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Nuclear Import Is Required for the Pro-apoptotic Function of the Golgi Protein p115

2008; Elsevier BV; Volume: 284; Issue: 3 Linguagem: Inglês

10.1074/jbc.m807263200

1083-351X

Shaeri Mukherjee, Dennis Shields,

Ubiquitin and proteasome pathways

During apoptosis the Golgi apparatus undergoes irreversible fragmentation. In part, this results from caspase-mediated cleavage of several high molecular weight coiled-coil proteins, termed golgins. These include GM130, golgin 160, and the Golgi vesicle tethering protein p115, whose caspase cleavage generates a C-terminal fragment (CTF) of 205 residues. Here we demonstrate that early during apoptosis, following the rapid cleavage of p115, endogenous CTF translocated to the cell nucleus and its nuclear import was required to enhance the apoptotic response. Expression of a series of deletion constructs identified a putative α-helical region of 26 amino acids, whose expression alone was sufficient to induce apoptosis; deletion of these 26 residues from the CTF diminished its proapoptotic activity. This region contains several potential SUMOylation sites and co-expression of SUMO together with the SUMO ligase, UBC9, resulted in SUMOylation of the p115 CTF. Significantly, when cells were treated with drugs that induce apoptosis, SUMOylation enhanced the efficiency of p115 cleavage and the kinetics of apoptosis. A construct in which a nuclear export signal was fused to the N terminus of p115 CTF accumulated in the cytoplasm and surprisingly, its expression did not induce apoptosis. In contrast, treatment of cells expressing this chimera with the antibiotic leptomycin induced its translocation into the nucleus and resulted in the concomitant induction of apoptosis. These results demonstrate that nuclear import of the p115 CTF is required for it to stimulate the apoptotic response and suggest that its mode of action is confined to the nucleus. During apoptosis the Golgi apparatus undergoes irreversible fragmentation. In part, this results from caspase-mediated cleavage of several high molecular weight coiled-coil proteins, termed golgins. These include GM130, golgin 160, and the Golgi vesicle tethering protein p115, whose caspase cleavage generates a C-terminal fragment (CTF) of 205 residues. Here we demonstrate that early during apoptosis, following the rapid cleavage of p115, endogenous CTF translocated to the cell nucleus and its nuclear import was required to enhance the apoptotic response. Expression of a series of deletion constructs identified a putative α-helical region of 26 amino acids, whose expression alone was sufficient to induce apoptosis; deletion of these 26 residues from the CTF diminished its proapoptotic activity. This region contains several potential SUMOylation sites and co-expression of SUMO together with the SUMO ligase, UBC9, resulted in SUMOylation of the p115 CTF. Significantly, when cells were treated with drugs that induce apoptosis, SUMOylation enhanced the efficiency of p115 cleavage and the kinetics of apoptosis. A construct in which a nuclear export signal was fused to the N terminus of p115 CTF accumulated in the cytoplasm and surprisingly, its expression did not induce apoptosis. In contrast, treatment of cells expressing this chimera with the antibiotic leptomycin induced its translocation into the nucleus and resulted in the concomitant induction of apoptosis. These results demonstrate that nuclear import of the p115 CTF is required for it to stimulate the apoptotic response and suggest that its mode of action is confined to the nucleus. In mammalian cells the Golgi apparatus is a highly polarized organelle comprising a series of stacked cisternae, which form a lace-like network in the perinuclear region of the cell. It receives de novo synthesized secretory and membrane proteins, as well as lipids from the endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; PARP, poly(ADP-ribose) polymerase; HA, hemagglutinin; NES, nuclear export signal; siRNA, small interfering RNA; GFP, green fluorescent protein; CTF, C-terminal fragment; SUMO, small ubiquitin-like modifier.; these cargo molecules are then modified, sorted, and transported to lysosomes, endosomes, secretory granules, and the plasma membrane. Although it is well established that the Golgi apparatus undergoes reversible disassembly during mitosis (1Shorter J. Warren G. Annu. Rev. Cell Dev. Biol. 2002; 18: 379-420Crossref PubMed Scopus (284) Google Scholar, 2Warren G. Malhotra V. Curr. Opin. Cell Biol. 1998; 10: 493-498Crossref PubMed Scopus (87) Google Scholar), indeed this appears to be a prerequisite for mitosis (3Sutterlin C. Hsu P. Mallabiabarrena A. Malhotra V. Cell. 2002; 109: 359-369Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), studies from several laboratories including our own, have also established a link between the Golgi apparatus and apoptosis (programmed cell death). During apoptosis, the Golgi apparatus undergoes extensive and irreversible fragmentation (4Maag R.S. Hicks S.W. Machamer C.E. Curr. Opin. Cell Biol. 2003; 15: 456-461Crossref PubMed Scopus (64) Google Scholar), the ER vesiculates (5Sesso A. Fujiwara D.T. Jaeger M. Jaeger R. Li T.C. Monteiro M.M. Correa H. Ferreira M.A. Schumacher R.I. Belisario J. Kachar B. Chen E.J. Tissue Cell. 1999; 31: 357-371Crossref PubMed Scopus (55) Google Scholar) and secretion is inhibited (6Lowe M. Lane J.D. Woodman P.G. Allan V.J. J. Cell Sci. 2004; 117: 1139-1150Crossref PubMed Scopus (73) Google Scholar). Golgi disassembly during apoptosis results, in part, from caspase-mediated cleavage of several golgins (7Barr F.A. Short B. Curr. Opin. Cell Biol. 2003; 15: 405-413Crossref PubMed Scopus (213) Google Scholar). Proteolysis of golgin 160 by caspase-2, as well as GRASP65, GM130, p115, syntaxin5, and giantin by caspases-3 and -7 contributes significantly to Golgi fragmentation (6Lowe M. Lane J.D. Woodman P.G. Allan V.J. J. Cell Sci. 2004; 117: 1139-1150Crossref PubMed Scopus (73) Google Scholar, 8Lane J.D. Lucocq J. Pryde J. Barr F.A. Woodman P.G. Allan V.J. Lowe M. J. Cell Biol. 2002; 156: 495-509Crossref PubMed Scopus (183) Google Scholar, 9Mancini M. Machamer C.E. Roy S. Nicholson D.W. Thornberry N.A. Casciola-Rosen L.A. Rosen A. J. Cell Biol. 2000; 149: 603-612Crossref PubMed Scopus (326) Google Scholar, 10Chiu R. Novikov L. Mukherjee S. Shields D. J. Cell Biol. 2002; 159: 637-648Crossref PubMed Scopus (126) Google Scholar, 11Walker A. Ward C. Sheldrake T.A. Dransfield I. Rossi A.G. Pryde J.G. Haslett C. Biochem. Biophys. Res. Commun. 2004; 316: 6-11Crossref PubMed Scopus (41) Google Scholar, 12Maag R.S. Mancini M. Rosen A. Machamer C.E. Mol. Biol. Cell. 2005; 16: 3019-3027Crossref PubMed Scopus (53) Google Scholar, 13Mukherjee S. Chiu R. Leung S.M. Shields D. Traffic. 2007; 8: 369-378Crossref PubMed Scopus (71) Google Scholar). Consistent with this idea, overexpression of caspase-resistant forms of golgin 160, GRASP65, or p115 has been shown to delay the kinetics of Golgi fragmentation during apoptosis (8Lane J.D. Lucocq J. Pryde J. Barr F.A. Woodman P.G. Allan V.J. Lowe M. J. Cell Biol. 2002; 156: 495-509Crossref PubMed Scopus (183) Google Scholar, 9Mancini M. Machamer C.E. Roy S. Nicholson D.W. Thornberry N.A. Casciola-Rosen L.A. Rosen A. J. Cell Biol. 2000; 149: 603-612Crossref PubMed Scopus (326) Google Scholar, 10Chiu R. Novikov L. Mukherjee S. Shields D. J. Cell Biol. 2002; 159: 637-648Crossref PubMed Scopus (126) Google Scholar). In addition, immunoreactive caspase-2, an upstream caspase, localizes to the Golgi apparatus (9Mancini M. Machamer C.E. Roy S. Nicholson D.W. Thornberry N.A. Casciola-Rosen L.A. Rosen A. J. Cell Biol. 2000; 149: 603-612Crossref PubMed Scopus (326) Google Scholar) and caspase-2-mediated cleavage of golgin 160 also appears to be an early event during apoptosis. Depending on the apoptotic stimulus, expression of a golgin 160 triple mutant resistant to caspase cleavage delays the onset of apoptosis (12Maag R.S. Mancini M. Rosen A. Machamer C.E. Mol. Biol. Cell. 2005; 16: 3019-3027Crossref PubMed Scopus (53) Google Scholar). Recently, our laboratory demonstrated that Golgi fragmentation is an early apoptotic event that occurs close to or soon after release of cytochrome c from mitochondria, an early indicator of apoptosis (13Mukherjee S. Chiu R. Leung S.M. Shields D. Traffic. 2007; 8: 369-378Crossref PubMed Scopus (71) Google Scholar). Together these observations demonstrate that specific Golgi proteins may function early during apoptosis, although their role in this process and the detailed molecular mechanism by which Golgi fragmentation occurs is not well understood. A key molecule in mediating Golgi fragmentation during apoptosis is the vesicle tethering protein p115 (10Chiu R. Novikov L. Mukherjee S. Shields D. J. Cell Biol. 2002; 159: 637-648Crossref PubMed Scopus (126) Google Scholar), a 962-residue peripheral membrane protein. p115 is an elongated homodimer consisting of two globular "head" domains, an extended "tail" region reminiscent of the myosin-II structure (14Sapperstein S.K. Walter D.M. Grosvenor A.R. Heuser J.E. Waters M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 522-526Crossref PubMed Scopus (174) Google Scholar), and 4 sequential coil-coil domains distal to the globular head region, the first of which, CC1, has been implicated in soluble NSF attachment protein receptors (SNARE) binding (15Shorter J. Beard M.B. Seemann J. Dirac-Svejstrup A.B. Warren G. J. Cell Biol. 2002; 157: 45-62Crossref PubMed Scopus (169) Google Scholar). Earlier in vitro studies on mitotic Golgi reassembly demonstrated that p115 interacts with GM130 and giantin and implicated it in Golgi cisternal stacking (16Shorter J. Warren G. J. Cell Biol. 1999; 146: 57-70Crossref PubMed Scopus (143) Google Scholar). Consistent with this idea, microinjection of anti-p115 antibodies caused Golgi fragmentation (17Alvarez C. Fujita H. Hubbard A. Sztul E. J. Cell Biol. 1999; 147: 1205-1222Crossref PubMed Scopus (102) Google Scholar). Based on data demonstrating p115 binding to GM130, giantin, GOS28, and syntaxin-5, Shorter et al. (15Shorter J. Beard M.B. Seemann J. Dirac-Svejstrup A.B. Warren G. J. Cell Biol. 2002; 157: 45-62Crossref PubMed Scopus (169) Google Scholar) suggested that p115 promotes formation of a GOS28-syntaxin-5 (v-/t-SNARE) complex and hypothesized that it coordinates the sequential tethering and docking of COPI vesicles to Golgi membranes. Interestingly, p115 has also been shown to be a Rab-1 effector that binds Rab-1-GTP directly and cross-linking experiments showed that it interacts with Syntaxin5, sly1, membrin, and rbet1 on microsomal membranes and COPII vesicles suggesting that p115-SNARE interactions may facilitate membrane "docking" (18Allan B.B. Moyer B.D. Balch W.E. Science. 2000; 289: 444-448Crossref PubMed Scopus (387) Google Scholar). More recent in vivo studies showed that inhibition of GM130 or giantin binding to p115 had little effect on Golgi morphology or reassembly following mitosis, suggesting its role in maintaining Golgi structure might be independent of GM130 binding (19Puthenveedu M.A. Linstedt A.D. J. Cell Biol. 2001; 155: 227-238Crossref PubMed Scopus (97) Google Scholar, 20Puthenveedu M.A. Linstedt A.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1253-1256Crossref PubMed Scopus (85) Google Scholar). Thus post-mitotic Golgi reassembly could be rescued by p115 lacking the C-terminal GM130 binding motif (residues 935–962) but not by a mutant lacking the SNARE interacting CC1 domain (20Puthenveedu M.A. Linstedt A.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1253-1256Crossref PubMed Scopus (85) Google Scholar). In addition, other studies have implicated GM130 and GRASP65 in Golgi ribbon formation and suggested that this may occur independently of interactions with p115 (21Puthenveedu M.A. Bachert C. Puri S. Lanni F. Linstedt A.D. Nat. Cell Biol. 2006; 8: 238-248Crossref PubMed Scopus (262) Google Scholar). Most significantly, knockdown of p115 using siRNA demonstrated that it is essential for maintaining Golgi structure, compartmentalization, and cargo traffic to the plasma membrane (20Puthenveedu M.A. Linstedt A.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1253-1256Crossref PubMed Scopus (85) Google Scholar, 22Sohda M. Misumi Y. Yoshimura S. Nakamura N. Fusano T. Sakisaka S. Ogata S. Fujimoto J. Kiyokawa N. Ikehara Y. Biochem. Biophys. Res. Commun. 2005; 338: 1268-1274Crossref PubMed Scopus (39) Google Scholar). Earlier work from our laboratory demonstrated that p115 is cleaved in vitro by caspase-8, an initiator caspase, as well as by the executioner caspase-3 (10Chiu R. Novikov L. Mukherjee S. Shields D. J. Cell Biol. 2002; 159: 637-648Crossref PubMed Scopus (126) Google Scholar, 13Mukherjee S. Chiu R. Leung S.M. Shields D. Traffic. 2007; 8: 369-378Crossref PubMed Scopus (71) Google Scholar). In response to apoptosis inducing drugs, p115 is cleaved in vivo at Asp757 to generate a 205-residue C-terminal fragment and an N-terminal polypeptide of 757 amino acids. Most significantly, expression of the p115 C-terminal fragment in otherwise healthy cells results in its translocation to the nucleus and the induction of apoptosis suggesting that this polypeptide plays a role in potentiating the apoptotic response. To further dissect p115 function during cell death, we have now determined the minimal domain in its C terminus that mediates apoptosis efficiently and analyzed the requirement of nuclear translocation in triggering the apoptotic response. Antibodies—Mouse monoclonal antibodies to p115 (7D1) were a gift from Dr. Gerry Waters (Merck, Rahway, NJ). Dr. Adam Linstedt (Carnegie Melon University, Pittsburgh, PA) provided a polyclonal antibody specific for the C terminus of p115. Rabbit anti-PARP (poly(ADP-ribose) polymerase) serum, specific for the cleaved p85 form of PARP was purchased from Cell Signaling (Danvers, MA). The following mouse monoclonal antibodies were used: anti-GM130 (BD Transduction Laboratories, San Diego, CA); anti-FLAG M2-peroxidase and anti-cytochrome c (BD Pharmingen, San Diego, CA); anti-HA-peroxidase (Roche Applied Science); and anti-Fas clone CH-11 (MBL International Corporation). Anti-active caspase-3 was purchased from Promega (San Luis Obispo, CA). EZview™ Red Anti-FLAG® M2 Affinity Gel was purchased from Sigma. Alexa Fluor goat anti-mouse and anti-rabbit antibodies were purchased from Molecular Probes, Inc. (Eugene, OR). A human p115 fragment (amino acids 645–962) was fused to the C terminus of glutathione S-transferase in expression vector pGEX2T. The recombinant protein was expressed in and purified from bacteria. The glutathione S-transferase motif was cleaved using thrombin, and the p115 fragment was injected into rabbits to raise the anti-p115 antibody, AE800. This antibody recognized full-length human p115 as well as the N-terminal (1–636) and C-terminal (758–962) polypeptides by both Western blot and immunofluorescence microscopy; it did not recognize rat p115 by immunofluorescence microscopy. Plasmid Constructs—The pSG5-FLAG-human p115 construct was provided by Dr. Yukio Ikehara (Fukuoka University School of Medicine, Fukuoka, Japan) and was used as a template to generate p115 constructs. For RNA interference, p115 was targeted by siRNA oligos as described (20Puthenveedu M.A. Linstedt A.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1253-1256Crossref PubMed Scopus (85) Google Scholar). pEGFPC1 plasmid was purchased from Clontech (Mountain View, CA). p3XFLAG-CMV™-7.1 expression vector was purchased from Sigma. pGEX2T vector was purchased from Amersham Biosciences. A GFP-NES-CTF (GFP-nuclear export signal tagged CTF) plasmid was generated in the laboratory using the nuclear export signal LQLPPLERLTL from the Rev protein of HIV-1. Cell Culture and Induction of Apoptosis—HeLa and COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm glutamine, 50 units/ml penicillin G sodium, 50 μg/ml streptomycin sulfate at 37 °C in a humidified incubator containing 5% CO2. Apoptosis was induced by treating cells with Anti-Fas clone CH-11 antibody (13Mukherjee S. Chiu R. Leung S.M. Shields D. Traffic. 2007; 8: 369-378Crossref PubMed Scopus (71) Google Scholar), etoposide (100 μm), or anisomycin (5 μg/ml) for the indicated times. Preparation of Cell Extracts and Western Blotting—Cells were lysed in medium containing 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 0.25% aprotinin, and a mixture of protease inhibitors (Roche). Thirty μg of each sample was loaded on a 10% polyacrylamide gel, followed by transfer to Immobilon-P membranes (Millipore Corp). Membranes were blocked in 5% nonfat milk in phosphate-buffered saline containing 0.1% Tween 20 (PBST) for 1 h. Subsequently, they were probed with appropriate antibodies in PBST, washed extensively, and the immunoreactive bands were visualized by Enhanced Chemiluminescence (Amersham Biosciences). Immunofluorescence Microscopy—Cells were grown on coverslips and treated as indicated, after which they were fixed in 3% paraformaldehyde and processed for immunofluorescence microscopy as described previously (13Mukherjee S. Chiu R. Leung S.M. Shields D. Traffic. 2007; 8: 369-378Crossref PubMed Scopus (71) Google Scholar). Confocal images were acquired by capturing Z-series images with a 0.25-μm step size on a Leica TCS SP2 AOBS confocal microscope (Leica, Dearfield, IL) using a ×63 oil immersion objective (1.4 N.A.). Laser lines at 405, 488, 546, and 633 nm were provided by 20 milliwatt Diode, 100 milliwatt Ar, 1.5 milliwatt HeNe, and 10 milliwatt HeNe lasers; sequential excitation by line and detection range settings were used to eliminate cross-talk between fluorophores. The images (1024 × 1024 pixel, 8 bit) were saved as TIFF files. The entire Z-series was projected using the maximum intensity method. Background was removed and contrast adjusted using Image J and Adobe Photoshop. Threshold background level for each antibody was selected, saved, and loaded in all experiments. SUMOylation Experiments—HeLa cells were grown on 10-cm dishes and transfected with 2 μg of plasmids encoding (FLAG)3-CTF, (HA)3-SUMO, and UBC9. At 18 h post-transfection, cells were lysed in 100 μl of lysis buffer (above) containing 5 mm N-ethylmaleimide to prevent de-SUMOylation and centrifuged at 1000 × g for 2 min. The postnuclear supernatant was pre-cleared three times for 1 h each at 4 °C with 30 μl of Protein G-Sepharose beads in a total volume of 500 μl of RIPA buffer containing 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 50 mm Tris-HCl, pH 8.0, 0.1% SDS and protease inhibitor mixture. Precleared lysates were incubated with 20 μl of EZview Red Anti-FLAG M2 Affinity Gel overnight at 4 °C. Following incubations, the beads were washed extensively in wash buffer containing 10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, 0.05% Triton X-100, and analyzed by SDS-PAGE and Western blotting. Intracellular Localization of Endogenous p115 CTF—Previous data from our laboratory showed that in cells overexpressing FLAG-tagged p115 CTF, the polypeptide accumulated almost exclusively in nuclei and its expression was sufficient to induce Golgi fragmentation and apoptosis (10Chiu R. Novikov L. Mukherjee S. Shields D. J. Cell Biol. 2002; 159: 637-648Crossref PubMed Scopus (126) Google Scholar). However, it was unknown whether nuclear accumulation of CTF was a consequence of overexpression, if the endogenous polypeptide were present in cell nuclei early during apoptosis, and if its nuclear translocation was a requirement for apoptosis. To address these questions, HeLa cells were treated with Fas antibody that cross-links the Fas receptor, after which they were analyzed by immunofluorescence microscopy using a polyclonal antibody specific for the C terminus of p115 (residues 758–962) and a monoclonal antibody to GM130 (Fig. 1). As expected, p115 and GM130 exhibited significant Golgi co-localization in control cells (Fig. 1, panels A–D). Upon Fas activation by the CH-11 antibody, a high level of immunoreactive p115 CTF was present in nuclei prior to significant fragmentation of the Golgi apparatus (Fig. 1A). To confirm the nuclear localization of p115 during the induction of apoptosis, at different times after Fas antibody treatment HeLa cells were co-stained for mitochondrial cytochrome c release, a characteristic marker of early apoptosis (23Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4060) Google Scholar), as well as for immunoreactive-p115 (Fig. 1B). Control cells stained with anti-cytochrome c antibodies exhibited the typical reticular staining of mitochondria and no nuclear localization of p115 was observed (Fig. 1, B, panels A–D, and C). In contrast, following 2 or 4 h of Fas activation, cytochrome c staining became quite diffuse consistent with its release from mitochondria, and nuclear p115 staining was evident (Fig. 1B, panels E–L). Most significantly, quantitation of the immunofluorescence microscopy data (Fig. 1C) showed that at 2 h following Fas activation, the cells showed little evidence of mitochondrial cytochrome c release or Golgi fragmentation, whereas ∼50% of treated cells exhibited immunoreactive-p115 in their nuclei, suggesting that the nuclear localization of p115 preceded the onset of apoptosis. As expected, at 4 h following treatment with Fas activating antibody, ∼80 to 90% of cells had characteristics of apoptosis, namely highly diffuse cytochrome c staining, condensed nuclei, and a disrupted Golgi apparatus (Fig. 1B); our previous data showed that PARP is also cleaved at this time (13Mukherjee S. Chiu R. Leung S.M. Shields D. Traffic. 2007; 8: 369-378Crossref PubMed Scopus (71) Google Scholar). To demonstrate that the early nuclear localization of p115 was not confined only to Fas-activated HeLa cells, COS-7 cells were treated with two well characterized activators of apoptosis, etoposide or anisomycin (supplement Fig. S1). In response to etoposide treatment, p115 had a diffuse, punctate appearance, whereas GM130 maintained its normal juxtanuclear localization; notably, p115 staining was evident in nuclei although the cells were not significantly apoptotic at this time. In cells treated with anisomycin the Golgi apparatus had a loose appearance; furthermore, p115 staining was evident in nuclei. Together, these results demonstrated that the endogenous p115 CTF translocated to the cell nucleus and in response to Fas or etoposide treatment, this preceded Golgi fragmentation. Because immunoreactive-GM130 was not observed in nuclei at this time (supplement Fig. S1), we concluded that the appearance of CTF in nuclei was a specific transport event and not a consequence of nuclear envelope breakdown. Together with our previous observations (13Mukherjee S. Chiu R. Leung S.M. Shields D. Traffic. 2007; 8: 369-378Crossref PubMed Scopus (71) Google Scholar), these data are consistent with the hypothesis that p115 cleavage and CTF nuclear translocation are early apoptotic events. Having demonstrated that the endogenous p115 CTF accumulated in cell nuclei during activation of apoptosis, our goal was to determine whether nuclear translocation was required for CTF-induced apoptosis. Consequently, it was important to determine whether CTF expression induced cytochrome c release or if it functioned independently of the mitochondrial pathway. To address these questions, COS-7 cells were transfected with either GFP alone or GFP-CTF and analyzed by immunofluorescence microscopy using a monoclonal antibody to GM130 or cytochrome c (Fig. 2). Cells transfected with GFP alone showed normal Golgi and cytochrome c staining, whereas ∼60% of those expressing GFP-CTF had a disrupted Golgi apparatus (Fig. 2, panels D–F; see also Fig. 7). Significantly, in these cells cytochrome c staining was quite diffuse consistent with its release into the cytoplasm and the mitochondria aggregated significantly (Fig. 2, panels J–L), indicating that the cells were in an early phase of apoptosis. This result was consistent with observations that mitochondrial aggregation precedes release of cytochrome c and is an upstream apoptotic event (13Mukherjee S. Chiu R. Leung S.M. Shields D. Traffic. 2007; 8: 369-378Crossref PubMed Scopus (71) Google Scholar, 24Haga N. Fujita N. Tsuruo T. Oncogene. 2003; 22: 5579-5585Crossref PubMed Scopus (77) Google Scholar).FIGURE 7Quantitation of Golgi fragmentation and apoptosis in response to expression of various p115 CTF truncation mutants. COS-7 cells were transfected with the indicated GFP-tagged CTF constructs and stained with GM130 and Hoechst to identify the Golgi apparatus and nuclei, respectively. Golgi fragmentation and apoptosis were quantified by counting ∼300 cells expressing each construct; each set of cells was counted by two individuals. Data are the average of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Based on these observations we speculated that the CTF might act on mitochondria directly to release cytochrome c thereby activating the apoptosome. However, because the data of Fig. 1 and our previous studies (10Chiu R. Novikov L. Mukherjee S. Shields D. J. Cell Biol. 2002; 159: 637-648Crossref PubMed Scopus (126) Google Scholar) showed that CTF was present in the nuclei of apoptotic cells, it was likely that the effect on mitochondria occurred as a consequence of nuclear translocation. To distinguish between these possibilities, we expressed a chimera comprising GFP-CTF fused to the HIV-1 Rev protein nuclear export signal (GFP-NES-CTF; Fig. 3). This construct, which was excluded from nuclei, accumulated in the perinuclear region of the cell and had a diffuse reticular appearance characteristic of the ER. Despite its abundant expression this chimera had no effect on the morphology of either the Golgi apparatus or mitochondria and we observed few apoptotic cells (Fig. 3, panels A–D and E--H). If CTF nuclear translocation were required to induce apoptosis, abrogation of the NES by treating these cells with the antibiotic leptomycin would lead to nuclear accumulation of CTF, induction of apoptosis, and Golgi fragmentation. Consistent with this idea, in cells treated with leptomycin (Fig. 3, panels I–L and M--P) the CTF accumulated in the nucleus, and within 4 h the Golgi apparatus was fragmented, cytochrome c staining became quite diffuse and mitochondrial aggregation was evident. Quantitative analysis (Fig. 3B) showed that less than 20% of GFP-NES-CTF expressing cells were apoptotic in the absence of leptomycin, whereas following drug treatment ∼55 to 60% became apoptotic within 4 h. Importantly, these results suggested that CTF did not interact with mitochondria directly to induce cytochrome c release, but were in agreement with our observation that CTF nuclear translocation was required to induce apoptosis. p115 Knockdown Delays Apoptosis—Our previous data are consistent with a model whereby caspase cleavage of p115 generates the CTF fragment that functions in enhancing or potentiating the apoptotic response. Consequently, depletion of p115 might transiently delay the onset of drug-induced apoptosis. To test this idea, cells depleted of p115 by using siRNA were incubated in the absence or presence of agonistic Fas antibody to induce apoptosis and the level of PARP, a marker of apoptosis was determined (Fig. 4). Fas treatment of cells transfected with mock siRNA led to efficient cleavage of full-length (∼110 kDa) PARP (∼66% of FL PARP was cleaved after 4 h) to release the p85 fragment (Fig. 4, panels A and B). In contrast, in cells with diminished p115 levels, ∼19% of full-length PARP was cleaved after 4 h compared with untreated cells (compare lanes 2 and 4 in Fig. 4, A and B) suggesting that the presence of p115 CTF enhanced apoptosis. Surprisingly, the apparent level of the p85 fragment did not appear significantly different in mock and p115 siRNA cells. This was likely due to differential recognition of FL PARP and the p85 fragment by the PARP antibody and to the possibility that the fragment was further cleaved at later stages of apoptosis, which would be delayed by the absence of p115 CTF. Similar results were obtained when cells were treated with anisomycin and apoptosis was induced via the intrinsic pathway (data not shown). Identification of the p115 CTF Minimal Apoptotic Domain— The preceding data demonstrated that generation of the CTF and its nuclear import stimulated the apoptotic response. To identify the minimal domain within the CTF that could induce apoptosis, a series of N- and C-terminal truncation mutants were generated (Fig. 5, panel A). Representative micrographs of three such GFP-tagged constructs (residues 758–859, 884–962, and 859–962) are shown (Fig. 5B); FLAG-tagged CTF constructs gave similar results (data not shown). All three chimeras translocated to the nucleus; however, neither GFP-CTF-(758–859) nor GFP-CTF-(884–962) caused significant apoptosis. Most importantly, expression of the CTF-(859–962) truncation mutant induced apoptosis. These experiments suggested that amino acids 859–884 were required to generate the apoptotic phenotype. Indeed, inclusion of amino acids 859–884 in any of the GFP-CTF constructs resulted in both Golgi fragmentation and cell death, whereby cells became rounded, shrunken and in many cases the Golgi apparatus was not evident (supplement Fig. S2). Confirmation that residues 859–884 were key in effecting apoptosis came from expression of a GFP construct comprising 859–884 alone (Fig. 6A, panels A–D). In cells expressing this construct, GM130 staining was diffuse and the normal tight Golgi morphology of COS-7 cells was absent. Additionally, cells expressing a construct encoding amino acids 859–962 fused to GFP were positive for staining with an antibody that recognizes only active caspase-3 and not the precursor