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Brief Communication 377

The GRIP domain – a novel Golgi-targeting domain found in several coiled-coil proteins and Ben J. Nichols

Many large coiled-coil proteins are being found to the cytoplasmic face of Golgi membranes. For a third associated peripherally with the cytoplasmic face of the coiled-coil Golgi protein, GM130, it has been shown that organelles of the secretory pathway. Various roles have sequences at its extreme carboxyl terminus specify target- been proposed for these proteins, including the ing to the Golgi [6]. The carboxy-terminal regions of docking of donor vesicles or organelles to an acceptor golgin-245 and golgin-97 do not show any homology to this organelle prior to fusion, and, in the case of the Golgi region of GM130. We noticed, however, that the two pro- apparatus, the stacking of the cisternae [1–5]. Such teins are distantly related to each other in their carboxy-ter- critical roles require accurate recruitment to the correct minal non-coiled-coil portions. GenBank database searches organelle. For the endosomal coiled-coil protein EEA1, with the iterative program PSI-BLAST [11] revealed the targeting requires a carboxy-terminal FYVE domain, existence of a family of proteins that show homology over a which interacts with Rab5 and phosphatidylinositol region of 50–60 amino acids (Figure 1a), with the con- 3-phosphate (PI(3)P), whereas the Golgi protein GM130 verged searches showing a significance of p < 10–7 for each interacts with Golgi membranes via the protein member. In all but one case the conserved sequence is at GRASP65 [3,6,7]. In this paper, we show that two other the carboxyl terminus of a protein that is predicted to form mammalian Golgi coiled-coil proteins, golgin-245/p230 almost entirely coiled-coils (Figure 1b). The only excep- and golgin-97, have a conserved domain of about 50 tion to this is Ran-binding protein 2α (RanBP2α), and the amino acids at their carboxyl termini. This ‘GRIP’ provenance of this protein is unclear (see Figure 1 legend). domain is also found at the carboxyl terminus of We propose to call the conserved sequence a GRIP several other large coiled-coiled proteins of unknown domain, after the four proteins in which it is found and function, including two human proteins and proteins in about which something has been reported (golgin-97, the genomes of Caenorhabditis elegans and yeasts. RanBP2α, Imh1p, and p230/golgin-245). The GRIP domains from several of these proteins, including that from the yeast protein Imh1p, were To examine the possibility that the function of the GRIP sufficient to specify Golgi targeting in mammalian cells domain is to specify Golgi localisation, the GRIP domains when fused to green fluorescent protein (GFP). This from golgin-245 and golgin-97 were fused to the carboxyl result suggests that this small domain functions to terminus of GFP and expressed in COS cells. Figure 2b recruit specific coiled-coil proteins to the Golgi by shows that when living cells were viewed two days after recognising a determinant that has been well conserved transfection, bright fluorescence was visible in ribbons in eukaryotic evolution. clustered adjacent to the nucleus, clearly distinct from the diffuse cytoplasmic signal seen with GFP chimeras that do Address: MRC Laboratory of , Hills Road, Cambridge CB2 2QH, UK. not carry targeting information. Double-label immunoflu- orescence of fixed and permeabilised cells confirmed that Correspondence: Sean Munro this perinuclear fluorescence corresponds to the Golgi E-mail: [email protected] apparatus (Figure 2c). The GFP chimeras colocalised with Received: 21 December 1998 markers for either the early Golgi (the KDEL-receptor Revised: 16 February 1999 ERD2) or the late Golgi (TGN46). Upon scattering of the Accepted: 23 February 1999 Golgi by treatment with the microtubule-depolymerising Published: 29 March 1999 drug nocodazole, the GFP chimeras remained colocalised with Golgi markers. The drug brefeldin A (BFA) causes Current Biology 1999, 9:377–380 the Golgi stack to fuse with the http://biomednet.com/elecref/0960982200900377 and disappear; when cells expressing the GFP–golgin-245 © Elsevier Science Ltd ISSN 0960-9822 chimera were treated with BFA for 20 minutes, the GFP signal became diffuse, consistent with loss of a Golgi local- isation (Figure 2b). The GFP chimeras did not colocalise Results and discussion with endocytosed transferrin, indicating that the peri- Golgin-245 (also called p230) and golgin-97 were originally nuclear localisation does not reflect an association with the identified as self-antigens recognised by autoantisera from recycling endosome compartment (Figure 2c). Together, patients with Sjögren’s syndrome [8–10]. They are large these results show that the GRIP domains of the Golgi proteins predicted to form extensive coiled-coils with short proteins golgin-245 and golgin-97 are capable of mediating non-coiled-coil regions at either end, and both are localised targeting to the Golgi. We also examined the GRIP 378 Current Biology, Vol 9 No 7

Figure 1

Structure and occurrence of the GRIP (a) domain. (a) Alignment of the carboxy-terminal Hs Golgin-245 2013 GGNLYHTDVSLFGEPTEFEYLRKVLFEYMMGRET.....KTMAKVITTVLKFPDDQTQKILEREDARLMSWLRSSS* Hs Golgin-97 678 APSVTNNTDLTDAREINFEYLKHVVLKFMSCRES...EAFHLIKAVSVLLNFSQEEENMLKETLEYKMSWFGSKPAPKGSIRPSISNPRIPWS* portions of the indicated proteins. Residues Hs KIAA0336 1501 RLERNQEREKSAAN...LEYLKNVLLQFIFLKPG..SERERLLPVINTMLQLSPEEKGKLAAVAQGEEENASRSSGWASYLHSWSGLR* Hs AC000357 ... QSHIEKNIRDQSREGANLEYLKNIIYRFLTLPDS..LGRQQTLTAILTILHFSPEEKQVIMRLPTSASWWPSGKR* that are identical (yellow) or related (grey) in Sc Imh1p 851 KIRSSSSLELDSEKNEKIAYIKNVLLGFLEHKEQ....RNQLL PVISMLLQLDSTDEKRLVMSLK* Ce F59A2.6 1049 TGRTKSMELHGIADPAEAEYLRNVLYRYMTNRESLGKESVTLARVIGTVARFDESQMKNVISSEESRQASWAAAVGGTVSHALNR* five or more of the nine sequences are shaded Ce T05G5.9 620 RLEREEKREAELSNEKNMEYLKNVFVQFLKPESVP.AERDQLVIVLQRVLHLSPKEVEILKAASAHMATAQAGSWSSYFSGWSG* Ce C15C7.2 561 KVRVLEGKLQMLSSTSEIDYLRNIFTQFLHSMGSPNAASKAILKAMGSVLKVPMAEMKIIDKK* (Hs, Homo sapiens; Sc, Saccharomyces Sp SPAC27D7.02 690 SVEVNSKHPGSDDMLIDKEYTR NILFQFLEQRDR....RPEIVNLLSILLDLSEEQKQKLLSVKY* cerevisiae; Sp, Schizosaccharomyces pombe; (b) Human Ce, C. elegans). The tyrosine conserved in all Golgin-245/p230 of the sequences is indicated by an Golgin-97 arrowhead. KIAA0336 is a human cDNA of KIAA0336 unknown function (accession number RanBP2α AB002334). AC000357 is an open-reading AC000357 frame from a human cosmid with the same C. elegans accession number; several corresponding F59A2.6 expressed sequence tags (ESTs), for example T05G5.9 Z43507, indicate that it is expressed. The C15C7.2 asterisk indicates the carboxyl teminus of the Yeast protein. (b) Location of the GRIP domain in Imh1p (S. cerevisiae) the indicated proteins. GRIP domains are SPAC27D7.02 (S. pombe) shown in yellow, and regions predicted to 100 amino acids form coiled-coils are shown in green. Current Biology RanBP2α is encoded by a partial cDNA and its carboxy-terminal 125 amino acids are very from the middle of the large non-coiled-coil fusion, and hence of unclear functional closely related (five mismatches) to the nuclear pore protein RanBP2 [20]. This significance (it is unlikely to represent a carboxyl terminus of KIAA0336, whereas the suggests that it is the product of a hybrid cloning artifact because several human and rest of the protein is very similar to a portion gene formed by recent duplications and mouse ESTs span the junction). domain from the protein of unknown localisation that is In order to determine whether the GRIP domain of Imh1p encoded by the KIAA0336 cDNA. A fusion comprising provides a similar targeting function to those of the mam- GFP and the GRIP domain of KIAA0336 also localised to malian proteins, we initially expressed it as a GFP fusion the Golgi when expressed in COS cells (Figure 2b). This in COS cells. The fusion protein was partially distributed result reinforces the idea that the GRIP domain specifies a throughout the cytoplasm but was also clearly concen- Golgi localisation, and suggests that KIAA0336 encodes a trated in the Golgi region (Figure 3a), demonstrating that new member of the family of Golgi coiled-coil proteins. the Imh1p GRIP domain can serve as a Golgi targeting domain, and that what it recognises on the Golgi must The alignment of the GRIP domains in Figure 1b shows have been well conserved through eukaryotic evolution. only a single invariant residue, a tyrosine near the amino When live yeast cells expressing the GFP–Imh1p-GRIP- terminus of the domain. Figure 2b shows that when this domain chimera were examined, some of the protein could tyrosine is mutated to an alanine in the GRIP domains of be seen in punctate cytoplasmic structures characteristic of golgin-97 and golgin-245, the Golgi localisation of the cor- the yeast Golgi (Figure 3b) [13]. The punctate fluores- responding GFP chimeras is abolished. Western blotting cence was not affected by deletion of the endogenous showed that the wild-type and mutant chimeras were IMH1 gene — if anything, the signal became brighter and intact and accumulated to similar levels (data not shown). more distinct (Figure 3b and data not shown). This This suggests that the Golgi targeting of the GRIP demonstrates that the ability of the Imh1p GRIP domain domain is mediated by a sequence-specific interaction, to target to a specific part of the cell does not simply rather than reflecting an association based on a global reflect an interaction with endogenous Imh1p. We have physical property such as charge. Indeed, all of the GRIP- found that, in both yeast and mammalian cells, the level of domain sequences have a similar predicted secondary Golgi association of the GFP–GRIP-domain chimeras is structure of three short α helices, suggesting that the reduced upon fixation and permeabilisation. This has pre- domain forms a well-organised structure. vented us from directly determining the yeast Golgi com- partment with which the Imh1p chimera is associated. We The genome of the yeast S. cerevisiae encodes only one observed, however, that in a strain with a temperature-sen- protein with a GRIP domain, namely the coiled-coil sitive allele of SEC14 the punctate fluorescence signal was protein Imh1p. The IMH1 gene was originally isolated as a lost at the non-permissive temperature. The sec14 muta- suppressor of the temperature-sensitive lethality of a tion selectively perturbs the function of the late Golgi, mutation in the small GTP-binding protein Ypt6p, a close blocking transport to the plasma membrane, and does not relative of the mammalian protein Rab6 [12]. The function affect other membrane-trafficking steps [14]. This sug- and intracellular location of Imh1p are unknown, however. gests that the Imh1p chimera may be associated with this Brief Communication 379

Figure 2

Expression of GFP–GRIP-domain fusions in COS cells. (a) Structure of the GFP–GRIP- (a) N GFP GRIP C (c) GFP fusion Organelle marker domain fusions used in this study. Myc (b) Fluorescence confocal micrographs of live COS cells expressing GFP fused to the GRIP (b) domain of the indicated protein. The fusions contained the carboxy-terminal 83, 100 and 90 residues of golgin-245, golgin-97 and KIAA0336, respectively. The lower panels Golgin-97 ERD2 show COS cells expressing the indicated fusions in which the conserved tyrosine residue in the GRIP domain has been mutated to an alanine. (c) Immunofluorescence Golgin-245 Golgin-97 confocal micrographs of fixed and permeabilised COS cells expressing GFP fused to the GRIP domain of the indicated KIAA0336 TGN46 protein. Cells were stained with rabbit antisera to the indicated Golgi proteins [21,22], or with Alexa-588–streptavidin (Molecular Probes) to detect biotinylated transferrin (Sigma), which was added at 50 µg/ml for 20 min prior to fixation. Similar results were KIAA0336 Golgin-245 + BFA obtained with all of the GRIP domains when Golgin-245 TGN46 + nocodazole compared to all of the markers, and representative combinations are shown. The structure of the GFP–Myc portion of the fusion constructs, COS cell transfection, and protein localisation have all been described previously [23]. Cells were visualised with a Biorad MRC600 confocal microscope. BFA was used at 10 µg/ml for 20 min and Golgin-245 Y2177A Golgin-97 Y697A Golgin-97 Transferrin nocodazole was used at 10 µM for 120 min. Current Biology

late Golgi compartment. In addition, Figure 3c shows that Golgi-specific PIP has not yet been identified. Precise when yeast membranes were pelleted by centrifugation, a organelle specification may require a combinatorial recogni- portion of the GFP–Imh1p-GRIP-domain chimera tion of multiple structures, and indeed the FYVE domain remained in the supernatant with the cytosolic protein also associates with the small GTPase Rab5 [3]. Thus it is phosphoglycerate kinase, but the remainder pelleted with possible that a Golgi-specific Rab such as Rab6 may bind to the P100 membranes that contain the Golgi, indicative of a the GRIP domain, although again there is no similarity peripheral association with Golgi membranes [15]. between the GRIP domain and previously identified Rab6- binding proteins [18,19]. Imh1p shows a genetic interaction The results described here characterise a small domain with the yeast Rab6 homologue Ypt6p, with an increase in from two known coiled-coil proteins of the Golgi that can IMH1 copy number suppressing the temperature-sensitive specify a Golgi localisation and presumably serves in the lethality, but not the protein-trafficking defects, of a muta- recruitment of a family of such proteins to the Golgi. The tion in YPT6 [12,24]. This mutation truncates Ypt6p after domain is also present in several coiled-coil proteins of only 63 residues of 215, however, suggesting that Imh1p is unknown function from humans and worms, and so identi- able to exert an effect in the absence of functional Ypt6p. fies these as strong candidates for Golgi proteins. The The conservation of the GRIP domain in both yeasts and results raise the question of the nature of the receptor for mammalian cells should nonetheless help in the search for the GRIP domain on Golgi membranes. Immunoprecipita- the ligand of the domain, and thereby reveal more about tion of the GFP chimeras from COS cells that had been the recruitment and function of the growing number of labelled with [35S]methionine and solubilised in Triton large coiled-coil proteins found on Golgi membranes. The X-100 did not reveal any stoichiometrically associated GRIP domain has also been identified by F.A. Barr and by protein(s) (data not shown). Thus the domain may bind to a Gleeson and colleagues (this issue). Golgi-specific lipid, in a manner analogous to the binding of the FYVE domain to PI(3)P on endosomal membranes Acknowledgements We thank Hugh Pelham and James Whyte for comments on the manuscript, [7,16,17]. It should be noted, however, that the GRIP Vaz Ponnambalam and Hans-Dieter Söling for antibodies, and our col- domain shows no homology to the FYVE domain, and a leagues for invaluable advice and reagents. 380 Current Biology, Vol 9 No 7

Figure 3

Expression of GFP fused to the GRIP domain of the yeast protein Imh1p. (a) GFP–Imh1p TGN46 (b) (a) Immunofluorescence confocal micrographs obtained as described in Figure 2c except that the GRIP domain is the carboxy-terminal 89 residues of Imh1p. (b) Fluorescence micrographs of live yeast either without (no GFP) or expressing the Imh1p GRIP-domain GFP–Imh1p fusions from a TPI promoter in a CEN plasmid. No GFP in ∆imh1 Strains are either wild-type SEY6210 [15], the same strain with the endogenous IMH1 gene deleted (∆imh1), sec14-3 grown at 25°C (c) (permissive temperature), or sec14-3 after P13 P100 S100 shifting from 25oC to 37°C for 25 min, as indicated. GFP fusions contained the carboxy- Anti-GFP GFP–Imh1p GFP–Imh1p terminal 89 or 109 residues of Imh1p in ∆imh1 in sec14ts 25ºCin sec14ts 37ºC and sec14-3 strains, respectively. Cells were 5 µm photographed with a Princeton Instruments Anti-PGK CCD camera on a Zeiss microscope. (c) Immunoblot of membranes from SEY6210 cells expressing the 89 residue Imh1p–GFP Anti-Tlg1p fusion that were pelleted at 13,000 × g (P13), × or at 100,000 g (P100) from the P13 Relative loading 5 5 1 supernatant. The P100 supernatant (S100) Current Biology was also blotted. Fractions were prepared as described previously [15]. The lower presumably because of proteolysis. Blots were protein phosphoglycerate kinase (PGK, GFP–Imh1p band appears after cell lysis, probed with antibodies to GFP, the cytosolic Molecular Probes), or the Golgi tSNARE Tlg1p.

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