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Originally published In Press as doi:10.1074/jbc.M705794200 on August 20, 2007

J. Biol. Chem., Vol. 282, Issue 41, 29874-29881, October 12, 2007
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Identification of a Redox-sensitive Cysteine in GCP60 That Regulates Its Interaction with Golgin-160*

Juan I. Sbodio and Carolyn E. Machamer1

From the Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, July 16, 2007 , and in revised form, August 15, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Golgins were initially identified as antigens from patients with autoimmune disease (reviewed in Ref. 1). 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. 2). 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. 3), and RNA interference experiments have implicated different golgins in specific membrane traffic steps (4–7). On the other hand, cleavage of golgin proteins such as Giantin (8), GM130 (9), p115 (10), and golgin-160 (11) 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 (10). 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 (12) and the beta1-adrenergic receptor (13). Golgin-160 was also shown to be involved in targeting the glucose transporter GLUT4 to the insulin-responsive secretory vesicles in adipocytes (14). The N-terminal head domain contains Golgi targeting information, a cryptic nuclear localization signal (NLS),2 and three aspartate residues that can be targeted by caspases during apoptosis (11, 15). 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 (11). 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 (15). 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 (16).

Golgin-160 also interacts with GCP60 (Golgi complex-associated protein of 60 kDa) (17). GCP60, initially identified as a Giantin interactor, was proposed to play a role in maintenance of the Golgi structure (18). Recently, GCP60 (also known as ACBD3) was also shown to be involved in regulating signaling during asymmetric cell division in neuronal progenitor cells (19). 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 (18, 20). Human GCP60 is 96% identical to human PAP7, a peripheral-type benzodiazepine receptor and cAMP-dependent protein kinase regulatory subunit RI-{alpha} interactor (21). GCP60/PAP7 is highly expressed in steroid-producing tissues, such as testis and ovary (21), and is also expressed in all the regions of the brain examined (22). GCP60/PAP7 localizes predominantly to the Golgi region (17, 18). It is also present at mitochondria in steroid hormone-producing cells where it was implicated in sterol trafficking (23). Recently, GCP60/PAP7 was also shown to be involved in iron homeostasis (22).

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 (17). 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 (15). 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 (17). 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 (17).

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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression Constructs—The Myc-tagged golgin-160-(140–311) construct has been described previously (15). The pEGFP-GCP60 and pEGFP-GCP60-(328–528) have been described (17). 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 (17, 24). 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 (15). 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 M N-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 (17). 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 x 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).


Figure 1
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  		FIGURE 1.
GCP60 interacts preferentially with the golgin-160-(140–311) fragment. A, schematic representation of full-length golgin-160 and the potential fragments of the golgin-160 head domain that would be generated by single or multiple caspase cleavage events. B, HeLa cells were transfected with a plasmid encoding GFP-GCP60-(328–528). Aliquots of cell lysates were incubated with GST fused to different golgin-160 fragments (as indicated by the residue numbers) or GST alone. Bound proteins were separated by SDS-PAGE, and GFP-GCP60-(328–528) was detected by immunoblotting with anti-GFP antibodies. C, GST fusion proteins used for the binding assays are shown in a Coomassie Blue-stained gel. Positions of the molecular mass markers (in kDa) are shown on the left.

 
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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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) (11). 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 (17). 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 (17). 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 (17). 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.3 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, beta-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, beta-mercaptoethanol, and iodoacetamide observations implicate cysteine residues as important mediators of the GCP60-golgin-160 binding.


Figure 2
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  		FIGURE 2.
Interaction of GCP60 with golgin-160-(140–311) is sensitive to reducing agents. A, HeLa cells were transfected with a plasmid encoding GFP-GCP60-(328–528) and cells were lysed in buffers containing different concentrations of DTT as indicated. Aliquots of cell lysates were incubated with GST fused to golgin-160-(140–311) or GST alone. Bound proteins were separated by SDS-PAGE, and GFP-GCP60-(328–528) was detected by immunoblotting with anti-GFP antibodies. B, lysates from HeLa cells transfected with plasmids encoding GFP-GCP60-(328–528) were incubated with GST fused to golgin-160-(140–311) or GST alone. Bound proteins were washed and incubated with lysis buffer with or without DTT for 30 min at 4 °C. Bound proteins were washed and resolved as in A. C, HeLa cells were transfected with a plasmid encoding GFP-GCP60-(328–528). Aliquots of cell lysates were incubated with GST fused to golgin-160-(140–311) or GST alone in the presence or absence of 1 mM DTT or 2 mM beta-mercaptoethanol (BME). Bound proteins were washed and resolved as in A.

 


Figure 3
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  		FIGURE 3.
Treatment with iodoacetamide prevents GCP60 interaction with golgin-160. HeLa cells were transfected with a plasmid encoding GFP-GCP60-(328–528). Cells were treated or not with 10 mM iodoacetamide (IAA), followed by lysis with or without 1 mM DTT. Aliquots of cell lysates were incubated with GST fused to golgin-160-(140–311) or GST alone. Bound proteins were separated by SDS-PAGE, and GFP-GCP60-(328–528) was detected by immunoblotting with anti-GFP antibodies.

 
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) (17, 18, 21). 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 (15). We showed that overexpression of GCP60 increases Golgi retention of these fragments, including a fragment representing the full-length head domain of golgin-160 (17). This suggests that GCP60 might interact, at least weakly, with the full-length form of golgin-160 in vivo (17). Interaction with Giantin was proposed to be responsible for Golgi localization of GCP60 (18); 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% (13). 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 effect of the GCP60-C463S mutant on the localization of golgin-160-(140–311) in vivo.


Figure 4
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  		FIGURE 4.
Mutation of cysteine 463 in GCP60 prevents interaction with golgin-160-(140–311). A, schematic representation of full-length GCP60 and the C-terminal fragment used for the binding assays showing the cysteine residues. B, HeLa cells were transfected with plasmids encoding GFP-GCP60-(328–528), GFP-GCP60-(328–528)C487S, or GFP-GCP60-(328–528)C463S. Cells were lysed with or without 1 mM DTT. The lysates were incubated with GST fused to golgin-160-(140–311) or GST alone. Bound proteins were separated by SDS-PAGE, and GFP-GCP60-(328–528) was detected by immunoblotting with anti-GFP antibodies.

 
Like GCP60-(328–528)C463S, the full-length form of the mutant did not interact with golgin-160-(140–311) in vitro (Fig. 6A). However, the mutant form of GCP60 localized to the Golgi like the wild-type protein, as expected from the results above (Fig. 6C). This suggests that mutation of Cys-463 in GCP60 does not affect its interaction with Giantin. To investigate whether the mutation of Cys-463 blocked GFP-GCP60 interaction with golgin-160-(140–311) in vivo, we assayed for Golgi retention of a Myc-tagged form of the golgin-160 fragment in the presence of GFP-GCP60, GFP-GCP60-C463S, or GFP alone (Fig. 6C). Quantification of this observation is described under "Experimental Procedures" and is presented in Fig. 6D. The results show that golgin-160-(140–311) was retained at the Golgi complex more efficiently when overexpressed together with wild-type GCP60 than when it was overexpressed with either the C463S mutant or GFP alone (Fig. 6D). These data suggest that the interaction of the mutant GCP60 with golgin-160-(140–311) in vivo is diminished as compared with wild-type GCP60 and cannot prevent the golgin-160 fragment from translocating to the nucleus as effectively as wild-type GCP60.


Figure 5
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  		FIGURE 5.
Depletion of golgin-160 does not affect localization of GCP60 to the Golgi complex. HeLa cells were transfected with or without golgin-160 siRNA; at 72 h post-siRNA transfection, cells were transfected with a plasmid encoding GFP-GCP60. The cells were fixed, permeabilized, and stained for GFP (GCP60), golgin-160, and DNA (Hoechst 33258).

 
Cysteine 463 of GCP60 Is Redox-sensitive—When GCP60 was incubated in the presence of reducing agents, its interaction with golgin-160 was prevented or reversed (Fig. 2). To investigate the potential redox sensitivity of cysteine 463 in GCP60, we measured the binding of golgin-160-(140–311) and GCP60 under reducing or oxidizing conditions. We purified GFP-GCP60-(328–528) using golgin-160-(140–311), washed away unbound proteins, and aliquoted purified GFP-GCP60-(328–528) bound to golgin-160-(140–311) into different tubes. The aliquots were treated with or without DTT, followed by H2O2 and successive washes to get rid of unbound proteins and the excess oxidizing agent. A second DTT treatment was performed on one of the H2O2-treated aliquots followed by washing as described under "Experimental Procedures." The results confirmed that DTT treatment disrupted the interaction as compared with the untreated samples (Fig. 7A, lane 3 versus 4). On the other hand, when the DTT-treated sample was incubated with H2O2, the interaction was partially restored (Fig. 7, lane 5). This restored binding was specific to oxidized cysteine 463, because the wild-type GFP-GCP60-(328–528) but not the C463S mutant showed binding after treatment (Fig. 7, A, lane 5, versus B, lane 5). This restored interaction was also reversed by a second DTT treatment (Fig. 7A, lane 5 versus 6). Together, these results implicate cysteine 463 in GCP60 as a redox-sensitive cysteine that mediates GCP60-golgin-160 interaction when it is oxidized. The most likely form of Cys-463 is sulfenic acid (Cys-SOH) given its readily reversible nature (25).


Figure 6
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  		FIGURE 6.
Full-length GCP60-(C463S) interacts poorly with golgin-160-(140–311) in vitro and in vivo. A, HeLa cells were transfected with plasmids encoding GFP-GCP60 or GFP-GCP60(C463S). Aliquots of cell lysates were incubated with GST fused to golgin-160-(140–311) or GST alone. Bound proteins were separated by SDS-PAGE, and the GFP-GCP60 proteins were detected by immunoblotting with anti-GFP antibodies. B, GST fusion proteins used for the binding assays are shown in a Coomassie Blue-stained gel. Positions of the molecular mass markers (in kDa) are shown on the left. C, HeLa cells were transfected with plasmids encoding GFP-GCP60, GFP-GCP60(C463S), or GFP alone, and Myc-tagged golgin-160(140–311). The cells were fixed, permeabilized, and stained for GFP (GCP60), Myc (golgin-160(140–311)), and DNA (Hoechst 33258). D, cells expressing both constructs were scored according to the localization of the golgin-160 fragment as described under "Experimental Procedures." The data are presented as the average of four experiments ± S.E. p ≤ 0.001 for the increase of the golgin-160 fragment at the Golgi in GCP60 wild-type as compared with the mutant.

 
In Vitro S-Nitrosylation of GCP60 Enhances Its Interaction with Golgin-160-(140–311)—Regulation of protein function can be achieved by oxidization or S-nitrosylation of the same cysteine residues (reviewed in Ref. 26), and both modifications are readily reversible by DTT (25). To investigate the possibility that GCP60 could also be S-nitrosylated, we purified GFP-GCP60-(328–528) with golgin-160-(140–311) (Fig. 8A) and eluted it with DTT (Fig. 8B, lane 1). Reduced GCP60 was isolated from excess DTT by acetone precipitation and incubated with fresh golgin-160-(140–311) fused to GST or GST alone in the presence of the nitric oxide donor, DEA-NONOate, the solvent NaOH, or DEA-NONOate followed by DTT (Fig. 8B) as described under "Experimental Procedures." Treatment with DEA-NONOate, but not NaOH, enhanced GCP60 binding to golgin-160-(140–311) fused to GST but not to GST alone (Fig. 8, lane 5 versus lanes 3 and 4). This enhanced binding was also reversed by DTT treatment (Fig. 8, lane 5 versus 6). These data suggest that GCP60 can be S-nitrosylated in vitro and that this modification can promote GCP60-golgin-160-(140–311) interaction.


Figure 7
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  		FIGURE 7.
Treatment with hydrogen peroxide restores binding between reduced GCP60 and golgin-160. A, HeLa cells were transfected with a plasmid encoding GFP-GCP60-(328–528). Aliquots of cell lysates were incubated with golgin-160-(140–311) fused to GST or GST alone. GST-golgin-160-(140–311) beads bound to GFP-GCP60-(328–528) were washed and aliquoted. Aliquots were treated or not with 1 mM DTT, followed by incubation or not with 2mM H2O2, as indicated. Beads with bound proteins were washed three times, and one sample was treated again with 1 mM DTT followed by three more washes. Bound proteins were separated by SDS-PAGE, and GFP-GCP60-(328–528) was detected by immunoblotting with anti-GFP antibodies. B, HeLa cells were transfected with a plasmid encoding GFP-GCP60-(328–528)C463S. Aliquots of cell lysates were incubated with golgin-160-(140–311) fused to GST or GST alone as in A. Golgin-160-(140–311) beads were aliquoted (without being washed), and the aliquots were treated or not with 1mM DTT, followed by incubation or not with 2 mM H2O2. After three washes, bound proteins were separated by SDS-PAGE, and GFP-GCP60-(328–528) and GFP-GCP60-(328–528)C463S were detected by immunoblotting with anti-GFP antibodies.

 


Figure 8
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  		FIGURE 8.
S-Nitrosylation of reduced GCP60 restores its binding with golgin-160 in vitro. A, HeLa cells were transfected with a plasmid encoding GFP-GCP60-(328–528). Aliquots of cell lysates were incubated with golgin-160-(140–311) fused to GST or GST alone. Bound proteins were separated by SDS-PAGE, and GFP-GCP60-(328–528) was detected by immunoblotting with anti-GFP antibodies. B, GFP-GCP60-(328–528) bound to GST-golgin160-(140–311) beads (from A) was eluted with DTT, acetone-precipitated, and resuspended in lysis buffer with no DTT. Reduced GCP60-(328–528) was aliquoted in tubes containing fresh golgin-160-(140–311) fused to GST or GST alone, treated or not with 10 mM DEA-NONOate or NaOH for 1 h, and washed three times. One of the samples treated with DEA-NONOate was subsequently incubated with DTT for 30 min and washed three times. Bound proteins were separated by SDS-PAGE, and GFP-GCP60-(328–528) was detected by immunoblotting with anti-GFP antibodies.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Golgin-160, initially identified from patients with autoimmune disease, localizes to the cytoplasmic side of the Golgi (27, 28). It has the characteristic coiled-coil structure of golgin proteins in the C-terminal two-thirds of the protein, whereas the N-terminal portion, or head, contains a cryptic nuclear localization signal, Golgi targeting information, and three aspartates that can be cleaved by caspases during apoptosis (11, 15). Cleavage of the golgin-160 head domain generates distinct fragments that can translocate to the nucleus after exposure of the cryptic NLS (15). Expression of a caspase-resistant form of golgin-160 disrupts apoptosis induced by some pro-apoptotic stimuli (16), suggesting that golgin-160 fragments may transduce distinct apoptotic signals. Recently, we identified GCP60 as an interactor of golgin-160 (17). We showed that a caspase generated golgin-160 fragment, golgin-160-(140–311), exhibited a preferential interaction for GCP60 in vitro (17). This preferential interaction was also reflected in vivo because overexpression of GCP60 increased the Golgi retention of golgin-160-(140–311), preventing nuclear accumulation (17).

GCP60 localizes to the Golgi through its interaction with Giantin, and has been proposed to play a role in maintaining Golgi structure (18). The C-terminal half of the protein, which was predicted to be involved in protein-protein interaction through a Golgi dynamics domain, contains the binding sequence for Giantin and golgin-160 (17, 18, 20). The N-terminal sequence of GCP60 contains an acyl-CoA-binding site and an aspartate, Asp-15, that can be cleaved by caspase-3 in vitro (17, 18). The GCP60 cleavage product that could be generated by caspase-3 is predicted to show the same binding pattern for golgin-160-(140–311) as the full-length form, because the shorter fragment GCP60-(328–528) binds as well as full-length protein (17). GCP60 is 96% identical to PAP7, a protein identified through its interaction with peripheral type benzodiazepine receptor and cAMP-dependent protein kinase A regulatory subunit RI-{alpha} (21). These two interactors also bind to the C-terminal region of GCP60/PAP7 (21) confirming the role of this portion of GCP60/PAP7 in protein-protein interactions.

In this study, we characterized the interaction of golgin-160-(140–311) with GCP60 using the truncated form of GCP60, GCP60-(328–528). We found that GCP60 prefers to interact with golgin-160-(140–311) as compared with any of the other golgin-160 fragments that could be generated by caspase cleavage (Fig. 1). We also found this interaction to be sensitive to reducing and cysteine-alkylating agents (Fig. 2 and Fig. 3). This allowed us to identify a single cysteine residue in GCP60, cysteine 463, that is critical for the interaction. When cysteine 463 was mutated to serine, the in vitro interaction was abolished (Fig. 4). In vivo, the cysteine to serine mutant fails to retain golgin-160-(140–311) at the Golgi as well as the wild-type protein, allowing the fragment to translocate to the nucleus (Fig. 6). Because reducing agents affect this interaction, we investigated if oxidizing agents could restore the interaction. Indeed, when reduced GCP60 was treated with H2O2, the interaction was reestablished (Fig. 7). This dual regulation of the interaction (GCP60 redox state and cleavage of golgin-160) might be a way to specifically control the binding and therefore golgin-160-(140–311) localization, under special circumstances. In addition, the redox-sensitive cysteine residue in GCP60 is conserved in other mammals where golgin-160 is also conserved (e.g. mouse and rat), suggesting that this type of regulation is not unique to humans.

The cytoplasm is generally considered a reducing environment. However, reactive oxygen species are continuously produced by cells under aerobic conditions (reviewed in Ref. 29). Although reactive oxygen species are destructive at high levels, they can act as signaling molecules at low levels (reviewed in Ref. 30). In particular, H2O2 has been shown to influence many signaling pathways (reviewed in Ref. 31). Oxidation of cysteine residues depends mainly on the reactivity (low pKa) of the particular thiol (32). Thiol oxidation can lead to the formation of sulfenic acid (Cys-SOH), sulfinic acid (Cys-SO2H), or sulfonic acid (Cys-SO3H). Cys-SOH is the only oxidation state that is readily reversible (25). This property of Cys-SOH in cytoplasmic proteins is thus useful as a redox sensor in cell signaling (33). For example, oxidation of the active site cysteine in several protein-tyrosine phosphatases reversibly blocks activity, thus regulating signaling through tyrosine kinase receptors (34, 35). In HeLa cells, Cys-463 in GCP60 is likely to be in the sulfenic acid form when it interacts with golgin-160 given the reversibility of the interaction (Fig. 7). Because efficient interaction appears to require both cleavage of golgin-160 to produce the 140–311 fragment and oxidation of Cys-463 in GCP60, this suggests GCP60 might play a role in sensing redox changes at Golgi membranes under specific circumstances. The Golgi retention in vivo of the golgin-160 fragments when GCP60 is overexpressed might be due to the weak interaction observed under reducing conditions, oxidation of Cys-463 under normal conditions, or oxidative stress induced by transfecting the cells. This redox regulation of the interaction might also suggest that the weaker Golgi retention we observed previously for other golgin-160 fragments, such as 60–311 (17), might become more efficient under increased oxidizing conditions in the cytoplasm.

Another post-translational modification of cysteine residues that we investigated is S-nitrosylation. It has been shown for N-ethylmaleimide-sensitive factor, a protein involved in exocytosis, that the same cysteine residue can be S-nitrosylated or oxidized (36, 37). In this case, the final effect of these modifications is inhibition of exocytosis, although the mechanism of inhibition is different for the two modifications (36, 37). When reduced GCP60 was treated with the nitric oxide donor, DEA-NONOate, GCP60 binding to golgin-160-(140–311) was increased (Fig. 8). This suggests that GCP60 can be oxidized or S-nitrosylated to promote its interaction with golgin-160. In addition to N-ethylmaleimide-sensitive factor, S-nitrosylation has been shown to affect a number of protein targeting and trafficking steps (22, 38, 39).

In summary, the data presented here suggest that the interaction of golgin-160-(140–311) with GCP60 is highly coordinated. Cleavage of golgin-160, modification of cysteine 463 in GCP60, and the expression levels of GCP60 all contribute to the regulation of the interaction, which alters golgin-160-(140–311) localization. Because these two proteins are ubiquitously expressed, it will be very important to study the significance of this interaction in cells that are more suitable for modulating the interaction by either H2O2 or nitric oxide production, like endothelial or macrophage cells. The study of this interaction will help elucidate the function of the golgin-160 fragment in transmitting and/or sensing oxidative or nitrosative stresses that may lead to cell death.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM42522 (to C. E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Cell Biology, The Johns Hopkins University School of Medicine, 725 Wolfe St., Baltimore, MD 21205. Tel.: 410-955-1809; Fax: 410-955-4129; E-mail: machamer{at}jhmi.edu.

2 The 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. Back

3 J. I. Sbodio and C. E. Machamer, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank the members of the Machamer laboratory for helpful discussions and comments on the manuscript. We also thank Dr. Charles J. Lowenstein for useful discussions.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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