Identification of a Redox-sensitive Cysteine in GCP60 That Regulates Its Interaction with Golgin-160
2007; Elsevier BV; Volume: 282; Issue: 41 Linguagem: Inglês
10.1074/jbc.m705794200
ISSN1083-351X
AutoresJuan I. Sbodio, Carolyn E. Machamer,
Tópico(s)ATP Synthase and ATPases Research
ResumoGolgin-160 is ubiquitously expressed in vertebrates. It localizes to the cytoplasmic side of the Golgi and has a large C-terminal coiled-coil domain. The noncoiled-coil N-terminal head domain contains Golgi targeting information, a cryptic nuclear localization signal, and three caspase cleavage sites. Caspase cleavage of the golgin-160 head domain generates different fragments that can translocate to the nucleus by exposing the nuclear localization signal. We have previously shown that GCP60, a Golgi resident protein, interacts weakly with the golgin-160 head domain but has a strong interaction with one of the caspase-generated golgin-160 fragments (residues 140–311). This preferential interaction increases the Golgi retention of the golgin-160 fragment in cells overexpressing GCP60. Here we studied the interaction of golgin-160-(140–311) with GCP60 and identified a single cysteine residue in GCP60 (Cys-463) that is critical for the interaction of the two proteins. Mutation of the cysteine blocked the interaction in vitro and disrupted the ability to retain the golgin-160 fragment at the Golgi in cells. We also found that Cys-463 is redox-sensitive; in its reduced form, interaction with golgin-160 was diminished or abolished, whereas oxidation of the Cys-463 by hydrogen peroxide restored the interaction. In addition, incubation with a nitric oxide donor promoted this interaction in vitro. These findings suggest that nuclear translocation of golgin-160-(140–311) is a highly coordinated event regulated not only by cleavage of the golgin-160 head but also by the oxidation state of GCP60. Golgin-160 is ubiquitously expressed in vertebrates. It localizes to the cytoplasmic side of the Golgi and has a large C-terminal coiled-coil domain. The noncoiled-coil N-terminal head domain contains Golgi targeting information, a cryptic nuclear localization signal, and three caspase cleavage sites. Caspase cleavage of the golgin-160 head domain generates different fragments that can translocate to the nucleus by exposing the nuclear localization signal. We have previously shown that GCP60, a Golgi resident protein, interacts weakly with the golgin-160 head domain but has a strong interaction with one of the caspase-generated golgin-160 fragments (residues 140–311). This preferential interaction increases the Golgi retention of the golgin-160 fragment in cells overexpressing GCP60. Here we studied the interaction of golgin-160-(140–311) with GCP60 and identified a single cysteine residue in GCP60 (Cys-463) that is critical for the interaction of the two proteins. Mutation of the cysteine blocked the interaction in vitro and disrupted the ability to retain the golgin-160 fragment at the Golgi in cells. We also found that Cys-463 is redox-sensitive; in its reduced form, interaction with golgin-160 was diminished or abolished, whereas oxidation of the Cys-463 by hydrogen peroxide restored the interaction. In addition, incubation with a nitric oxide donor promoted this interaction in vitro. These findings suggest that nuclear translocation of golgin-160-(140–311) is a highly coordinated event regulated not only by cleavage of the golgin-160 head but also by the oxidation state of GCP60. Golgins were initially identified as antigens from patients with autoimmune disease (reviewed in Ref. 1Nozawa K. Fritzler M.J. Chan E.K. Autoimmun. Rev. 2005; 4: 35-41Crossref PubMed Scopus (34) Google Scholar). These proteins, which are not related in sequence, share a long coiled-coil domain that can form a rod-like structure and are localized to the cytoplasmic face of the Golgi (reviewed in Ref. 2Barr F.A. Short B. Curr. Opin. Cell Biol. 2003; 15: 405-413Crossref PubMed Scopus (213) Google Scholar). Golgins have been implicated in Golgi complex structure and function. Phosphorylation of some golgins is required for mitotic disassembly of the Golgi complex (reviewed in Ref. 3Short B. Haas A. Barr F.A. Biochim. Biophys. Acta. 2005; 1744: 383-395Crossref PubMed Scopus (195) Google Scholar), and RNA interference experiments have implicated different golgins in specific membrane traffic steps (4Diao A. Rahman D. Pappin D.J. Lucocq J. Lowe M. J. Cell Biol. 2003; 160: 201-212Crossref PubMed Scopus (193) Google Scholar, 5Lu L. Tai G. Hong W. Mol. Biol. Cell. 2004; 15: 4426-4443Crossref PubMed Scopus (146) Google Scholar, 6Yoshino A. Setty S.R. Poynton C. Whiteman E.L. Saint-Pol A. Burd C.G. Johannes L. Holzbaur E.L. Koval M. McCaffery J.M. Marks M.S. J. Cell Sci. 2005; 118: 2279-2293Crossref PubMed Scopus (78) Google Scholar, 7Reddy J.V. Burguete A.S. Sridevi K. Ganley I.G. Nottingham R.M. Pfeffer S.R. Mol. Biol. Cell. 2006; 17: 4353-4363Crossref PubMed Scopus (101) Google Scholar). On the other hand, cleavage of golgin proteins such as Giantin (8Lowe M. Lane J.D. Woodman P.G. Allan V.J. J. Cell Sci. 2004; 117: 1139-1150Crossref PubMed Scopus (68) Google Scholar), GM130 (9Nozawa K. Casiano C.A. Hamel J.C. Molinaro C. Fritzler M.J. Chan E.K. Arthritis Res. 2002; 4: R3Crossref PubMed Scopus (60) Google Scholar), p115 (10Chiu R. Novikov L. Mukherjee S. Shields D. J. Cell Biol. 2002; 159: 637-648Crossref PubMed Scopus (122) Google Scholar), and golgin-160 (11Mancini 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 (318) Google Scholar) during apoptosis disrupts the ribbon-like organization of the Golgi stacks characteristic of mammalian cells. Expression of noncleavable forms of p115 or golgin-160 delays Golgi disassembly during apoptosis, whereas expression of a potential C-terminal caspase-cleavage product of p115, which translocates to the nucleus, induces apoptosis (10Chiu R. Novikov L. Mukherjee S. Shields D. J. Cell Biol. 2002; 159: 637-648Crossref PubMed Scopus (122) Google Scholar). These observations link cleavage of these Golgi proteins to apoptotic signaling as well as Golgi structure. Golgin-160 is predicted to have a noncoiled-coil "head" domain (N-terminal one-third) and a long coiled-coil (C-terminal two-thirds). Golgin-160 has been shown to promote the cell surface expression of a subset of potassium channels (12Bundis F. Neagoe I. Schwappach B. Steinmeyer K. Cell. Physiol. Biochem. 2006; 17: 1-12Crossref PubMed Scopus (20) Google Scholar) and the β1-adrenergic receptor (13Hicks S.W. Horn T.A. McCaffery J.M. Zuckerman D.M. Machamer C.E. Traffic. 2006; 7: 1666-1677Crossref PubMed Scopus (37) Google Scholar). Golgin-160 was also shown to be involved in targeting the glucose transporter GLUT4 to the insulin-responsive secretory vesicles in adipocytes (14Williams D. Hicks S.W. Machamer C.E. Pessin J.E. Mol. Biol. Cell. 2006; 17: 5346-5355Crossref PubMed Scopus (46) Google Scholar). The N-terminal head domain contains Golgi targeting information, a cryptic nuclear localization signal (NLS), 2The abbreviations used are: NLS, nuclear localization signal; GCP60, Golgi complex-associated protein of 60 kDa; GFP, green fluorescent protein; GST, glutathione S-transferase; DTT, dithiothreitol; DEA-NONOate, diethylamine NONOate; PBS, phosphate-buffered saline; siRNA, small interfering RNA. 2The abbreviations used are: NLS, nuclear localization signal; GCP60, Golgi complex-associated protein of 60 kDa; GFP, green fluorescent protein; GST, glutathione S-transferase; DTT, dithiothreitol; DEA-NONOate, diethylamine NONOate; PBS, phosphate-buffered saline; siRNA, small interfering RNA. and three aspartate residues that can be targeted by caspases during apoptosis (11Mancini 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 (318) Google Scholar, 15Hicks S.W. Machamer C.E. J. Biol. Chem. 2002; 277: 35833-35839Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Caspase-2 has been shown to cleave golgin-160 at aspartate 59, whereas caspase-3 cleaves it at aspartate 139. In addition, aspartate 311 can be cleaved by either caspase-2, -3, or -7 (11Mancini 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 (318) Google Scholar). Cleavage of the golgin-160 head domain by caspases generates different fragments exposing the cryptic NLS, promoting nuclear translocation of the fragments that contain it (15Hicks S.W. Machamer C.E. J. Biol. Chem. 2002; 277: 35833-35839Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The function of these golgin-160 fragments remains unknown, but cells expressing caspase-resistant golgin-160 were less sensitive to pro-apoptotic drugs that induce ER stress or ligate death receptors, indicating a potential role in signaling stress in the secretory pathway (16Maag R.S. Mancini M. Rosen A. Machamer C.E. Mol. Biol. Cell. 2005; 16: 3019-3027Crossref PubMed Scopus (52) Google Scholar). Golgin-160 also interacts with GCP60 (Golgi complex-associated protein of 60 kDa) (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). GCP60, initially identified as a Giantin interactor, was proposed to play a role in maintenance of the Golgi structure (18Sohda M. Misumi Y. Yamamoto A. Yano A. Nakamura N. Ikehara Y. J. Biol. Chem. 2001; 276: 45298-45306Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Recently, GCP60 (also known as ACBD3) was also shown to be involved in regulating signaling during asymmetric cell division in neuronal progenitor cells (19Zhou Y. Atkins J.B. Rompani S.B. Bancescu D.L. Petersen P.H. Tang H. Zou K. Stewart S.B. Zhong W. Cell. 2007; 129: 163-178Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The GCP60 sequence predicts a central coiled-coil domain, an acyl-CoA binding domain, and a Golgi dynamics domain, also found in other Golgi proteins and proposed to be involved in protein-protein interactions (18Sohda M. Misumi Y. Yamamoto A. Yano A. Nakamura N. Ikehara Y. J. Biol. Chem. 2001; 276: 45298-45306Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 20Anantharaman V. Aravind L. Genome Biol. 2002; 3 (research0023)Google Scholar). Human GCP60 is 96% identical to human PAP7, a peripheral-type benzodiazepine receptor and cAMP-dependent protein kinase regulatory subunit RI-α interactor (21Li H. Degenhardt B. Tobin D. Yao Z.X. Tasken K. Papadopoulos V. Mol. Endocrinol. 2001; 15: 2211-2228Crossref PubMed Scopus (136) Google Scholar). GCP60/PAP7 is highly expressed in steroid-producing tissues, such as testis and ovary (21Li H. Degenhardt B. Tobin D. Yao Z.X. Tasken K. Papadopoulos V. Mol. Endocrinol. 2001; 15: 2211-2228Crossref PubMed Scopus (136) Google Scholar), and is also expressed in all the regions of the brain examined (22Cheah J.H. Kim S.F. Hester L.D. Clancy K.W. Patterson III, S.E. Papadopoulos V. Snyder S.H. Neuron. 2006; 51: 431-440Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). GCP60/PAP7 localizes predominantly to the Golgi region (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 18Sohda M. Misumi Y. Yamamoto A. Yano A. Nakamura N. Ikehara Y. J. Biol. Chem. 2001; 276: 45298-45306Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). It is also present at mitochondria in steroid hormone-producing cells where it was implicated in sterol trafficking (23Liu J. Li H. Papadopoulos V. J. Steroid Biochem. Mol. Biol. 2003; 85: 275-283Crossref PubMed Scopus (72) Google Scholar). Recently, GCP60/PAP7 was also shown to be involved in iron homeostasis (22Cheah J.H. Kim S.F. Hester L.D. Clancy K.W. Patterson III, S.E. Papadopoulos V. Snyder S.H. Neuron. 2006; 51: 431-440Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). We identified GCP60 as a golgin-160 interacting protein using the golgin-160 head domain as bait in a yeast two-hybrid screen. However, GCP60 shows a preferential interaction with the caspase-generated golgin-160 fragment, 140–311, compared with the golgin-160 head domain or the 60–311 fragment (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Golgin-160-(140–311), which could be generated by cleavage by caspase-3 alone or in combination with caspase-2 or -7, translocates to the nucleus when exogenously expressed (15Hicks S.W. Machamer C.E. J. Biol. Chem. 2002; 277: 35833-35839Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Interestingly, overexpression of golgin-160-(140–311) with GCP60 reduces nuclear translocation and increases its Golgi retention, suggesting that this interaction also occurs in vivo (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In addition, cells overexpressing GCP60 are more sensitive to apoptosis induced by staurosporine, suggesting a possible pro-survival role of this golgin-160 fragment in the nucleus (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Here we studied the interaction of golgin-160-(140–311) with GCP60. We identified a redox-sensitive cysteine in GCP60 that regulates the interaction. Because this interaction prevents this golgin-160 fragment from translocating to the nucleus, these findings will contribute to our understanding of the function of golgin-160-(140–311) in the nucleus and the consequences of redox changes at Golgi complex membranes. Expression Constructs—The Myc-tagged golgin-160-(140–311) construct has been described previously (15Hicks S.W. Machamer C.E. J. Biol. Chem. 2002; 277: 35833-35839Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The pEGFP-GCP60 and pEGFP-GCP60-(328–528) have been described (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The pEGFP-GCP60-(328–528)C463S and pEGFP-GCP60-(328–528)C487S were generated by site-directed mutagenesis (QuikChange, Stratagene, La Jolla, CA), and base changes were confirmed by sequencing using the dideoxy sequencing method. Glutathione S-transferase (GST) fusion proteins of golgin-160, 1–393, 60–311, 60–139, and 140–311, have been described previously (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 24Hicks S.W. Machamer C.E. J. Biol. Chem. 2005; 280: 28944-28951Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The GST-golgin-160-(1–139) and -(1–311) constructs were made by PCR amplification and cloned into the BamHI site of pGEX-4T (Amersham Biosciences). The GST-golgin-160-(60–393) and -(140–393) constructs were made by digestion of GST-golgin-160-(60–311) and -(140–311), respectively, with BamHI and EcoRI and cloned into BamHI-EcoRI-digested pGEX-golgin-160-(1–393). Cells and Antibodies—HeLa cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum (Atlanta Biologicals, Norcross, GA) and 0.1 mg/ml normocin-O (InvivoGen, San Diego) at 37 °C in 5% CO2. The anti-N-terminal golgin-160 antibody has been described previously (15Hicks S.W. Machamer C.E. J. Biol. Chem. 2002; 277: 35833-35839Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Polyclonal rabbit anti-GFP antibodies were from Molecular Probes (Eugene, OR), and anti-GFP mouse antibodies were from Roche Applied Science as were the monoclonal anti-Myc antibodies. The Texas Red-conjugated goat anti-rabbit IgG was from Jackson ImmunoResearch (West Grove, PA), and the Alexa 488-conjugated goat anti-mouse IgG was from Molecular Probes, Inc. (Eugene, OR). Indirect Immunofluorescence Microscopy—HeLa cells cultured on coverslips (70–80% confluent) were transfected with 1 μg of DNA per 35-mm dish using FuGENE 6 (Roche Applied Science) as recommended by the manufacturer. At 18–20 h post-transfection, cells were rinsed in phosphate-buffered saline (PBS), fixed in 3% paraformaldehyde in PBS for 10 min, rinsed in PBS containing 10 mm glycine (Gly/PBS) for 5 min, permeabilized in 0.5% Triton X-100 in Gly/PBS for 3 min, and rinsed in Gly/PBS. The coverslips were then incubated in primary antibodies for 20 min, washed twice with Gly/PBS, and incubated with secondary antibodies. After washing, the cells were incubated with Hoechst 33258 (Sigma) for 3 min, washed, and mounted in glycerol containing 0.1 mN-propyl gallate. The images were collected on an Axioskop microscope (Zeiss, Thornwood, NY) equipped with epifluorescence and a Sensys CCD camera (Photometrics, Tucson, AZ) using IP Lab software (Signal Analytics, Vienna, VA). The scoring of cells for Fig. 6C has been described in detail previously (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Briefly, a nuclear classification was assigned to those cells with exclusively nuclear staining or nuclear staining equal to or stronger than Golgi staining. Golgi classification was assigned to those cells where the Golgi staining was stronger than nuclear staining. Approximately 150 cells were counted for each transfection condition in four independent experiments. The samples were coded so that scoring was performed without knowing their identities. Binding Assays—The GST constructs were expressed in Escherichia coli BL21-codon plus (Stratagene) and purified on glutathione-Sepharose 4B as recommended by the manufacturer (Amersham Biosciences). For the binding assays, 10 μgof purified GST or GST fusion proteins were rebound to glutathione beads and incubated overnight with transfected HeLa cell lysates, obtained as follows. Cells cultured in 60-mm plates (70–80% confluent) were transfected using FuGENE 6 as described above. The cells were lysed in detergent solution (62.5 mm EDTA, 50 mm Tris-HCl, pH 8, 0.4% deoxycholate, 1.0% Nonidet P-40) and protease inhibitors (catalog number P8340, Sigma), incubated on ice for 15 min, and spun at 14,000 × g at 4 °C for 15 min. Lysates were incubated with the beads overnight and washed three times in detergent solution. Bound proteins were eluted in sample buffer, boiled, resolved by SDS-PAGE, and detected by immunoblotting. Briefly, electrophoresed proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA), and the membranes were blocked in 5% milk/TBS-T (0.1% Tween 20, 150 mm NaCl, 10 mm Tris-HCl, pH 7.4). Blots were incubated overnight with the appropriate primary antibody at 4 °C and with secondary antibody for 60 min at room temperature. The primary antibodies were diluted in TBS-T containing 4% bovine serum albumin and 0.02% sodium azide, whereas the horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Amersham Biosciences) were used at a 1:2000 dilution in TBS-T only. Membranes were analyzed using chemiluminescence (ECL, Amersham Biosciences) and measured using x-ray films or by the VersaDoc Imaging System (Bio-Rad) using Quantity One software (Bio-Rad). Oxidation Assay—HeLa cells transfected with plasmids encoding GFP-GCP60-(328–528) or GFP-GCP60-(328–528)C463S were lysed in detergent solution and the lysates were incubated overnight with golgin-160-(140–311) fused to GST or GST alone that had been previously rebound to glutathione beads as described above. For the wild-type GCP60 C-terminal fragment, after the overnight binding, the beads were washed three times and aliquoted into four 1.5-ml tubes. One tube was washed three more times, and bound proteins were eluted in sample buffer. The other three tubes were treated with 1 mm DTT for 30 min with rocking at 4 °C. After the DTT incubation, one tube was washed three times, and bound proteins were eluted in sample buffer. The two tubes left were incubated with 2 mm hydrogen peroxide (H2O2) (Sigma) for 1 h at room temperature with rocking and washed three times with detergent solution, and one tube received sample buffer to elute the bound proteins, whereas the other tube was treated with 1 mm DTT at 4 °C for 30 min. After the second DTT incubation, the tube was washed three times, and sample buffer was added to elute bound proteins. Eluted proteins were boiled, resolved by SDS-PAGE, and detected by immunoblotting as described above. S-Nitrosylation Assay—HeLa cells transfected with a plasmid encoding GFP-GCP60-(328–528) were lysed in detergent solution and bound to golgin-160-(140–311) fused to GST overnight. The bound GFP-GCP60 C-terminal fragment was washed three times and eluted from GST-golgin-160 beads with detergent solution containing 1 mm DTT for 1 h. The eluate was precipitated by adding 10 volumes of–20 °C acetone for 20 min at–20 °C and rinsed once with–20 °C acetone. The pellet was resuspended in detergent solution and aliquoted into five tubes containing 10 μg of golgin-160-(140– 311) fused to GST or GST alone rebound to glutathione beads. Some samples received diethylamine (DEA)-NONOate (Sigma) to a final concentration of 10 mm or equal volume of the solvent, 10 mm NaOH, and were incubated at room temperature protected from light with rocking for 1 h. The samples were washed three times, and one of the DEA-NONOate-treated samples received 1 mm DTT, and it was incubated for 30 min at room temperature and protected from light, after which it was washed three times with detergent solution. Bound proteins were eluted in sample buffer, boiled, resolved in SDS-PAGE, and detected by immunoblotting as described above. GCP60 Interacts Preferentially with the Golgin-160-(140–311) Fragment in Vitro—We previously showed that the N-terminal domain of golgin-160 could be targeted by caspases-2, -3, and -7 during apoptosis, generating different fragments (Fig. 1A) (11Mancini 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 (318) Google Scholar). Recently, we showed that GCP60 interacts better with golgin-160-(140–311) when compared with the 60–311 fragment and the full-length head, 1–393 (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The golgin-160-(140–311) fragment could be generated by cleavage by caspase-3 alone, cleavage by caspase-3 and -2, or cleavage by caspase-3 and -7 on the same golgin-160 molecule (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This led to the idea that the GCP60 binding domain in golgin-160 is either masked in its full-length form and becomes exposed after cleavage or that a conformational change in golgin-160-(140–311) favors the interaction. To investigate whether other golgin-160 fragments generated by a single cleavage event would also show a preferred interaction with GCP60, we produced GST fusion proteins representing fragments cleaved once with caspase-2 at Asp-59 (residues 60–393), or at Asp-311 (residues 1–311), and fragments cleaved once with caspase-3 at Asp-139 (residues 1–139 and 139–393) (Fig. 1A). Binding assays were performed as described under "Experimental Procedures" using lysates from cells expressing GFP-GCP60-(328–528), a C-terminal fragment of GCP60 that shows the same binding preference for golgin-160-(140–311) as the full-length form (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The results show that even though all the golgin-160 fragments that have residues 140–311 in their sequence interacted, none of them interacted as strongly as that of the fusion protein representing golgin-160-(140–311) (Fig. 1B). These data suggest that cleavage at both Asp-139 and Asp-311 on the same golgin-160 molecule is necessary to promote a robust interaction with GCP60. Dithiothreitol and Iodoacetamide Prevent GCP60-Golgin-160 Interaction—Dithiothreitol (DTT), a reducing agent often used to mimic the cytosolic environment in cell lysates, prevents disulfide bonds from forming under oxidizing conditions normally present during the processing of samples. We noticed that the golgin-160-GCP60 interaction was sensitive to strong reducing conditions. 3J. I. Sbodio and C. E. Machamer, unpublished observations. To examine if milder reducing conditions would also interfere with this interaction, we performed binding assays using different DTT concentrations. As shown in Fig. 2A, the presence of 1 or 0.5 mm DTT in the lysis buffer was enough to prevent interaction. Even when the DTT concentration was as low as 0.1 mm, the interaction was strongly diminished, as compared with binding in the absence of DTT (Fig. 2A). This interaction could also be disrupted if DTT was added after binding (Fig. 2B) or if another reducing agent, β-mercaptoethanol, was used (Fig. 2C). The data suggest that reducing agents can inhibit or reverse the interaction of golgin-160-(140–311) and GCP60. To investigate the potential role of cysteine residues in this protein-protein interaction, we treated HeLa cells transfected with a plasmid encoding GFP-GCP60-(328–528) prior to lysis with iodoacetamide, a cysteine-alkylating agent, and performed binding assays using golgin-160-(140–311) fused to GST or GST alone. Fig. 3 shows that preincubation of cells with iodoacetamide prevented the interaction of golgin-160-(140–311) and GCP60-(328–528). Together, the DTT, β-mercaptoethanol, and iodoacetamide observations implicate cysteine residues as important mediators of the GCP60-golgin-160 binding. Mutation of Cysteine 463 in GCP60 Disrupts Its Interaction with Golgin-160 in Vitro—To date, all known GCP60 interactors have been shown to bind to the C-terminal portion of the protein (Fig. 4A) (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 18Sohda M. Misumi Y. Yamamoto A. Yano A. Nakamura N. Ikehara Y. J. Biol. Chem. 2001; 276: 45298-45306Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 21Li H. Degenhardt B. Tobin D. Yao Z.X. Tasken K. Papadopoulos V. Mol. Endocrinol. 2001; 15: 2211-2228Crossref PubMed Scopus (136) Google Scholar). As shown above, treatment with DTT or iodoacetamide prevents GCP60-(328–528) interaction with golgin-160-(140–311). Golgin-160-(140–311) does not have cysteine residues in its sequence, so an intermolecular disulfide bond cannot form between these two sequences. On the other hand, GCP60-(328–528) has two cysteine residues, Cys-463 and Cys-487 (Fig. 4A). To investigate if the absence of DTT in the lysis buffer could induce formation of an intramolecular disulfide bond that promotes binding to golgin-160-(140–311), we mutated each cysteine residue individually to serine and assayed for binding in the presence or absence of DTT. Fig. 4B shows that when Cys-487 was mutated to Ser, the GCP60-(328–528) mutant bound golgin-160-(140–311) as well as wild-type GCP60, and this binding was prevented by 1 mm DTT. On the other hand, when Cys-463 was mutated to serine, the interaction was abolished regardless of the presence of DTT. These results suggest that interaction of GCP60 with golgin-160-(140–311) depends on cysteine 463 in GCP60. Mutation of Cysteine 463 in GCP60 Reduces Its Interaction with Golgin-160-(140–311) in Vivo—Caspase cleavage of golgin-160 generates distinct fragments that expose an otherwise cryptic NLS, allowing those fragments that contain the NLS to translocate to the nucleus (15Hicks S.W. Machamer C.E. J. Biol. Chem. 2002; 277: 35833-35839Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). We showed that overexpression of GCP60 increases Golgi retention of these fragments, including a fragment representing the full-length head domain of golgin-160 (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This suggests that GCP60 might interact, at least weakly, with the full-length form of golgin-160 in vivo (17Sbodio J.I. Hicks S.W. Simon D. Machamer C.E. J. Biol. Chem. 2006; 281: 27924-27931Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Interaction with Giantin was proposed to be responsible for Golgi localization of GCP60 (18Sohda M. Misumi Y. Yamamoto A. Yano A. Nakamura N. Ikehara Y. J. Biol. Chem. 2001; 276: 45298-45306Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar); however, golgin-160 might also play a role to its Golgi localization. Therefore, to evaluate the contribution of Cys-463 in GCP60 to golgin-160-(140–311) interaction in vivo, we first needed to determine whether golgin-160 contributes to Golgi localization of GCP60. To investigate this, we depleted golgin-160 using RNA interference. HeLa cells were transfected with small interfering RNA duplexes (siRNA) against golgin-160, using conditions previously shown to deplete golgin-160 more than 95% (13Hicks S.W. Horn T.A. McCaffery J.M. Zuckerman D.M. Machamer C.E. Traffic. 2006; 7: 1666-1677Crossref PubMed Scopus (37) Google Scholar). After 72 h the cells were transfected with a plasmid encoding GFP-GCP60. Immunofluorescence microscopy showed that when golgin-160 was knocked down to undetectable levels, GCP60 still localized to the Golgi, suggesting that interaction with golgin-160 is not required to localize GCP60 to the Golgi complex (Fig. 5). Thus, it was possible to evaluate the ef
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