Molecular Mechanism of Mitotic Golgi Disassembly and Reassembly Revealed by a Defined Reconstitution Assay
2007; Elsevier BV; Volume: 283; Issue: 10 Linguagem: Inglês
10.1074/jbc.m707715200
ISSN1083-351X
AutoresDanming Tang, Kari Mar, Graham Warren, Yanzhuang Wang,
Tópico(s)Retinal Development and Disorders
ResumoIn mammalian cells, flat Golgi cisternae closely arrange together to form stacks. During mitosis, the stacked structure undergoes a continuous fragmentation process. The generated mitotic Golgi fragments are distributed into the daughter cells, where they are reassembled into new Golgi stacks. In this study, an in vitro assay has been developed using purified proteins and Golgi membranes to reconstitute the Golgi disassembly and reassembly processes. This technique provides a useful tool to delineate the mechanisms underlying the morphological change. There are two processes during Golgi disassembly: unstacking and vesiculation. Unstacking is mediated by two mitotic kinases, cdc2 and plk, which phosphorylate the Golgi stacking protein GRASP65 and thus disrupt the oligomer of this protein. Vesiculation is mediated by the COPI budding machinery ARF1 and the coatomer complex. When treated with a combination of purified kinases, ARF1 and coatomer, the Golgi membranes were completely fragmented into vesicles. After mitosis, there are also two processes in Golgi reassembly: formation of single cisternae by membrane fusion, and restacking. Cisternal membrane fusion requires two AAA ATPases, p97 and NSF (N-ethylmaleimide-sensitive fusion protein), each of which functions together with specific adaptor proteins. Restacking of the newly formed Golgi cisternae requires dephosphorylation of Golgi stacking proteins by the protein phosphatase PP2A. This systematic study revealed the minimal machinery that controls the mitotic Golgi disassembly and reassembly processes. In mammalian cells, flat Golgi cisternae closely arrange together to form stacks. During mitosis, the stacked structure undergoes a continuous fragmentation process. The generated mitotic Golgi fragments are distributed into the daughter cells, where they are reassembled into new Golgi stacks. In this study, an in vitro assay has been developed using purified proteins and Golgi membranes to reconstitute the Golgi disassembly and reassembly processes. This technique provides a useful tool to delineate the mechanisms underlying the morphological change. There are two processes during Golgi disassembly: unstacking and vesiculation. Unstacking is mediated by two mitotic kinases, cdc2 and plk, which phosphorylate the Golgi stacking protein GRASP65 and thus disrupt the oligomer of this protein. Vesiculation is mediated by the COPI budding machinery ARF1 and the coatomer complex. When treated with a combination of purified kinases, ARF1 and coatomer, the Golgi membranes were completely fragmented into vesicles. After mitosis, there are also two processes in Golgi reassembly: formation of single cisternae by membrane fusion, and restacking. Cisternal membrane fusion requires two AAA ATPases, p97 and NSF (N-ethylmaleimide-sensitive fusion protein), each of which functions together with specific adaptor proteins. Restacking of the newly formed Golgi cisternae requires dephosphorylation of Golgi stacking proteins by the protein phosphatase PP2A. This systematic study revealed the minimal machinery that controls the mitotic Golgi disassembly and reassembly processes. The interphase Golgi apparatus, as seen by light or fluorescence microscopy, is a compact juxta-nuclear reticulum, located most often in the peri-centriolar region of the cell (1Louvard D. Reggio H. Warren G. J. Cell Biol. 1982; 92: 92-107Crossref PubMed Scopus (226) Google Scholar). Electron microscopy (EM) 2The abbreviations used are:EMelectron microscopyARF1ADP-ribosylation factor-1ICinterphase cytosolMCmitotic cytosolplkPolo-like kinase-1NSFN-ethylmaleimide-sensitive fusion proteinPP2Aprotein serine/threonine phosphatase type 2APTPprotein-tyrosine phosphataseSNAPsoluble NSF attachment proteinTGNtrans-Golgi network.2The abbreviations used are:EMelectron microscopyARF1ADP-ribosylation factor-1ICinterphase cytosolMCmitotic cytosolplkPolo-like kinase-1NSFN-ethylmaleimide-sensitive fusion proteinPP2Aprotein serine/threonine phosphatase type 2APTPprotein-tyrosine phosphataseSNAPsoluble NSF attachment proteinTGNtrans-Golgi network. shows that it is comprised of discrete Golgi stacks linked together by tubules that connect equivalent cisternae in adjacent stacks (2Rambourg A. Clermont Y. Eur. J. Cell Biol. 1990; 51: 189-200PubMed Google Scholar). At the onset of mitosis, the characteristic stacked organization of the Golgi apparatus undergoes extensive fragmentation (3Robbins E. Gonatas N.K. J. Histochem. Cytochem. 1964; 12: 704-711Crossref PubMed Scopus (148) Google Scholar, 4Lucocq J.M. Pryde J.G. Berger E.G. Warren G. J. Cell Biol. 1987; 104: 865-874Crossref PubMed Scopus (128) Google Scholar). The mitotic Golgi fragments generated by this disassembly process are subsequently distributed to daughter cells, where they are reassembled into new Golgi stacks after mitosis. So far, the mechanism that controls the Golgi disassembly and reassembly processes is not well understood. electron microscopy ADP-ribosylation factor-1 interphase cytosol mitotic cytosol Polo-like kinase-1 N-ethylmaleimide-sensitive fusion protein protein serine/threonine phosphatase type 2A protein-tyrosine phosphatase soluble NSF attachment protein trans-Golgi network. electron microscopy ADP-ribosylation factor-1 interphase cytosol mitotic cytosol Polo-like kinase-1 N-ethylmaleimide-sensitive fusion protein protein serine/threonine phosphatase type 2A protein-tyrosine phosphatase soluble NSF attachment protein trans-Golgi network. Biochemical reconstitution experiments have provided powerful tools with which to dissect biological processes. Two basic experimental approaches have been taken to reconstitute mitotic Golgi disassembly and reassembly. One involves semipermeabilized cells, in which cells are permeabilized gently with detergent (e.g. digitonin), washed with 1 m KCl to remove endogenous cytosol and peripheral membrane proteins, and then incubated in cytosol prepared from mitotic (or interphase) cells (5Kano F. Takenaka K. Yamamoto A. Nagayama K. Nishida E. Murata M. J. Cell Biol. 2000; 149: 357-368Crossref PubMed Scopus (79) Google Scholar, 6Colanzi A. Deerinck T.J. Ellisman M.H. Malhotra V. J. Cell Biol. 2000; 149: 331-339Crossref PubMed Scopus (85) Google Scholar). Cells can then be processed directly for immunofluorescence or electron microscopy, or biochemical analysis of proteins. This approach has been used to test the mitotic kinases that regulate the Golgi disassembly process (5Kano F. Takenaka K. Yamamoto A. Nagayama K. Nishida E. Murata M. J. Cell Biol. 2000; 149: 357-368Crossref PubMed Scopus (79) Google Scholar, 6Colanzi A. Deerinck T.J. Ellisman M.H. Malhotra V. J. Cell Biol. 2000; 149: 331-339Crossref PubMed Scopus (85) Google Scholar). The second method involves purified Golgi membranes to which mitotic or interphase cytosol is added as above (7Misteli T. Warren G. J. Cell Biol. 1994; 125: 269-282Crossref PubMed Scopus (104) Google Scholar, 8Rabouille C. Kondo H. Newman R. Hui N. Freemont P. Warren G. Cell. 1998; 92: 603-610Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 9Rabouille C. Misteli T. Watson R. Warren G. J. Cell Biol. 1995; 129: 605-618Crossref PubMed Scopus (113) Google Scholar). After incubation, membranes are separated from cytosol by centrifugation through a sucrose cushion. Membranes can then be processed for biochemical analysis of proteins and morphological analysis of the membranes. This approach has contributed to the discovery and examination of much of the currently identified proteins that mediate Golgi membrane tethering (10Shorter J. Warren G. J. Cell Biol. 1999; 146: 57-70Crossref PubMed Scopus (141) Google Scholar, 11Satoh A. Wang Y. Malsam J. Beard M.B. Warren G. Traffic. 2003; 4: 153-161Crossref PubMed Scopus (103) Google Scholar), fusion (8Rabouille C. Kondo H. Newman R. Hui N. Freemont P. Warren G. Cell. 1998; 92: 603-610Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 12Kondo H. Rabouille C. Newman R. Levine T.P. Pappin D. Freemont P. Warren G. Nature. 1997; 388: 75-78Crossref PubMed Scopus (359) Google Scholar, 13Rabouille C. Levine T.P. Peters J.M. Warren G. Cell. 1995; 82: 905-914Abstract Full Text PDF PubMed Scopus (310) Google Scholar, 14Wang Y. Satoh A. Warren G. Meyer H.H. J. Cell Biol. 2004; 164: 973-978Crossref PubMed Scopus (128) Google Scholar), and Golgi cisternal stacking (15Barr F.A. Puype M. Vandekerckhove J. Warren G. Cell. 1997; 91: 253-262Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 16Shorter J. Watson R. Giannakou M.E. Clarke M. Warren G. Barr F.A. EMBO J. 1999; 18: 4949-4960Crossref PubMed Scopus (248) Google Scholar, 17Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. EMBO J. 2003; 22: 3279-3290Crossref PubMed Scopus (148) Google Scholar). Although the discovery of these proteins that are involved in regulation of Golgi membrane dynamics has contributed much to our understanding of the biogenesis of the Golgi apparatus, it is unclear whether these proteins are sufficient to control mitotic Golgi disassembly and reassembly, as all these studies used cytosol, or cell extract, in the reconstitution assays. Because cytosol contains many kinds of proteins, it is difficult to identify the minimal machinery or the key components that control Golgi disassembly during mitosis and reassembly afterward. This study describes for the first time an in vitro assay that reconstitutes the entire mitotic Golgi disassembly and reassembly processes using biochemically purified components. Our results show that the disassembly process is mediated by two independent but interactive processes: cisternal membrane unstacking mediated by mitotic kinases, and membrane vesiculation mediated by the COPI vesicle budding machinery. Post-mitotic Golgi reassembly also consists of two processes: membrane fusion mediated by two AAA ATPases, p97 and NSF, and cisternal membrane restacking, mediated by dephosphorylation of the Golgi stacking proteins by protein phosphatase PP2A. Our method provides a powerful tool to further dissect the molecular mechanism that regulates Golgi membrane dynamics during the cell cycle. Reagents—All reagents were from Sigma, Roche Applied Sciences, or Calbiochem, unless otherwise stated. The following antibodies were used: monoclonal antibodies against ARF1 (1D9, Abcam), Bet1 (BD Transduction Laboratories), β-COP (M3A5, T. Kreis), GM130 and Gos28 (BD Transduction Laboratories), GRASP65 (F. Barr), and α-tubulin (K. Gull); polyclonal antibodies against ARF1 (D. Shields and D. Sheff), β-COP (EAGE, T. Kreis), GM130 (MLO7, M. Lowe), GRASP55 (J. Seemann), GRASP65 (17Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. EMBO J. 2003; 22: 3279-3290Crossref PubMed Scopus (148) Google Scholar), α-Mannosidase I (J. Seemann) and II (K. Moremen), Golgin-84 (A. Satoh), rat serum albumin (G. Warren), syntaxin 5 (A. Price), and TGN38 (18Taguchi T. Pypaert M. Warren G. Traffic. 2003; 4: 344-352Crossref PubMed Scopus (17) Google Scholar). The polyclonal antibody for phosphorylated GM130 was generously provided by M. Lowe. Secondary antibodies for immunofluorescence and for Western blotting were from Molecular Probes and Jackson Immunoresearch Laboratories, respectively. Protein Expression and Purification—Golgi membranes were purified from rat liver (19Wang Y. Taguchi T. Warren G. Celis J. 3rd Ed. Cell Biology: A Laboratory Handbook. Elsevier Science, San Diego2006Google Scholar). Interphase (IC) and mitotic (MC) cytosol were prepared from HeLa S3 cells (7Misteli T. Warren G. J. Cell Biol. 1994; 125: 269-282Crossref PubMed Scopus (104) Google Scholar, 17Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. EMBO J. 2003; 22: 3279-3290Crossref PubMed Scopus (148) Google Scholar). The histone kinase activity of mitotic cytosol was 20–25-fold higher than that of interphase cytosol (20Lowe M. Rabouille C. Nakamura N. Watson R. Jackman M. Jamsa E. Rahman D. Pappin D.J. Warren G. Cell. 1998; 94: 783-793Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Cdc2 kinase (complexed with cyclin B1) and plk kinases were expressed and purified as described, and kinase activity was measured (17Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. EMBO J. 2003; 22: 3279-3290Crossref PubMed Scopus (148) Google Scholar). Coatomer complex was purified from rabbit cytosol (21Pavel J. Harter C. Wieland F.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2140-2145Crossref PubMed Scopus (69) Google Scholar). Myristoylated ARF1 was co-expressed in bacteria with a second plasmid encoding the yeast N-myristoyltransferase in BL21 bacterial cells and purified as previously described (22Randazzo P.A. Weiss O. Kahn R.A. Methods Enzymol. 1995; 257: 128-135Crossref PubMed Scopus (45) Google Scholar, 23Xiang Y. Seemann J. Bisel B. Punthambaker S. Wang Y. J. Biol. Chem. 2007; 282: 21829-21837Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Recombinant His-tagged NSF was purified as previously described (24Whiteheart S.W. Rossnagel K. Buhrow S.A. Brunner M. Jaenicke R. Rothman J.E. J. Cell Biol. 1994; 126: 945-954Crossref PubMed Scopus (338) Google Scholar). Recombinant His-tagged α-SNAP (soluble NSF attachment protein) and γ-SNAP were expressed in bacteria and purified with nickel beads (13Rabouille C. Levine T.P. Peters J.M. Warren G. Cell. 1995; 82: 905-914Abstract Full Text PDF PubMed Scopus (310) Google Scholar). Recombinant p97 and p47 were expressed in bacteria and purified by chromatography using nickel beads (25Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (289) Google Scholar). p97 was also purified by chromatography from rat liver cytosol (12Kondo H. Rabouille C. Newman R. Levine T.P. Pappin D. Freemont P. Warren G. Nature. 1997; 388: 75-78Crossref PubMed Scopus (359) Google Scholar, 25Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (289) Google Scholar). p115 was purified from rat liver cytosol by chromatography as previously described (10Shorter J. Warren G. J. Cell Biol. 1999; 146: 57-70Crossref PubMed Scopus (141) Google Scholar, 26Levine T.P. Rabouille C. Kieckbusch R.H. Warren G. J. Biol. Chem. 1996; 271: 17304-17311Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). GRASP65 Dephosphorylation—Purified protein serine/threonine phosphatases PP1, PP2A, PP2B, PP2C, and protein tyrosine phosphatase (PTP) were purchased from Upstate Biotech. Purified PP1 originated from skeletal muscle and contained multiple PP1 isoforms. PP2A was purified from human red blood cells as a heterodimer of A and C subunits; the trimeric form of PP2A, called PP2A1, was purified from rabbit skeletal muscles and contained ACBα subunits. PP2B/calcineurin was purified from bovine brain. Recombinant PP2Cα and protein tyrosine phosphatase 1B (PTP-1B) were expressed in bacteria and purified by chromatography. GRASP65 phosphorylation was achieved by treating Golgi membranes with mitotic cytosol, as in the Golgi disassembly assay. Briefly, 5 μg of purified rat liver Golgi membranes were mixed with 500 μg of mitotic cytosol in MEB buffer (50 mm Tris-HCl, pH 7.4, 0.2 m sucrose, 50 mm KCl, 20 mm β-glycerophosphate, 15 mm EGTA, 10 mm MgCl2, 2 mm ATP, 1 mm GTP, 1 mm glutathione, and protease inhibitors) and an ATP regeneration system (10 mm creatine phosphate, 1 mm ATP, 20 μg/ml creatine kinase, 20 ng/ml cytochalasin B) in a 50-μl reaction. After 20 min of incubation at 37 °C, the membranes were pelleted through a 0.4 m sucrose layer at 55,000 rpm for 30 min in a TLA55 rotor (Beckman). To dephosphorylate GRASP65, the membrane pellets were directly resuspended in KHM buffer (20 mm Hepes-KOH, pH7, 0.2 m sucrose, 60 mm KCl, 5 mm Mg(OAc)2, 2 mm ATP, 1 mm GTP, 1 mm glutathione, protease inhibitors) containing 100 μg interphase cytosol, or 0.5 units of purified phosphatases (except for PP2A1, for which 0.125 milliunits was used), and then incubated at 30 °C for 60 min. The membranes were solubilized in SDS buffer and analyzed by Western blotting for GRASP65. GRASP65 phosphorylation and dephosphorylation were analyzed by the migration shift of GRASP65 on SDS-PAGE (17Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. EMBO J. 2003; 22: 3279-3290Crossref PubMed Scopus (148) Google Scholar). To express isoform-specific PP2A, sf9 cells were co-infected by baculoviruses (generously provided by Gary Thomas, the Vollum Institute, Oregon) encoding the A, Bα, B′α, and C subunits. Cell lysates of the infected cells were prepared as described (27Molloy S.S. Thomas L. Kamibayashi C. Mumby M.C. Thomas G. J. Cell Biol. 1998; 142: 1399-1411Crossref PubMed Scopus (82) Google Scholar). Each lysate exhibited similar activity toward phosphorylase a (28Lowe M. Gonatas N.K. Warren G. J. Cell Biol. 2000; 149: 341-356Crossref PubMed Scopus (125) Google Scholar), and Western blotting confirmed that the appropriate subunits were expressed at similar levels by the recombinant baculoviruses in each combinatorial infection. Relative equal amounts of the lysate (20 μl each) with AC, ACBα, ACB′α subunits expressed were used to treat 5 μg of mitotic Golgi fragments prepared as described above. The same amount of lysate from non-infected cells was used as a control. Okadaic acid was purchased from Calbiochem, inhibitor-2 from Upstate Biotech, and microcystin LR from Sigma. Golgi Disassembly and Reassembly Assay—All recombinant proteins used in this study were wild type. Although the nonhydrolyzable GTP analogue GTPγS could enhance the Golgi disassembly, the resulting Golgi fragments could not be reassembled; thus GTPγS was not used in this study. Mitotic Golgi disassembly assay was performed as previously described (9Rabouille C. Misteli T. Watson R. Warren G. J. Cell Biol. 1995; 129: 605-618Crossref PubMed Scopus (113) Google Scholar, 17Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. EMBO J. 2003; 22: 3279-3290Crossref PubMed Scopus (148) Google Scholar, 23Xiang Y. Seemann J. Bisel B. Punthambaker S. Wang Y. J. Biol. Chem. 2007; 282: 21829-21837Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 29Malsam J. Satoh A. Pelletier L. Warren G. Science. 2005; 307: 1095-1098Crossref PubMed Scopus (153) Google Scholar). Briefly, Golgi membranes (200 μg) were mixed with 10 mg of mitotic cytosol, or with purified coatomer (100 μg), recombinant myristoylated ARF1 (50 μg), 1 mm GTP, and an ATP-regenerating system in MEB buffer, in a final volume of 1000 μl. After incubation for 20 min at 37 °C, mitotic Golgi fragments (MGFs) were isolated and soluble factors were removed by centrifugation (55,000 rpm for 30 min in a TLA55 rotor) through a 0.4 m sucrose cushion in KHM buffer onto a 6-μl2 m sucrose cushion. The membranes were resuspended in KHM buffer, and aliquots were succeeded to fixation and EM processing, or to reassembly reactions described below. For Golgi reassembly, 20 μg of MGFs were treated with the following combinations of proteins in 30-μl reactions: 1) 400 μl of interphase cytosol; 2) 3 μg of NSF (100 ng/μl), 0.75 μg of α-SNAP (25 ng/μl), 0.75 μg of γ-SNAP (25 ng/μl) (8Rabouille C. Kondo H. Newman R. Hui N. Freemont P. Warren G. Cell. 1998; 92: 603-610Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), 0.9 μg of p115 (30 ng/μl) (10Shorter J. Warren G. J. Cell Biol. 1999; 146: 57-70Crossref PubMed Scopus (141) Google Scholar), and 2 units of PP2A (0.07 units/μl); 3) 3 μg of p97 (100 ng/μl), 0.75 μg of p47 (14Wang Y. Satoh A. Warren G. Meyer H.H. J. Cell Biol. 2004; 164: 973-978Crossref PubMed Scopus (128) Google Scholar), 0.9 μg of p115 (30 ng/μl), and 2 units of PP2A (0.07 units/μl). Reactions were incubated at 37 °C, and membranes were pelleted and processed for EM. The percentage of membranes in cisternae or in vesicles was determined by the intersection method (10Shorter J. Warren G. J. Cell Biol. 1999; 146: 57-70Crossref PubMed Scopus (141) Google Scholar, 17Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. EMBO J. 2003; 22: 3279-3290Crossref PubMed Scopus (148) Google Scholar). Cisternae were defined as long membrane profiles with a length more than four times their width, with the width no more than 80 nm. Vesicles were defined as round profiles of ∼70 nm in diameter. The average size of the vesicles was determined by measuring the diameter of 50 vesicles. Results were quantitated from three independent experiments and the statistical significance was assessed by Student's t test. Analysis of the Contents in Golgi Remnants and Vesicles—To determine which proteins were sorted into the vesicles, Golgi disassembly reactions described above using 200 μg of Golgi treated with ARF1 and coatomer in the presence or absence of mitotic kinases were supplemented with 250 mm KCl in cold assay buffer to stop the reaction and release the vesicles. The reactions were directly loaded onto a step gradient comprised of 1.0 ml 0.5 m, 1.5 ml 0.8 m, 2 ml 1.2 m, 2 ml 1.4 m, and 2 ml 1.6 m sucrose in assay buffer containing 250 mm KCl (23Xiang Y. Seemann J. Bisel B. Punthambaker S. Wang Y. J. Biol. Chem. 2007; 282: 21829-21837Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Membranes were centrifuged to equilibrium at 200,000 × g, 4°C for 3 h in a near vertical rotor (NVT65, Beckman Instruments, 50,000 rpm). COPI-coated vesicles typically peaked at 1.2–1.3 m sucrose, while uncoated Golgi remnants peaked at about 0.8 m sucrose (23Xiang Y. Seemann J. Bisel B. Punthambaker S. Wang Y. J. Biol. Chem. 2007; 282: 21829-21837Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Fractions were diluted 3-fold with a buffer without sucrose and membranes in each fraction were pelleted by centrifugation in a Beckman TLA55 rotor at 55,000 rpm for 60 min followed by Western blotting analysis. The soluble proteins, most of which were found in the top two fractions, were not pelleted after this centrifugation. This allowed us to determine the distribution of only the proteins that were associated with Golgi remnants and vesicles. Six categories of proteins were analyzed: 1) COPI coat proteins ARF1 and β-COP; 2) Golgi matrix proteins GM130, GRASP65, GRASP55, and Golgin-84; 3) Golgi enzymes α-mannosidase I and II; 4) Golgi SNAREs Syntaxin 5, Gos28 and Bet1; 5) the trans-Golgi protein TGN38; and 6) a secretory cargo protein rat serum albumin (RSA). For morphological analysis, fractions containing the Golgi remnants (no. 1–3) and vesicles (no. 7–9) were pooled, diluted 3-fold using a buffer without sucrose, and pelleted by centrifugation. Membrane samples were fixed and processed for EM. Defined Golgi Disassembly—During mitosis, the Golgi apparatus undergoes continuous fragmentation, which requires ARF1 and COPI budding (23Xiang Y. Seemann J. Bisel B. Punthambaker S. Wang Y. J. Biol. Chem. 2007; 282: 21829-21837Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). To test whether COPI vesicle budding activity is sufficient to fragment the entire Golgi stack, we treated purified Golgi membranes with purified ARF1 and coatomer (Fig. 1B). This treatment led to fragmentation of a large fraction (40%, Fig. 1F) of the Golgi membranes into vesicles. However, a significant amount of the membranes remained as short cisternae concentrated in ministacks (Fig. 1B, arrows). These ministacks did not diminish even when increased amounts of ARF1 and coatomer were used (not shown), suggesting that vesicle budding alone is not sufficient to complete the fragmentation process of the entire Golgi stack. This result indicates that fragmentation of the Golgi during mitosis may require a COPI-independent fragmentation process, as previously proposed (7Misteli T. Warren G. J. Cell Biol. 1994; 125: 269-282Crossref PubMed Scopus (104) Google Scholar). However, since most of the observed cisternae were found in ministacks, it is possible that unstacking of the cisternae may help to improve the fragmentation process. Previous work showed that Golgi membrane stacking is mediated by GRASP65, whose oligomerization functions as a glue to hold the cisternal membrane together (17Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. EMBO J. 2003; 22: 3279-3290Crossref PubMed Scopus (148) Google Scholar, 30Wang Y. Satoh A. Warren G. J. Biol. Chem. 2005; 280: 4921-4928Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Oligomerization of GRASP65 is regulated by phosphorylation, which is mediated by two mitotic kinases, cdc2 (in complex with cyclin B1) and plk. GRASP65 phosphorylation disrupts its oligomerization and thus causes cisternal membrane unstacking (17Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. EMBO J. 2003; 22: 3279-3290Crossref PubMed Scopus (148) Google Scholar). In addition, cdc2 and plk also mimic mitotic cytosol to phosphorylate GM130, another Golgi matrix protein whose phosphorylation during mitosis is involved in mitotic Golgi disassembly (20Lowe M. Rabouille C. Nakamura N. Watson R. Jackman M. Jamsa E. Rahman D. Pappin D.J. Warren G. Cell. 1998; 94: 783-793Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In this study, when the Golgi membranes were treated with these mitotic kinases, the Golgi membrane became unstacked and thus generated a large amount of single cisternae. However, this treatment did not vesiculate the Golgi cisternal membranes (Fig. 1C). Quantitation results showed that about 15% of the membranes were in vesicles when the Golgi membranes were treated with both mitotic kinases; thus, there is no change compared with that of untreated membranes (Fig. 1F). To test whether unstacking and vesiculation have a synergistic effect in terms of Golgi fragmentation, both sets of components, ARF1/coatomer and kinases, were combined and used to treat Golgi membranes. As expected, the result showed that fragmentation was complete; essentially all membranes were fragmented into vesicles except the elements of the trans-Golgi network (TGN) indicated by the electron-dense contents of lipoproteins (Fig. 1D, asterisks). The size of the vesicles generated by purified components was 70 ± 9 nm (Fig. 1D); no significant change was seen compared with an average diameter of 72 ± 6 nm for those generated by mitotic cytosol (Fig. 1E). Quantitation of the results showed that about 79% of membranes were vesiculated by the treatment with ARF1/coatomer in the presence of mitotic kinases cdc2 and plk, a significant increase compared with treatment with ARF1 and coatomer alone (40%), or with only the two mitotic kinases (15%) (Fig. 1F), suggesting that vesiculation and unstacking are independent but synergistic. The Golgi disassembly after this treatment was also slightly higher than with mitotic cytosol, which had ∼50% of membranes in vesicles (Fig. 1, E and F). Detailed examination of EM images at higher magnification indicated that most of the vesicles, generated by treatment with either mitotic cytosol or with ARF1 and coatomer in the presence or absence of mitotic kinases, were coated (data not shown). These results suggest that the mitotic Golgi fragmentation consists of two independent but interactive processes: unstacking, mediated by mitotic kinases that phosphorylate Golgi stacking proteins, and vesiculation, mediated by ARF1 and the COPI coat protein complex coatomer. Golgi Enzymes and SNARE Proteins Are Enriched in Vesicles—To determine which proteins are enriched in the vesicles generated by treatment with ARF1, coatomer and mitotic kinases, we fractionated the fragmented Golgi membranes on a 0.5–1.6 m sucrose gradient, as described earlier (23Xiang Y. Seemann J. Bisel B. Punthambaker S. Wang Y. J. Biol. Chem. 2007; 282: 21829-21837Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). After centrifugation to equilibrium, membrane-bound proteins in each fraction were collected by high-speed centrifugation. Previous work showed that coated vesicles are enriched in a fraction equivalent to the density of 1.2 m sucrose (29Malsam J. Satoh A. Pelletier L. Warren G. Science. 2005; 307: 1095-1098Crossref PubMed Scopus (153) Google Scholar, 31Nickel W. Malsam J. Gorgas K. Ravazzola M. Jenne N. Helms J.B. Wieland F.T. J. Cell Sci. 1998; 111: 3081-3090Crossref PubMed Google Scholar), which is fraction 8 (23Xiang Y. Seemann J. Bisel B. Punthambaker S. Wang Y. J. Biol. Chem. 2007; 282: 21829-21837Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) in this gradient, while the Golgi remnants remained on the top 3 fractions of the gradient with the density of about 0.8 m sucrose, which is consistent with the density of the Golgi membranes (23Xiang Y. Seemann J. Bisel B. Punthambaker S. Wang Y. J. Biol. Chem. 2007; 282: 21829-21837Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 32Serafini T. Rothman J.E. Methods Enzymol. 1992; 219: 286-299Crossref PubMed Scopus (25) Google Scholar). To determine which proteins had been recruited into the vesicles, a number of proteins were tested by Western blotting for their distributions in the gradient. Both ARF1 and the β-COP subunit of the coatomer were found in fractions 7–9, indicating the location of the coated vesicles in the gradient, although a subpopulation of both proteins were found on the top of the gradient where the Golgi remnants were enriched (Fig. 2A). Perhaps these were membranes undergoing a budding process. There are indications that the Golgi matrix proteins, which include the Golgin and GRASP families of cisternal stacking proteins (33Pfeffer S.R. J. Cell Biol. 2001; 155: 873-875Crossref PubMed Scopus (42) Google Scholar), play a critical role in Golgi structure formation as well as in Golgi inheritance (34Shorter J. Warren G. Annu. Rev. Cell Dev. Biol. 2002; 18: 379-420Crossref PubMed Scopus (278) Google Scholar, 35Seemann J. Pypaert M. Taguchi T. Malsam J. Warren G. Science. 2002; 295: 848-851Crossref PubMed Scopus (109) Google Scholar). Two Golgi matrix
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