THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 4, Issue of January 28, pp. 2831–2836, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Amino-terminal Domain of the Golgi Protein Giantin Interacts Directly with the Vesicle-tethering Protein p115* (Received for publication, November 10, 1999) Giovanni M. Lesa‡§¶, Joachim Seemann‡ʈ**, James Shorter‡ ‡‡, Joe¨l Vandekerckhove§§¶¶, and Graham Warren‡**ʈʈ From the ‡Cell Biology Laboratory and §Molecular Neuropathobiology Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, WC2A 3PX, United Kingdom and the §§Departments of Biochemistry and Medical Protein Research (VIB), Universiteit Gent, Ledeganckstraat 35, B-9000 Gent, Belgium Giantin is thought to form a complex with p115 and munc-18/sec1 group of proteins (22, 23). In vitro studies em- Golgi matrix protein 130, which is involved in the reas- ploying yeast vacuoles suggest that after SNARE pairing a sembly of Golgi cisternae and stacks at the end of mito- phosphatase-dependent (24) and a Ca2ϩ/calmodulin-dependent sis. The complex is involved in the tethering of coat step (25) are required for fusion. protomer I vesicles to Golgi membranes and the initial It has become increasingly clear that before SNARE pairing stacking of reforming cisternae. Here we show that the COP vesicles become tethered to target membranes. Tethering NH2-terminal 15% of Giantin suffices to bind p115 in is thought to be mediated by rod-like proteins with extensive vitro and in vivo and to block cell-free Golgi reassembly. coiled-coil (23, 26–28). One such protein is p115, which was Because Giantin is a long, rod-like protein anchored to identified originally because it is required for transport within the membrane by its extreme COOH terminus, these the Golgi apparatus (29). Subsequently, it was implicated in results support the idea of a long, flexible tether linking fusion of transcytotic vesicles with the plasma membrane (30) vesicles and cisternae. and in docking COPI vesicles to Golgi membranes (31). The p115 yeast homolog, Uso1p, is involved in endoplasmic reticu- lum to Golgi transport (32) and is essential for docking of In eukaryotic cells proteins and lipids are conveyed to differ- endoplasmic reticulum-derived vesicles (33). Both proteins ent intracellular compartments via vesicular carriers. Coated form dimers with two globular heads, a long coiled-coil domain vesicles carrying selected cargo bud from the donor compart- and a short acidic tail (34, 35). ment and fuse with the appropriate acceptor compartment Further understanding of p115 function came from studying delivering their content (1). COPII1 vesicles transport newly the Golgi apparatus during the cell cycle. During interphase synthesized proteins from the endoplasmic reticulum to the p115 binds Golgi membranes via the Golgi matrix protein Golgi apparatus (2–4). COPI vesicles bud from the Golgi and GM130 (36). At the onset of mitosis cdc2/cyclin B phosphoryl- transport back to the endoplasmic reticulum molecules that ates GM130, and this inhibits the binding of p115 to the Golgi need to be salvaged or recycled (5–7). In addition, COPI vesicles apparatus in vitro (36–38) and in vivo (39). This may explain have been shown to be involved in anterograde transport (8– the extensive vesiculation of the Golgi complex observed during 10) and, more recently, in endocytosis (11–13). mitosis (40–42). The loss of p115 would prevent the tethering Correct intracellular transport demands that both COPI and and hence fusion of COPI vesicles. Continued budding in the COPII vesicles are targeted to and fuse with specific mem- absence of fusion would help to vesiculate the Golgi apparatus branes. Membrane fusion requires NSF, a set of soluble pro- (42–44). teins, the SNAPs, and pairing between two sets of cognate Another receptor for p115 is Giantin (31, 36). In addition to proteins, the SNAREs, one set being on the vesicle (v-SNARE), residing on Golgi membranes, Giantin is also incorporated into the other on the target membrane (t-SNARE) (14–17). This budding COPI vesicles (31), whereas GM130 is largely ex- association is thought to be positively regulated by the Ypt/Rab cluded. In vitro binding experiments showed that p115 binds family of GTPases (18–21) and negatively regulated by the Giantin on COPI vesicles and GM130 on Golgi membranes. It has therefore been suggested that p115 tethers COPI vesicles * The costs of publication of this article were defrayed in part by the by connecting GM130 on the Golgi membrane to Giantin on the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to COPI vesicle (31). Experiments employing a Golgi reassembly indicate this fact. assay (45, 46) lend support to this idea. They showed that p115, ¶ Supported by a traveling research fellowship from the Wellcome GM130, and Giantin play a crucial role in Golgi cisternal re- Trust. growth (47), and more recently they have been implicated in ʈ Supported by the Deutsche Forschungsgemeinschaft. stacking Golgi cisternae (48). ** Present address: Dept. of Cell Biology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520 8002. Because tethers operate before SNARE pairing (33), one ‡‡ Supported by a predoctoral research fellowship from the Imperial could imagine that they act at a greater distance. They would Cancer Research Fund. perhaps allow the vesicle to sample the membrane before ¶¶ Supported by a grant from the Fund for Scientific Research of bringing it closer to permit v- and t-SNARE interaction. Such a Flanders. ʈʈ To whom correspondence should be addressed. Tel.: 203-785-5058; sampling function would be critically dependent on the length Fax: 203-785-4301; E-mail: [email protected]. of the tethering complex. Both GM130 and Giantin appear to be 1 The abbreviations used are: COPII, coat protomer II; COPI, coat long, rod-like proteins. GM130 is about 50 nm in length by protein complex I; NSF, N-ethylmaleimide-sensitive factor; SNAP, sol- negative staining,2 whereas Giantin has a predicted length, uble NSF attachment protein; SNARE, SNAP receptor; GM130, Golgi matrix protein 130; PAGE, polyacrylamide gel electrophoresis; kb, ki- lobase(s); PCR, polymerase chain reaction; MALDI, matrix-assisted laser desorption and ionization. 2 M. Lowe, R. Newman, and G. Warren, unpublished observation. This paper is available on line at http://www.jbc.org 2831 2832 NH2-terminal Giantin Binds p115 based on sedimentation analysis, of 250 nm (49, 50). Further- more, GM130 binds to Golgi membranes at its COOH-terminal end and to p115 at its NH2-terminal end. Giantin is anchored by its COOH terminus to the membrane, but the binding site for p115 has not been mapped. Given the importance of this site in conceptualizing the tethering complex we have mapped the p115 binding site on Giantin and analyzed its properties in vitro and in vivo. EXPERIMENTAL PROCEDURES Antibodies—The following antibodies were used in this study: mouse monoclonal 4H1 against p115 (29), mouse monoclonal against GM130 (Transduction Laboratories, Lexington, KY), rabbit polyclonal anti-myc tag (Santa Cruz Biotechnology, Santa Cruz, CA), Texas Red-X goat anti-mouse (Molecular Probes, Eugene, OR), and Alexa Fluor 488 goat anti-rabbit (Molecular Probes). SDS-PAGE and Western Blotting—Proteins were solubilized in SDS- PAGE sample buffer, boiled for 4 min, and analyzed on 6, 10, or 12% SDS-polyacrylamide gels (51, 52). For Western blotting, proteins were transferred onto Hybond C blots (Amersham Pharmacia Biotech, Upp- sala, Sweden) using a semidry blotter. Blocking and antibody incuba- tions were performed in phosphate-buffered saline containing 5% (w/v) low fat skim milk powder and 2% (v/v) polyoxyethylenesorbitan mono- FIG. 1. Schematic representation of the Giantin fragments laurate (Tween 20). Secondary antibodies were horseradish peroxidase used to map the binding site for p115. The fragments were gener- conjugates (Tago, Buckingam, U. K.) and were detected with an ECL kit ated by in vitro transcription/translation using [35S]methionine as the (Amersham Pharmacia Biotech). label with the exception of Gtn1125–1695, which was prepared as a Constructs for in Vitro Transcription/Translation—The construct recombinant protein from E. coli. Each fragment was analyzed for used for in vitro transcription/translation of full-length Giantin was binding to biotinylated p115 immobilized on neutravidin beads and pGCP364/pSG5, kindly provided by Dr. Y. Ikehara (Fukuoka Univer- compared with binding of full-length Giantin. The results from a num- sity, Japan). pGCP364/pSG5 contains the full-length cDNA of rat Gi- ber of experiments (some of which are presented in Fig. 2) are summa- antin cloned into the EcoRI restriction site of the eukaryotic expression rized on the right. ϩϩϩϩ indicates binding of full-length Giantin to vector pSG5 (Stratagene, La Jolla, CA). Gtn450–3187 was obtained by immobilized p115; ϩϩϩ, strong binding; ϩ, moderate binding; Ϯ, poor in vitro transcription/translation of pGL88, which contains an ϳ8.5-kb binding; Ϫ, no detectable binding; N.T., not tested. XbaI fragment from pGCP364/pSG5 in the cloning vector pSTBlue-1 (Novagen, Madison, WI) oriented with its 5Ј-end toward the T7 pro- Pfu turbo DNA polymerase to PCR amplify a ϳ1.7-kb fragment from moter. Plasmids encoding for Gtn448, Gtn105–448, Gtn186, Gtn293– pGCP364/pSG5 using primers GL98, 5Ј-GACTCAGGATCCAAT- 448, Gtn301, and Gtn187–448 were obtained by PCR using primers GAAGCTTCAAGAAGCCTTAATTTCC-3Ј, which contains a BamHI re- with BamHI and EcoRI restriction sites, the high fidelity DNA polym- striction site and a starting codon, and GL99, 5Ј-CCTGAGCTTCTAC- erase Pfu turbo (Stratagene), and pGCP364/pSG5 as a template. Each CTGAGAATTCAGATTACGAGTCTCTTC-3Ј, which contains an EcoRI PCR product was subcloned in pBluescript II (Stratagene) between the restriction site.