisterna reveals that GRASP65 (Figure 5E, shaded bars) is present over the first cisterna, while GRASP55 (Figure 5E, open bars) is mainly present over the second and third cisternae. For the double labellings, the distribution of gold particles for GRASP65 (34 gold particles) and GRASP55 (97 gold particles) over 14 Golgi regions with a defined cis to trans polarity were quantitated. Plotting these distributions as a function of cisterna reveals that GRASP65 (Figure 5F, shaded bars) is present over the first cisterna, while GRASP55 (Figure 5F, open bars) is mainly present over the second and third cisternae. From these data it appears that GRASP65 is located at the cis-face of the Golgi, while GRASP55 is found more over the stack. This is consistent for GRASP65 with the localization of its binding partner GM130, to the cis/medial-Golgi at the electron microscope level (Nakamura et al., 1995). Figure 5.Localization of GRASP55 and GRASP65 within the Golgi apparatus by cryo-electron microscopy. HeLa cells transfected with rat GRASP65 and GRASP55–GFP were processed for cryo-electron microscopy. Cryosections were labelled with a rabbit polyclonal FBA31 or the 7E10 monoclonal antibody to rat GRASP65, or a polyclonal antibody to GFP to detect GRASP55–GFP. A panel of images is shown: (A) single FBA31 labelling for GRASP65; (B) single GFP labelling for GRASP55; (C and D) double labelling for GRASP65 with 7E10, large gold particles marked by arrows, and GRASP55 with anti-GFP, small gold particles. N marks the nucleus in (C) and the scale bar denotes 0.25 μM. Distributions of gold particles over Golgi cisternae were quantitated for GRASP65 and GRASP55 in both single (E) and double (F) labelling experiments. The percentage of gold particles is plotted for each Golgi cisterna from cis (1) to trans (5). Download figure Download PowerPoint Figure 6.GRASP65 and GRASP55 binding to GM130. Transcription-translation assays were performed using plasmids encoding GM130, GRASP65 or GRASP55 alone, or GRASP65 and GRASP55 with full-length GM130 or the N- and C-terminal deletions ΔN441 and ΔC887. Immunoprecipitations were performed using antibodies to GM130 with either 5 or 10 μl for single and co-in vitro translations, respectively. Aliquots of the total (A) and immunoprecipitated (B) material were analysed by SDS–PAGE and autoradiography. The positions of GRASP65 and GRASP55 are marked by an open and closed triangle, respectively, in (A) and (B). (C) Golgi membranes (10 μg), were extracted in buffer 20 mM HEPES-KOH pH 7.3, 200 mM KCl, 0.5% (w/v) Triton X-100. This extract was then fractionated by gel filtration over Superose 6, collecting 1 ml fractions. Aliquots of each fraction were analysed by SDS–PAGE and Western blotting with antibodies to either GM130 MLO-7 (open triangles), GRASP65 7E10 (open squares) or GRASP55 FBA32 (closed squares). The distribution of each marker is plotted as a percentage in a given fraction of the total signal for that marker. Two-hybrid assays were carried out using GRASP65 (D) and GRASP55 (E) as the bait constructs. Wild-type GM130 C-terminal domain, a C-terminal deletion ΔC887 and three point mutants M984A, V985A and I986A were used as prey constructs. Controls using only GRASP65, GRASP55 and GM130 together with the appropriate empty vector are shown in (F). To test for two-hybrid interactions, strains were streaked on to synthetic media lacking leucine, tryptophan, histidine and adenine. Representative examples of such plates are shown in (D), (E) and (F). The strength of these two-hybrid interactions was quantitated using liquid assays for β-galactosidase (G). Strains were Western blotted for the GRASP65 and GRASP55 prey constructs, and the GM130 bait constructs (G). Download figure Download PowerPoint GRASP65 and GRASP55 binding to GM130 Given the high degree of homology between the two proteins in the region previously shown to be important for binding to GM130, it seemed likely that, like GRASP65, GRASP55 would interact with GM130. To test this, the same in vitro transcription–translation system developed to demonstrate and characterize the interaction between GRASP65 and GM130 was used (Barr et al., 1998). In vitro translation reactions were carried out with plasmids encoding wild-type, and ΔN441 and ΔC887 deletions of GM130, GRASP65 and GRASP55 as indicated in the legend to Figure 6. Aliquots of the total reactions were analysed by SDS–PAGE and autoradiography, the position of GRASP65 is marked by an open triangle and that of GRASP55 by a filled triangle (Figure 6A and B). To assay for an interaction between GM130 and GRASP65 or GRASP55, immunoprecipitations were performed from these in vitro translation reactions with antibodies to GM130. Analysis of the bound material revealed that antibodies to GM130 precipitated GM130 (Figure 6B, lane 1) but not GRASP65 or GRASP55 (Figure 6B, lanes 8 and 9). If GRASP65 was translated together with GM130 it was found to be co-precipitated by antibodies to GM130 (Figure 6B, lane 2), whereas under the same conditions GRASP55 was not co-precipitated with GM130 (Figure 6B, lane 3). Deletion of the N-terminus of GM130, ΔN441, had no effect on the binding of GRASP65 to GM130 (Figure 6B, lane 4), while deletion of three amino acids from the C-terminus of GM130, ΔC887, abolished this interaction (Figure 6B, lane 6). Again, GRASP55 was unable to bind either of these truncated forms of GM130 (Figure 6B, lanes 5 and 7). Therefore, while GRASP65 can specifically bind to the C-terminus of GM130, GRASP55 is unable to under these conditions. To investigate this further, we decided to see if GRASP55 exists in a complex with GM130 and GRASP65 when isolated from stacked Golgi membranes. Golgi membranes were extracted in a salt- and detergent-containing buffer, fractionated by gel filtration, and the distributions of GRASP65, GRASP55 and GM130 determined by Western blotting (Figure 6C) GM130 (Figure 6C, open triangles) and GRASP65 (Figure 6C, open squares) were found to co-fractionate by gel filtration, existing as a complex of ∼1200 kDa, as reported previously (Barr et al., 1998). In contrast, GRASP55 (Figure 6C, closed squares) behaved as a 200 kDa protein by gel filtration, clearly resolved from GRASP65 and GM130. These observations are consistent with GRASP55 existing as part of a complex discrete from that containing GM130 and GRASP65. Because the residues known to be important for GM130 binding in GRASP65 are conserved in GRASP55, the inability to detect an interaction between GRASP55 and GM130 was unexpected. The two most likely possible explanations of this are that GRASP55 either does not bind to GM130, or that it binds to GM130 with much lower affinity than GRASP65. We decided to use the yeast two-hybrid to see if there is a low affinity interaction between GRASP55 and GM130 that was not detected with biochemical approaches. The interaction between GRASP65 and GM130 was used as a control, since this has been well characterized by other methods. A GRASP65 construct was transformed into a yeast reporter strain together with plasmids for wild-type GM130, or forms unable to bind (GM130ΔC983, M984A) or reduced in their ability to bind (GM130V985A, I986A) GRASP65 (Figure 6D). These strains were tested for the ability to grow under conditions that select for the activation of the reporter genes, indicative of a two-hybrid interaction (Figure 6D). Wild-type GM130 or mutants (Figure 6D, GM130V985A, I986A) with 50% reduced binding to GRASP65 were able to grow, indicating that these proteins could interact with GRASP65. Mutations that abolish the ability of GM130 to bind to GRASP65 (Figure 6D, GM130ΔC983, M984A) could not grow, indicating there was no longer any interaction between these forms of GM130 and GRASP65. A GRASP55 construct was transformed into a yeast reporter strain together with the plasmids for GM130 used above (Figure 6E). Wild-type GM130 or mutants (Figure 6E, GM130V985A, I986A) with 50% reduced binding to GRASP65 were able to grow, indicating that these proteins could interact with GRASP55. Mutations that abolish the ability of GM130 to bind to GRASP65 (Figure 6E, GM130ΔC983, M984A) could not grow, indicating that there was no longer any interaction between these forms of GM130 and GRASP55. Controls where either the empty bait or prey plasmids were tested for their ability to activate wild-type GM130, GRASP55 or GRASP65 constructs were all unable to grow on selective media (Figure 6F). These results demonstrate that GRASP55 can interact with GM130, and it does so via the same C-terminal signal as that recognized
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