Three Homologous ArfGAPs Participate in Coat Protein I-mediated Transport
2009; Elsevier BV; Volume: 284; Issue: 20 Linguagem: Inglês
10.1074/jbc.m900749200
ISSN1083-351X
AutoresAkina Saitoh, Hye‐Won Shin, Akane Yamada, Satoshi Waguri, Kazuhisa Nakayama,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoArfGAP1 is a prototype of GTPase-activating proteins for ADP-ribosylation factors (ARFs) and has been proposed to be involved in retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER) by regulating the uncoating of coat protein I (COPI)-coated vesicles. Depletion of ArfGAP1 by RNA interference, however, causes neither a discernible phenotypic change in the COPI localization nor a change in the Golgi-to-ER retrograde transport. Therefore, we also examined ArfGAP2 and ArfGAP3, closely related homologues of ArfGAP1. Cells in which ArfGAP1, ArfGAP2, and ArfGAP3 are simultaneously knocked down show an increase in the GTP-bound ARF level. Furthermore, in these cells proteins resident in or cycling through the cis-Golgi, including ERGIC-53, β-COP, and GM130, accumulate in the ER-Golgi intermediate compartment, and Golgi-to-ER retrograde transport is blocked. The phenotypes observed in the triple ArfGAP knockdown cells are similar to those seen in β-COP-depleted cells. Both the triple ArfGAP- and β-COP-depleted cells accumulate characteristic vacuolar structures that are visible under electron microscope. Furthermore, COPI is concentrated at rims of the vacuolar structures in the ArfGAP-depleted cells. On the basis of these observations, we conclude that ArfGAP1, ArfGAP2, and ArfGAP3 have overlapping roles in regulating COPI function in Golgi-to-ER retrograde transport. ArfGAP1 is a prototype of GTPase-activating proteins for ADP-ribosylation factors (ARFs) and has been proposed to be involved in retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER) by regulating the uncoating of coat protein I (COPI)-coated vesicles. Depletion of ArfGAP1 by RNA interference, however, causes neither a discernible phenotypic change in the COPI localization nor a change in the Golgi-to-ER retrograde transport. Therefore, we also examined ArfGAP2 and ArfGAP3, closely related homologues of ArfGAP1. Cells in which ArfGAP1, ArfGAP2, and ArfGAP3 are simultaneously knocked down show an increase in the GTP-bound ARF level. Furthermore, in these cells proteins resident in or cycling through the cis-Golgi, including ERGIC-53, β-COP, and GM130, accumulate in the ER-Golgi intermediate compartment, and Golgi-to-ER retrograde transport is blocked. The phenotypes observed in the triple ArfGAP knockdown cells are similar to those seen in β-COP-depleted cells. Both the triple ArfGAP- and β-COP-depleted cells accumulate characteristic vacuolar structures that are visible under electron microscope. Furthermore, COPI is concentrated at rims of the vacuolar structures in the ArfGAP-depleted cells. On the basis of these observations, we conclude that ArfGAP1, ArfGAP2, and ArfGAP3 have overlapping roles in regulating COPI function in Golgi-to-ER retrograde transport. The ADP-ribosylation factors (ARFs) 3The abbreviations used are: ARF, ADP-ribosylation factor; COPI, coat protein I; ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; GAP, GTPase-activating protein; KDELR, KDEL receptor; siRNA, small interfering RNA; RNAi, RNA interference; VSVG, vesicular stomatitis virus G protein; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GFP, green fluorescent protein; EGFP, enhanced GFP; HA, hemagglutinin; si-, small interfering-. 3The abbreviations used are: ARF, ADP-ribosylation factor; COPI, coat protein I; ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; GAP, GTPase-activating protein; KDELR, KDEL receptor; siRNA, small interfering RNA; RNAi, RNA interference; VSVG, vesicular stomatitis virus G protein; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GFP, green fluorescent protein; EGFP, enhanced GFP; HA, hemagglutinin; si-, small interfering-. are a family of small GTPases. Once associated with organellar membranes in their GTP-bound form, these proteins trigger formation of coated carrier vesicles, e.g. coat protein I (COPI)-coated vesicles. ARFs cycle between a GDP-bound inactive state and a GTP-bound active state; in the latter form they recruit various effectors, including the COPI coat (1Nickel W. Wieland F.T. Histochem. Cell Biol. 1998; 109: 477-486Crossref PubMed Scopus (33) Google Scholar, 2Donaldson J.G. Honda A. Weigert R. Biochim. Biophys. Acta. 2005; 1744: 364-373Crossref PubMed Scopus (96) Google Scholar). Exchange of bound GDP for GTP is catalyzed by guanine-nucleotide exchange factors, which constitute a large family of proteins that share a Sec7-like catalytic domain (3Gillingham A.K. Munro S. Annu. Rev. Cell Dev. Biol. 2007; 23: 579-611Crossref PubMed Scopus (438) Google Scholar, 4Shin H.-W. Nakayama K. J. Biochem. (Tokyo). 2004; 136: 761-767Crossref PubMed Scopus (59) Google Scholar). GTP hydrolysis in turn is stimulated by GTPase-activating proteins (GAPs), which constitute a large family that share a zinc finger-like catalytic domain (3Gillingham A.K. Munro S. Annu. Rev. Cell Dev. Biol. 2007; 23: 579-611Crossref PubMed Scopus (438) Google Scholar, 5Nie Z. Randazzo P.A. J. Cell Sci. 2006; 119: 1203-1211Crossref PubMed Scopus (103) Google Scholar).COPI-coated vesicles mediate retrograde transport from the cis-Golgi or endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) to the ER and probably intra-Golgi transport as well. In budding yeasts two ARF-GAPs, Gcs1 and Glo3, have been shown to play overlapping roles in COPI-mediated transport processes (6Lewis S.M. Poon P.P. Singer R.A. Johnston G.C. Spang A. Mol. Biol. Cell. 2004; 15: 4064-4072Crossref PubMed Scopus (49) Google Scholar, 7Poon P.P. Cassel D. Spang A. Rotman M. Pick E. Singer R.A. Johnston G.C. EMBO J. 1999; 18: 555-564Crossref PubMed Scopus (132) Google Scholar). According to the prevailing view, ARF-GAPs (in particular, ArfGAP1, which is the founding member of mammalian ARF-GAPs and the counterpart of yeast Gcs1) (8Cukierman E. Huber I. Rotman M. Cassel D. Science. 1995; 270: 1999-2002Crossref PubMed Scopus (268) Google Scholar) either induce dissociation of the coat from COPI-coated vesicles or antagonize formation of vesicles (for review, see Ref. 5Nie Z. Randazzo P.A. J. Cell Sci. 2006; 119: 1203-1211Crossref PubMed Scopus (103) Google Scholar). This view is based on several lines of evidence; first, blocking GTP hydrolysis on ARF1 by adding GTPγS or a GTPase-defective ARF1 mutant inhibits uncoating of COPI-coated vesicles in a cell-free reconstitution system (9Tanigawa G. Orci L. Amherdt M. Ravazzola M. Helms J.B. Rothman J.E. J. 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A. 2003; 100: 8253-8257Crossref PubMed Scopus (54) Google Scholar); finally, ArfGAP1-mediated GTP hydrolysis is stimulated by the addition of the COPI coat in vitro (13Szafer E. Rotman M. Cassel D. J. Biol. Chem. 2001; 276: 47834-47839Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14Goldberg J. Cell. 1999; 96: 893-902Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar).However, additional evidence suggests roles of ArfGAP1 beyond that of a simple inactivator of ARFs (for review, see Ref. 5Nie Z. Randazzo P.A. J. Cell Sci. 2006; 119: 1203-1211Crossref PubMed Scopus (103) Google Scholar); first, GTP hydrolysis on ARF is required for proper sorting of cargo molecules into COPI-coated vesicles (15Lanoix J. Ouwendijk J. Lin C.-C. Stark A. Love H.D. Ostermann J. Nilsson T. EMBO J. 1999; 18: 4935-4948Crossref PubMed Scopus (177) Google Scholar, 16Nickel W. Malsam J. Gorgas K. Ravazzola M. Helms J.B. Wieland F.T. J. 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Cell Biol. 2005; 168: 1053-1063Crossref PubMed Scopus (64) Google Scholar, 22Majoul I. Straub M. Hell S.W. Duden R. Söling H.-D. Dev. Cell. 2001; 1: 139-153Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar); finally, Antonny and co-workers (23Bigay J. Casella J.-F. Drin G. Mesmin B. Antonny B. EMBO J. 2005; 24: 2244-2253Crossref PubMed Scopus (271) Google Scholar, 24Bigay J. Gounon P. Robineau S. Antonny B. Nature. 2003; 426: 563-566Crossref PubMed Scopus (258) Google Scholar) have proposed a model in which ArfGAP1 and Gcs1 sense the curvature of budding vesicles through a motif outside of their catalytic domain.Despite the critical roles of ArfGAP1 in COPI-coated vesicle formation, most of the available data regarding their function have been obtained by in vitro experiments. We, therefore, attempted to determine the function of ArfGAP1 in the cell by exploiting RNA interference (RNAi). However, we could not detect any phenotypic change in ArfGAP1 knockdown cells. Because there are two poorly characterized mammalian ArfGAPs, ArfGAP2 and ArfGAP3 (25Kahn R.A. Bruford E. Inoue H. Logsdon Jr., J.M. Nie Z. Premont R.T. Randazzo P.A. Satake M. Theibert A.B. Zapp M.L. Cassel D. J. Cell Biol. 2008; 182: 1039-1044Crossref PubMed Scopus (97) Google Scholar), both of which are more similar to Glo3 than Gcs1 (26Yahara N. Sato K. Nakano A. J. Cell Sci. 2006; 119: 2604-2612Crossref PubMed Scopus (20) Google Scholar, 27Frigerio G. Grimsey N. Dale M. Majoul I. Duden R. Traffic. 2007; 8: 1644-1655Crossref PubMed Scopus (43) Google Scholar, 28Liu X. Zhang C. Xing G. Chen Q. He F. FEBS Lett. 2001; 490: 79-83Crossref PubMed Scopus (30) Google Scholar, 29Zhang C. Yu Y. Zhang S. Liu M. Xing G. Wei H. Bi J. Liu X. Zhou G. Dong C. Hu Z. Zhang Y. Luo L. Wu C. Zhao S. He F. Genomics. 2000; 63: 400-408Crossref PubMed Scopus (18) Google Scholar), we then set out to determine the intracellular roles of these ArfGAPs. Here, we show that ArfGAP1, ArfGAP2, and ArfGAP3 play overlapping roles in COPI-mediated transport and in maintaining Golgi organization.EXPERIMENTAL PROCEDURESAntibodies, Reagents, and Plasmids—Antiserum to human ArfGAP1 was raised in rabbits against a synthetic peptide (amino acid residues 377-390) conjugated to keyhole limpet hemocyanin and affinity-purified using the immunized peptide immobilized on Sulfolink beads (Pierce). Antisera to human ArfGAP2 and ArfGAP3 were raised against their polypeptide regions (amino acid residues 329-430 and 329-426, respectively) fused to glutathione S-transferase and affinity-purified using the fusion proteins immobilized on Sulfolink beads. Monoclonal mouse anti-ERGIC-53 antibody (30Schweizer A. Fransen J.A. Bächi T. Ginsel L. Hauri H.-P. J. Cell Biol. 1988; 107: 1643-1653Crossref PubMed Scopus (374) Google Scholar) was originally provided by Hans-Peter Hauri (University of Basel) and later purchased from Alexis Biochemicals. Polyclonal rabbit antibodies to golgin-97 (31Yoshimura S. Yamamoto A. Misumi Y. Sohda M. Barr F.A. Fujii G. Shakoori A. Ohno H. Mihara K. Nakamura N. J. Biochem. (Tokyo). 2004; 135: 201-216Crossref PubMed Scopus (43) Google Scholar) and to GM130 (32Sohda M. Misumi Y. Yoshimura S. Nakamura N. Fusano T. Ogata S. Sakisaka S. Ikehara Y. Traffic. 2007; 8: 270-284Crossref PubMed Scopus (61) Google Scholar) were kindly provided by Nobuhiro Nakamura (Kanazawa University, Japan) and Yoshio Misumi (Fukuoka University), respectively. Monoclonal mouse antibodies to golgin-245, GM130, and green fluorescent protein (GFP) were purchased from BD Biosciences. Monoclonal mouse anti-ARF1 antibody (3F1) was from Affinity Bioreagents. Polyclonal rabbit and monoclonal mouse anti-β-COP antibodies were from Affinity Bioreagents and Sigma-Aldrich, respectively. Polyclonal rabbit anti-syntaxin 5 antibody was from Synaptic Systems. AlexaFluor- and horseradish peroxidase-conjugated secondary antibodies were from Molecular Probes and Jackson Immuno-Research Laboratories, respectively.An expression vector for N-terminal EGFP-tagged VSVG tsO45 (pCIpreEGFP-VSVG) was constructed by exchanging the mature VSVG tsO45 segment from pcDNA3-VSVG-EGFP (33Kasai K. Shin H.-W. Shinotsuka C. Murakami K. Nakayama K. J. Biochem. (Tokyo). 1999; 125: 780-789Crossref PubMed Scopus (44) Google Scholar) for the cation-independent mannose 6-phosphate receptor cDNA segment of pCIpreEGFP-CIMPRtail (34Waguri S. Dewitte F. Le Borgne R. Rouillé Y. Uchiyama Y. Dubremetz J.-F. Hoflack B. Mol. Biol. Cell. 2003; 14: 142-155Crossref PubMed Scopus (147) Google Scholar). Construction of an expression vector for C-terminal HA-tagged ARFRP1 was described previously (35Shin H.-W. Kobayashi H. Kitamura M. Waguri S. Suganuma T. Uchiyama Y. Nakayama K. J. Cell Sci. 2005; 118: 4039-4048Crossref PubMed Scopus (56) Google Scholar). An expression vector for N-terminal EGFP+VSVG-tagged KDELR (pCIpreEGFP-VSVG-KDELR) was constructed by exchanging a cDNA segment of the mature human KDELR2 region (kindly provided by Victor Hsu, Harvard Medical School) (36Yang J.-S. Lee S.Y. Spanó S. Gad H. Zhang L. Nie Z. Bonazzi M. Corda D. Luini A. Hsu V.W. EMBO J. 2005; 24: 4133-4143Crossref PubMed Scopus (80) Google Scholar, 37Cole N.B. Ellenberg J. Song J. DiEuliis D. Lippincott-Schwartz J. J. Cell Biol. 1998; 140: 1-15Crossref PubMed Scopus (169) Google Scholar) for the region covering the transmembrane and cytoplasmic regions of VSVG in pCIpreEGFP-VSVG.Cell Culture, RNAi Suppression, VSVG Transport Experiments, and Immunofluorescence Analysis—Culture of HeLa cells and transfection of expression plasmids were performed as described previously (35Shin H.-W. Kobayashi H. Kitamura M. Waguri S. Suganuma T. Uchiyama Y. Nakayama K. J. Cell Sci. 2005; 118: 4039-4048Crossref PubMed Scopus (56) Google Scholar, 38Shin H.-W. Morinaga N. Noda M. Nakayama K. Mol. Biol. Cell. 2004; 15: 5283-5294Crossref PubMed Scopus (104) Google Scholar). Knockdown of ArfGAP1, ArfGAP2, ArfGAP3, or β-COP was performed as previously described (39Ishizaki R. Shin H.-W. Iguchi-Ariga S.M.M. Ariga H. Nakayama K. Genes Cells. 2006; 11: 949-959Crossref PubMed Scopus (21) Google Scholar). Briefly, pools of siRNAs directed for the mRNA regions covering nucleotide residues 328-947, 400-1542, 397-1530, and 321-1040 (when the A residue of the initiation Met codon is assigned as residue 1) were prepared using a BLOCK-iT RNAi TOPO Transcription kit and a BLOCK-iT Dicer RNAi kit (Invitrogen). Cells were transfected with the siRNAs using Lipofectamine 2000 (Invitrogen) and incubated overnight. The transfected cells were then transferred to a culture dish containing coverslips, further incubated for up to 120 and 48 h in the case of ArfGAP knockdown and β-COP knockdown, respectively, and processed for immunofluorescence and immunoblot analyses and transport assays.Transport of EGFP-VSVG from the ER to the cell surface was examined as described previously (35Shin H.-W. Kobayashi H. Kitamura M. Waguri S. Suganuma T. Uchiyama Y. Nakayama K. J. Cell Sci. 2005; 118: 4039-4048Crossref PubMed Scopus (56) Google Scholar). HeLa cells transfected with pCIpreEGFP-VSVG were incubated at 40 °C overnight, then at 32 °C for up to 60 min. Retrograde transport of the EGFP-VSVG-KDELR chimera was assayed as follows; HeLa cells transfected with pCIpreEGFP-VSVG-KDELR were incubated at 40 °C overnight and at 20 °C for 3 h to accumulate the KDELR chimera in the Golgi. The cells were then incubated at 40 °C again for 2 h and processed for immunofluorescence analysis. Internalization of extracellularly applied transferrin was examined as described previously (38Shin H.-W. Morinaga N. Noda M. Nakayama K. Mol. Biol. Cell. 2004; 15: 5283-5294Crossref PubMed Scopus (104) Google Scholar).Determination of Intracellular Level of GTP-bound ARF—To determine the level of GTP-bound ARF, lysates of control cells or of siRNA-treated cells were subjected to pulldown assays using the GAT domain of GGA1, as described previously (40Shin H.-W. Shinotsuka C. Nakayama K. Methods Enzymol. 2005; 404: 206-215Crossref PubMed Scopus (18) Google Scholar, 41Takatsu H. Yoshino K. Toda K. Nakayama K. Biochem. J. 2002; 365: 369-378Crossref PubMed Scopus (94) Google Scholar). Briefly, lysates were pulled down with the glutathione S-transferase-GGA1(GAT) domain, pre-bound to glutathione-Sepharose 4B beads (GE Healthcare Biosciences), and bound materials were electrophoresed on a 12.5% SDS-polyacrylamide gel and subjected to immunoblot analysis with anti-ARF1 antibody (3F1).Electron Microscopy—For conventional electron microscopy, control and siRNA-treated cells were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 m phosphate buffer. They were post-fixed with 1% OsO4, embedded in Epon812, and sectioned as previously described (42Waguri S. Kohmura M. Gotow T. Watanabe T. Ohsawa Y. Kominami E. Uchiyama Y. Arch. Histol. Cytol. 1999; 62: 423-434Crossref PubMed Scopus (13) Google Scholar). Small vesicles/tubules near vacuoles were counted in 1.6 × 1.6-μm regions that were randomly selected within perinuclear region (40 areas from 20 cells). The count was normalized to the total area, excluding the vacuolar area, measured using the Meta-Morph image processing software (Universal Imaging Corporation; West Chester, PA), and expressed as the number of vesicles/tubules per 1 μm2. Immunoelectron microscopy on ultrathin cryosections was performed as described previously (35Shin H.-W. Kobayashi H. Kitamura M. Waguri S. Suganuma T. Uchiyama Y. Nakayama K. J. Cell Sci. 2005; 118: 4039-4048Crossref PubMed Scopus (56) Google Scholar, 42Waguri S. Kohmura M. Gotow T. Watanabe T. Ohsawa Y. Kominami E. Uchiyama Y. Arch. Histol. Cytol. 1999; 62: 423-434Crossref PubMed Scopus (13) Google Scholar).RESULTSKnockdown of ArfGAP1 Alone Has No Discernible Effect—In the prevailing model (5Nie Z. Randazzo P.A. J. Cell Sci. 2006; 119: 1203-1211Crossref PubMed Scopus (103) Google Scholar) ArfGAP1 is involved in cargo selection and formation of COPI-coated vesicles, as well as in their uncoating through inactivating ARFs. To explore the role of ArfGAP1 in COPI-mediated transport, we looked for phenotypic changes in cells depleted of ArfGAP1 by RNAi. By transfecting HeLa cells with a pool of siRNAs targeted against ArfGAP1, the ArfGAP1 protein was depleted by more than 95% (supplemental Fig. S1A, top panel), and a typical Golgi-like staining for ArfGAP1 was eliminated (supplemental Fig. S1B). We then compared subcellular localization of various proteins between cells depleted of ArfGAP1 (supplemental Fig. S2B) and control cells transfected with siRNAs for LacZ (supplemental Fig. S2A). However, we failed to detect a significant difference between the knockdown and control cells in the localization of proteins. The proteins examined included: β-COP (top panels), a COPI subunit; ERGIC-53 (middle panels), which is a major type I membrane protein containing a C-terminal di-lysine motif and which cycles between the cis-Golgi and ER (43Appenzeller-Herzog C. Hauri H.-P. J. Cell Sci. 2006; 119: 2173-2183Crossref PubMed Scopus (308) Google Scholar); GM130 (bottom panels), a cis-Golgi matrix protein (44Nakamura N. Rabouille C. Watson R. Nilsson T. Hui N. Slusarewicz P. Kreis T.E. Warren G. J. Cell Biol. 1995; 131: 1715-1726Crossref PubMed Scopus (666) Google Scholar). We also examined the effects of ArfGAP1 knockdown on retrograde and anterograde transport of model cargo proteins. However, ArfGAP1 knockdown affected neither retrieval of a VSVG-KDELR construct from the cis-Golgi to the ER nor anterograde transport of a GFP-VSVG construct from the ER to the plasma membrane through the Golgi (data not shown; see below).Simultaneous Depletion of ArfGAP1, ArfGAP2, and ArfGAP3 Disorganizes the Golgi Structure—We then examined a possibility that there might be another ARFGAP(s) that also participates in the COPI-mediated process. In mammals there are less well characterized paralogs (ArfGAP2 and ArfGAP3) of ArfGAP1. At the sequence level, ArfGAP1 is similar to yeast Gcs1, whereas ArfGAP2 and ArfGAP3 are more similar to Glo3 (26Yahara N. Sato K. Nakano A. J. Cell Sci. 2006; 119: 2604-2612Crossref PubMed Scopus (20) Google Scholar, 27Frigerio G. Grimsey N. Dale M. Majoul I. Duden R. Traffic. 2007; 8: 1644-1655Crossref PubMed Scopus (43) Google Scholar, 28Liu X. Zhang C. Xing G. Chen Q. He F. FEBS Lett. 2001; 490: 79-83Crossref PubMed Scopus (30) Google Scholar, 29Zhang C. Yu Y. Zhang S. Liu M. Xing G. Wei H. Bi J. Liu X. Zhou G. Dong C. Hu Z. Zhang Y. Luo L. Wu C. Zhao S. He F. Genomics. 2000; 63: 400-408Crossref PubMed Scopus (18) Google Scholar).We raised and affinity-purified polyclonal antibodies to ArfGAP2 and ArfGAP3 and used them to compare protein localization with that of various marker proteins. As shown in supplemental Fig. S1A, the antibodies each recognize a single band in immunoblot analysis; band intensities were specifically decreased by the appropriate siRNA treatment. When HeLa cells were doubly stained for any one of the ArfGAPs and GM130, the staining for ArfGAPs overlapped almost completely with the GM130 staining in the perinuclear Golgi region (supplemental Fig. S3, A-C). In contrast, the ArfGAP staining was juxtaposed to, but not significantly overlapping with staining for golgin-245 (supplemental Fig. S3, D-F), a protein associated with the trans-Golgi (45Gleeson P.A. Anderson T.J. Stow J.L. Griffiths G. Toh B.-H. Matheson F. J. Cell Sci. 1996; 109: 2811-2821Crossref PubMed Google Scholar). To unequivocally show cis-Golgi localization of these ArfGAPs, HeLa cells were treated with nocodazole to fragment the Golgi structure; the fragmented Golgi structures are composed of mini-stacks and are suitable for examining cis and trans polarity (46Cole N.B. Sciaky N. Marotta A. Song J. Lippincott-Schwartz J. Mol. Biol. Cell. 1996; 7: 631-650Crossref PubMed Scopus (405) Google Scholar). In the fragmented Golgi structures, the staining for any ArfGAP overlapped almost completely with that for GM130 (supplemental Fig. S3, G-I) but juxtaposed to that for golgin-245 (supplemental Fig. S3, J-L), indicating that these three ArfGAPs are associated predominantly with the cis-Golgi.We then examined whether depletion of ArfGAP2 or ArfGAP3 affected the localization of Golgi proteins and transport between the ER and Golgi. Despite the successful depletion of ArfGAP2 or ArfGAP3 by RNAi (supplemental Fig. S1), however, we failed to detect any difference in either the subcellular localization of Golgi proteins examined, anterograde transport of GFP-VSVG from the ER to the Golgi, or retrograde transport of VSVG-KDELR from the cis-Golgi to the ER (data not shown) between control cells and cells knocked down of ArfGAP2 or ArfGAP3.We next examined effects of depletion of any pairwise combination of ArfGAPs but failed to detect any significant difference in the subcellular distribution of the Golgi proteins, including ERGIC-53, β-COP, and GM130, between control cells and double-depleted cells (ArfGAP1 + 2, ArfGAP1 + 3, or ArfGAP2 + 3) (Fig. 1).However, in a subpopulation (∼30%) of cells in which ArfGAP1, ArfGAP2, and ArfGAP3 were simultaneously knocked down, we found significant changes in the distribution of the Golgi proteins. ERGIC-53 disappeared from reticular ER-like structures and instead became predominant in punctate ERGIC-like structures (Fig. 1R). In contrast, the reticular staining for protein disulfide isomerase, an ER marker, was unchanged in the triple knockdown cells (Fig. 1Q), indicative of the integrity of the ER. These observations suggest that retrieval of ERGIC-53 from the ERGIC/cis-Golgi to the ER is blocked in cells simultaneously depleted of ArfGAP1, ArfGAP2, and ArfGAP3. Furthermore, β-COP and GM130 were redistributed from perinuclear Golgi-like structures to punctate ERGIC-like structures in a subpopulation of the triple knockdown cells (Fig. 1, S and T).To support the hypothesis that these phenotypic changes are indeed induced by ArfGAP depletion, we then compared levels of GTP-bound active ARF in the control and knockdown cells. To this end we employed a pulldown assay using the GAT domain of GGA1, which specifically interacts with GTP-bound but not GDP-bound ARF (38Shin H.-W. Morinaga N. Noda M. Nakayama K. Mol. Biol. Cell. 2004; 15: 5283-5294Crossref PubMed Scopus (104) Google Scholar, 47Santy L.C. Casanova J.E. J. Cell Biol. 2001; 154: 599-610Crossref PubMed Scopus (311) Google Scholar) under the assumption that GTP hydrolysis on ARF would be slowed (i.e. that the level of GTP-bound ARF would be increased) if the ArfGAP levels were decreased. As shown in Fig. 2, the level of GTP-bound ARF was not significantly changed in cells depleted of single ArfGAP (lanes 7-9) as compared with the control cells (lane 6). In striking contrast, the level of GTP-bound ARF was robustly increased in the triple ArfGAP knockdown cells (lane 10). Thus, the phenotypic changes observed in the triple knockdown cells are correlated with an increase in the level of GTP-bound ARF, i.e. with a decrease in the ArfGAP levels.FIGURE 2Increase in the GTP-bound ARF level in cells simultaneously knocked down of ArfGAP1, ArfGAP2, and ArfGAP3. Lysates were prepared from HeLa cells treated for 120 h with siRNAs for LacZ (lanes 1, 6, and 11) or for ArfGAP1 (lanes 2, 7, and 12), ArfGAP2 (lanes 3, 8, and 13), ArfGAP3 (lanes 4, 9, and 14), or ArfGAP1+ArfGAP2+ArfGAP3 (lanes 5, 10, and 15) and subjected to pulldown with glutathione S-transferase (GST)-GGA1(GAT) (lanes 6-10) or GST (lanes 11-15). The data are representatives of five independent experiments for the control and triple ArfGAP knockdown cells and two independent experiments for the single ArfGAP knockdown cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the course of these experiments, we noticed that the triple knockdown cells showed two distinct phenotypes in terms of subcellular distribution of ERGIC-53 and β-COP. As shown in Fig. 3A, a fraction of these cells showed a punctate ERGIC-like distribution of ERGIC-53 and β-COP (stage 1), whereas a minor fraction showed perinuclear aggregates of ERGIC-53 and β-COP (stage 2). Extending the period of siRNA treatment (96 to 120 h) resulted in a significant increase in the fraction of both the stage 1 (21-34%) and stage 2 (4-11%) cells (Fig. 3B), suggesting that the stage 2 phenotype is the more severe of the two. However, further siRNA treatment resulted in a decrease in the stage 1 and stage 2 populations (data not shown), probably because of the death of cells completely depleted of all the three ArfGAPs, as reported previously (27Frigerio G. Grimsey N. Dale M. Majoul I. Duden R. Traffic. 2007; 8: 1644-1655Crossref PubMed Scopus (43) Google Scholar), and because of selective growth of cells incompletely depleted of any of the three ArfGAPs. For the following experiments we, therefore, used cells treated for 120 h with siRNAs for ArfGAP1, ArfGAP2, and ArfGAP3.FIGURE 3Two distinct phenotypic stages in cells triple-depleted of ArfGAP1, ArfGAP2, and ArfGAP3. A, HeLa cells treated for 120 h with siRNAs for ArfGAP1+ArfGAP2+ArfGAP3 were double-stained for ERGIC-53 (a-c) and β-COP (a′-c′). Cells with normal distribution of ERGIC-53 and β-COP (a and a′) or with typical stage 1 (b and b′) or stage 2 (c and c′) distributions are shown. B, HeLa cells treated with siRNAs for ArfGAP1+ArfGAP2+ArfGAP3 for 96 or 120 h were classified as having normal, stage 1, and stage 2 distributions of ERGIC-53, and the number of cells with each distribution was counted. The number (n) is the sum of counted cells in three independent experiments. C, HeLa cells treated with siRNAs for ArfGAP1+ArfGAP2+ArfGAP3 for 96 h were transfected with an expression vector for C-terminal-HA-tagged ArfGAP2 (a-a″) or ArfGAP3 (b-b″) and incubated for 24 h, then triply stained for HA (a and b), ERGIC-53 (a′ and b′) and β-COP (a′ and b′). Cells overexpressing ArfGAP2-HA or ArfGAP3-HA are indicated by asterisks. D, the number of cells with normal, stage 1, and stage 2 distribution of ERGIC-53 in mock-transfected cells or cells overexpressing either ArfGAP2-HA or ArfGAP3-HA. The number (n) is the sum of counted cells in three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The stage 1 and 2 phenotype
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