A role for BARS at the fission step of COPI vesicle formation from Golgi membrane
2005; Springer Nature; Volume: 24; Issue: 23 Linguagem: Inglês
10.1038/sj.emboj.7600873
ISSN1460-2075
AutoresJia-Shu Yang, Stella Y. Lee, Stefania Spanò, Helge Gad, Leiliang Zhang, Zhongzhen Nie, Matteo Bonazzi, Daniela Corda, Alberto Luini, Victor W. Hsu,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoArticle17 November 2005free access A role for BARS at the fission step of COPI vesicle formation from Golgi membrane Jia-Shu Yang Jia-Shu Yang Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Stella Y Lee Stella Y Lee Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Stefania Spanò Stefania Spanò Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Helge Gad Helge Gad Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Leiliang Zhang Leiliang Zhang Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Zhongzhen Nie Zhongzhen Nie Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD, USA Search for more papers by this author Matteo Bonazzi Matteo Bonazzi Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Daniela Corda Daniela Corda Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Alberto Luini Alberto Luini Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Victor W Hsu Corresponding Author Victor W Hsu Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jia-Shu Yang Jia-Shu Yang Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Stella Y Lee Stella Y Lee Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Stefania Spanò Stefania Spanò Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Helge Gad Helge Gad Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Leiliang Zhang Leiliang Zhang Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Zhongzhen Nie Zhongzhen Nie Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD, USA Search for more papers by this author Matteo Bonazzi Matteo Bonazzi Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Daniela Corda Daniela Corda Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Alberto Luini Alberto Luini Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Victor W Hsu Corresponding Author Victor W Hsu Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Jia-Shu Yang1,2, Stella Y Lee1,2, Stefania Spanò3, Helge Gad3, Leiliang Zhang1,2, Zhongzhen Nie4, Matteo Bonazzi3, Daniela Corda3, Alberto Luini3 and Victor W Hsu 1,2 1Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA 2Department of Medicine, Harvard Medical School, Boston, MA, USA 3Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy 4Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD, USA *Corresponding author. Brigham and Women's Hospital, One Jimmy Fund Way, Smith 538, Boston, MA 02115, USA. Tel.: +1 617 525 1103; Fax: +1 617 525 1104; E-mail: [email protected] The EMBO Journal (2005)24:4133-4143https://doi.org/10.1038/sj.emboj.7600873 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The core complex of Coat Protein I (COPI), known as coatomer, is sufficient to induce coated vesicular-like structures from liposomal membrane. In the context of biological Golgi membrane, both palmitoyl-coenzyme A (p-coA) and ARFGAP1, a GTPase-activating protein (GAP) for ADP-Ribosylation Factor 1, also participate in vesicle formation, but how their roles may be linked remains unknown. Moreover, whether COPI vesicle formation from Golgi membrane requires additional factors also remains unclear. We now show that Brefeldin-A ADP-Ribosylated Substrate (BARS) plays a critical role in the fission step of COPI vesicle formation from Golgi membrane. This role of BARS requires its interaction with ARFGAP1, which is in turn regulated oppositely by p-coA and nicotinamide adenine dinucleotide, which act as cofactors of BARS. Our findings not only identify a new factor needed for COPI vesicle formation from Golgi membrane but also reveal a surprising mechanism by which the roles of p-coA and GAP are linked in this process. Introduction Coat proteins play a key role in intracellular transport by coupling the deformation compartmental membrane for vesicle formation with cargo sorting that involves coats binding to specific sequences in the cytoplasmic domain of cargo proteins for their proper packaging into nascent vesicles. Coat Protein I (COPI) is one of the best characterized coat complexes (Bonifacino and Lippincott-Schwartz, 2003). Upon activation of the small GTPase ADP-Ribosylation Factor 1 (ARF1), coatomer (the core complex of COPI) becomes recruited onto Golgi membrane (Donaldson et al, 1992). Incubation of an artificial liposomal membrane with purified coatomer and ARF1 has been shown to induce coated vesicular-like structures from a larger liposomal membrane (Spang et al, 1998; Bremser et al, 1999), providing evidence that coatomer represents the core machinery in COPI vesicle formation. To identify the additional factors critical for COPI vesicle formation from the biological Golgi membrane, reconstitutions have been performed by incubating isolated Golgi membrane with purified coatomer and ARF1. When this membrane was washed to release peripheral membrane factors, palmitoyl coenzyme-A (p-CoA) was revealed to play a role in COPI vesicle formation at the fission step (Ostermann et al, 1993). How p-coA accomplishes this role remains unclear, although one mechanism has been suggested to involve the transfer of the palmitoyl chain to target proteins and/or lipids (Pfanner et al, 1989). In this respect, an intriguing potential connection has been the recent finding that phospholipid acyltransferase activity participates in the fission of compartmental membranes. Endophilin has been identified to possess such an activity to mediate the fission of clathrin-coated vesicles (Schmidt et al, 1999). Moreover, Brefeldin-A (BFA)-induced ADP-Ribosylation Substrate (BARS) has been shown to possess such an activity, and also induce the fission of Golgi tubules (Weigert et al, 1999). This latter finding has been surprising, as BARS is a splice variant of C-Terminal Binding Protein 1 (CtBP1), which has been well characterized as a transcription corepressor (Chinnadurai, 2002). Nevertheless, recent studies have begun to validate the fission activity of BARS, as it is critical for Golgi fragmentation during mitosis (Hidalgo Carcedo et al, 2004), and participates in the formation of post-Golgi transport carriers (Bonazzi et al, 2005). In the context of COPI transport, ARFGAP1, a GTPase-activating protein (GAP) for ARF1, can substitute for p-coA in reconstituting the formation of COPI vesicles from Golgi membrane (Yang et al, 2002). Besides its known role as a negative regulator of ARF1, GAP was shown to possess a novel function as an ARF1 effector by acting as a component of the COPI coat complex (Yang et al, 2002; Lee et al, 2005). In the current study, we elucidate a role for BARS in the fission step of COPI vesicle formation, and also reveal a mechanism by which the roles of GAP and p-coA are linked in this process. Results BARS is critical for reconstituting COPI vesicles from Golgi membrane Using an established two-stage incubation system to reconstitute COPI vesicles (Ostermann et al, 1993), we previously showed that the addition of GAP at the second-stage incubation was sufficient for vesicle formation from Golgi membrane washed with 0.5 M KCl (Yang et al, 2002). However, when Golgi membrane was washed more stringently with 3 M KCl, we found that the addition of GAP at the second stage was no longer sufficient (Figure 1A). Analyzing the wash fraction by gel filtration, we found that one fraction restored vesicle formation (Figure 1B). As this fraction comigrated with the 43 kDa marker, and BARS is a 50 kDa protein that has been shown to have fission activity on Golgi membrane (Weigert et al, 1999), we next immunoblotted the different fractions for BARS, and found that only the active fraction contained a significant level of BARS (Figure 1B). Consistent with this result, BARS was detectable on unwashed Golgi membrane and on membranes washed with 0.5 M KCl, but the level became significantly reduced on Golgi membrane washed with 3 M KCl (Figure 1C). Confirming that BARS was critical in reconstituting COPI vesicles from Golgi membrane washed with 3 M KCl, we found that vesicle formation was restored, when recombinant BARS was incubated in conjunction with GAP during the second-stage incubation. Similar results were obtained using Golgi membrane isolated from CHO cells (Figure 1D) or from rat liver (Supplementary Figure 1A). Figure 1.BARS is a peripheral Golgi membrane protein critical for reconstituting COPI vesicles. (A) GAP is no longer sufficient to complete the second stage of the vesicle reconstitution system when Golgi membrane is washed with 3 M KCl. CHO Golgi membrane was subjected to salt washes as indicated and then used for the two-stage incubation system, followed by immunoblotting of pellet (P) and supernatant (S) for β-COP after the second-stage incubation. (B) The critical activity lost upon washing Golgi membrane with 3 M KCl contains BARS. The 3 M KCl wash fraction was analyzed by gel filtration with eluted fractions monitored for total proteins (top panel), assayed for activity in restoring COPI vesicle formation (middle panel), and immunoblotted for BARS (bottom panel). (C) Endogenous BARS on Golgi membrane is markedly reduced by 3 M KCl wash. CHO Golgi membrane is subjected to salt washes as indicated, followed by immunoblotting for proteins as indicated. The level of the transmembrane KDELR is used to assess the levels of Golgi membrane in each condition. (D) The addition of BARS restores the ability of GAP to complete the second-stage incubation using Golgi membrane washed with 3 M KCl. CHO Golgi membrane washed with 3 M KCl was used for the two-stage incubation system, followed by immunoblotting of pellet (P) and supernatant (S) for β-COP after the second-stage incubation. (E) Antibody against endogenous BARS on Golgi membrane inhibits the reconstitution of COPI vesicles. CHO Golgi membrane washed with 0.5 M KCl was incubated with an anti-BARS antibody or an anti-mannosidase I antibody (as control), and then subjected to the two-stage incubation system, followed by immunoblotting of pellet (P) and supernatant (S) for β-COP after the second-stage incubation. Download figure Download PowerPoint As confirmation that BARS was critical for the active fraction of the salt wash, we found that this activity was lost upon the immunodepletion of BARS (Supplementary Figure 1B). The addition of recombinant BARS to the depleted fraction restored vesicle formation (Supplementary Figure 1C), thus excluding the possibility that a copurified protein other than BARS was the critical factor in the active fraction. We also confirmed that endogenous BARS was critical for COPI vesicle formation, as the addition of an anti-BARS antibody blocked the reconstitution of COPI vesicles from Golgi membrane washed with 0.5 M KCl, which would have retained endogenous BARS (Figure 1E). We next characterized COPI vesicles reconstituted using the new method of washing Golgi membrane with 3 M KCl. We had shown previously by a rapid, high-speed ultracentrifugation procedure that the redistribution of coatomer from Golgi membrane washed with 0.5 M KCl during the second-stage incubation represented mostly COPI vesicles (Lee et al, 2005). Using the same approach, we found that the release of coatomer from the second-stage incubation using Golgi membrane washed with 3 M KCl also represented mostly COPI vesicles. First, coatomer from the supernatant fraction of this second-stage incubation was detected virtually all in the pelleted fraction upon high-speed centrifugation, while purified soluble coatomer, either alone (Figure 2A) or in combination with GAP and BARS (Supplementary Figure 1D), remained virtually all in the supernatant. Second, examination of the pelleted coatomer fraction by electron microscopy (EM) revealed mostly vesicles of approximately 80 nm diameter, which were positive for subunits of coatomer (Figure 2B). Figure 2.Characterization of vesicle reconstitution using Golgi membrane washed with 3 M KCl. (A) Coatomer released from Golgi membrane during the second-stage incubation is mainly membrane-bound. CHO Golgi membrane was washed with 3 M KCl, and then subjected to the two-stage incubation system. The supernatant from the second-stage incubation, or soluble coatomer as control, was subjected to ultracentrifugation at 200 000 g for 1 h to obtain pellet (P) and supernatant (S), followed by immunoblotting for β-COP. (B) The pellet fraction after ultracentrifugation of the supernatant derived from the second-stage incubation contains mostly COPI vesicles. The pellet fraction in (A) was analyzed by whole-mount EM with immunogold labeling using antibodies directed against subunits of COPI as indicated; bar, 50 nm. (C) Vesicles reconstituted from Golgi membrane washed with 3 M KCl have density characteristic of COPI vesicles. CHO Golgi membrane washed with 3 M KCl was used in the two-stage incubation system, and then the supernatant from the second stage was subjected to equilibrium centrifugation, followed by fractionation and immunoblotting for β-COP. (D) Golgi membrane washed with 3 M KCl selectively releases cargo proteins during the second-stage incubation. Golgi membrane was isolated from CHO cells stably transfected with myc-tagged KDELR, washed with 3 M KCl, and then used for the two-stage incubation system. The supernatant after the second-stage incubation was then immunoblotted for proteins as indicated, and, as comparison, 10% of Golgi membrane used for the reconstitution was also immunoblotted. (E) Vesicles derived from Golgi membrane washed with 3 M KCl have similar levels of KDELR as those derived from Golgi membrane washed with 0.5 M KCl. Golgi membrane was isolated from CHO cells stably transfected with myc-tagged KDELR, and then washed as indicated for use in the two-stage incubation system. The level of myc-tagged KDELR on vesicles was quantified by EM. The mean derived from three independent experiments is shown with standard error. As a reference point for cargo sorting, GTPγS is used for the two-stage incubation system using Golgi membrane washed with 0.5 M KCl, as has been shown previously to inhibit cargo sorting. (F) Golgi membrane washed with 3 M KCl reconstitutes a similar level of COPI vesicles as those washed with 0.5 M KCl. CHO Golgi membrane was subjected to different washes as indicated and then used for the two-stage incubation system with components added at the second stage as indicated. The incubation was then examined by EM to quantify the level of COPI-positive vesicles using antibodies directed against COPI subunits. The mean derived from three independent experiments is shown with standard error. Download figure Download PowerPoint The level of immunogold labeling on the reconstituted vesicles was also similar to that previously observed for COPI vesicles reconstituted by the more traditional methods of using either GTPγS or p-coA (Yang et al, 2002), or for COPI vesicles in vivo (Orci et al, 1997). Nevertheless, because the degree of immunogold labeling could vary due to multiple technical factors, we confirmed the coating density of the reconstituted vesicles by showing that they floated to 43% sucrose upon equilibrium centrifugation (Figure 2C), which was similar to COPI vesicles previously reconstituted using less stringently washed Golgi membrane (Yang et al, 2002). Moreover, as observed previously using GTP in the reconstitution (Yang et al, 2002), the new method of reconstituting COPI vesicles also allowed their subsequent uncoating, as indicated by coatomer at the bottom of the flotation gradient (>53% sucrose in Figure 2C). As an additional insight, we noted that, even though GAP and BARS were needed at similar levels to complete the second-stage incubation of the reconstitution system (Supplementary Figure 1E), gradient analysis showed that the distribution of BARS did not exhibit a similar peak that marked COPI vesicles as seen for coatomer and GAP (Supplementary Figure 1F). Thus, we concluded that BARS was unlikely to function as a structural coat component on COPI vesicles, as previously concluded for GAP (Yang et al, 2002). Assessing cargo sorting, we detected both the KDEL receptor (a cargo protein specific for COPI retrograde transport (Girod et al, 1999; White et al, 1999)) and membrin (a Golgi SNARE that interacts with ARF1 (Honda et al, 2005)), to be released from Golgi membrane during the second-stage incubation (Figure 2D). In contrast, fibronectin, a luminal secretory protein that also has a significant Golgi distribution (Ledger et al, 1980), remained with the Golgi membrane (Figure 2D). Confirming this suggested selectivity in cargo sorting into reconstituted COPI vesicles, we found by quantitative immunogold EM that KDELR was detected to a similar extent in vesicles reconstituted either by the new method (using Golgi membrane washed with 3 M KCl) or the previous method (using Golgi membrane washed with 0.5 M KCl) (Figure 2E). We also ruled out more conclusively that the 3 M KCl wash was too harsh to allow Golgi membrane to reconstitute COPI vesicles through physiologic mechanisms. First, examining the morphology of the Golgi membrane, we found that it was not significantly altered by the 3 M KCl wash (Supplementary Figure 2A). Second, this wash did not result in the luminal fibronectin being released into the wash fraction (Supplementary Figure 2B). Third, tethering proteins, such as p115, GRASP55, and GRASP65, were also mostly retained on Golgi membrane after washing with 3 M KCl (Supplementary Figure 2C). Fourth, the effect of washing Golgi membrane with 3 M KCl could be reproduced by washing Golgi membrane with 2 M urea (Supplementary Figure 2D). Notably, 2.5 M urea had been used previously to wash ER membrane in the reconstitution of COPII vesicles (Spang and Schekman, 1998), providing precedence for highly stringent washing still enabling the physiologic mechanisms of vesicle formation. Fifth, as control, BARS alone (Figure 1D) or incubation of ARFGAP1 in conjunction with the Golgi tethering complex COG as control (Supplementary Figure 2E) did not allow the completion of the second-stage incubation. Sixth, compared to reconstitution using Golgi membrane washed with 0.5 M KCl, we found by quantitative immunogold EM that the new reconstitution using Golgi membrane washed with 3 M KCl produced a similar level of COPI-positive vesicles (Figure 2F). Thus, we concluded that Golgi membrane washed with 3 M KCl was able to support the physiologic mechanisms of COPI vesicle formation. COPI vesicle formation does not require the acyltransferase activity of BARS To gain insight into how BARS functioned in COPI vesicle formation, we noted that the crystal structure of BARS had revealed that it contained at least two functional domains (Nardini et al, 2003), a central domain that bound nicotinamide adenine dinucleotide (NAD), referred as the NAD-binding domain (NBD), and the rest of the protein that comes together to form one domain, referred as the substrate-binding domain (SBD). In addition to these domains, we also generated other domain constructs: N-terminal portion (NTP, consisting of the N-terminal subdomain of SBD and NBD), C-terminal portion (CTP, consisting of the C-terminal subdomain of SBD and NBD), and C-terminal domain (CTD, representing the extreme C-terminal subdomain) (Figure 3A). Figure 3.COPI vesicle formation does not require the acyltransferase activity of BARS. (A) Schematic representation of the different domain mutants of BARS. (B) COPI vesicle formation in the presence of different BARS mutants. CHO Golgi membrane washed with 3 M KCl was used for the two-stage incubation system, with the second-stage incubation containing GAP and different forms of BARS as indicated. The level of β-COP released into the supernatant after this stage was quantified, and then normalized to that derived from using the wild-type BARS for incubation. The mean of this normalized value derived from three independent experiments is shown with standard error. (C) Acyltransferase activity of different mutant BARS. The level of phosphatidic acid (PA) generated by the transfer of fatty acyl-CoA onto lysophosphatidic acid, catalyzed by the different forms of BARS, was measured. The mean derived from three independent experiments is shown with standard error. (D) COPI vesicle formation requires an acyltransferase activity. CHO Golgi membrane washed with 0.5 M KCl was used for the two-stage incubation, with the second stage using components as indicated, followed by immunoblotting of pellet (P) and supernatant (S) for β-COP after the second-stage incubation. (E) Binding to GAP by different mutant BARS. GAP fused to GST that had been gathered onto glutathione beads was incubated with different forms of BARS in a pulldown assay. The level of different BARS on beads was quantified, normalized to the level of GAP, and then compared to that of the wild-type BARS. The mean derived from three independent experiments is shown with standard error. Download figure Download PowerPoint These domain mutants were initially examined for their ability to support vesicle formation from Golgi membrane washed with 3 M KCl. Quantifying this result, we found that NBD, NTP, and CTP were markedly impaired in vesicle formation as compared to the full-length BARS, while SBD and CTD still retained significant ability to support vesicle formation (Figure 3B). We ruled out that the CTD construct resulted in a misfolded hydrophobic protein that induced vesicle formation by a nonspecific process, as deliberate denaturation of the CTD mutant by boiling eliminated its activity in vesicle formation (Supplementary Figure 3A). We then assessed whether the different BARS mutants still possessed acyltransferase activity (Figure 3C), which was shown previously to be associated with the ability of BARS to induce fission of Golgi tubules (Weigert et al, 1999). Surprisingly, the SBD and CTD mutants, which still possessed significant ability to support vesicle formation, had markedly impaired acyltransferase activity (compare Figure 3B and C). Thus, we concluded that the acyltransferase activity of BARS was not the only mechanism by which it participated in COPI vesicle formation. However, we also noted that COPI vesicle formation had been shown previously to require an acyltransferase activity on Golgi membrane (Pfanner et al, 1989). To sort out an explanation, we found that an inhibitor of phospholipid acyltransferase activity on Golgi membrane, CI-976 (Drecktrah et al, 2003), also inhibited vesicle formation in our reconstitution system (Figure 3D). Thus, as this acyltransferase activity is known to be possessed by multiple proteins other than BARS, we concluded that, while such an activity was required to reconstitute COPI vesicles from Golgi membrane, this activity possessed specifically by BARS was not essential. As an insight into an alternate mechanism to explain how BARS participated in COPI vesicle formation, we explored whether BARS interacted with GAP in a pulldown assay (Supplementary Figure 3B). Quantifying the interactions, we found that SBD, CTP, and CTD bound to GAP similarly as the full-length protein, while NBD and NTP showed marked impairment (Figure 3E). The noted interactions were likely specific, as wild-type BARS did not exhibit significant binding to other ARF GAPs, such as ACAP1 and PAP (Supplementary Figure 3C). We also noted that the recruitment of BARS to Golgi membrane was not affected by the presence of GAP (Supplementary Figure 3D). Moreover, the CTD domain could interact directly with membrane lipids, as indicated by its direct binding to liposomal membrane (Supplementary Figure 3E). Thus, we concluded that the interaction between BARS and GAP likely occurred after BARS became membrane-bound. The CTP mutant accumulates COPI-positive buds on Golgi membrane by affecting the fission step To gain further insight into the role of the interaction between GAP and BARS, we initially tested whether this interaction affected the GAP catalytic activity. However, we observed no significant effect (Supplementary Figure 4A). Thus, as a clue to an alternate mechanism, we noted that, while the binding results of the different forms of BARS largely correlated with their ability to support vesicle formation, a notable exception was the CTP mutant, because it retained significant ability to bind GAP, but did not support vesicle formation. As this mutant BARS contained the CTD fragment, which was fully able to support vesicle formation, an intriguing possibility was that the additional sequences in CTP would render this mutant acting dominant negatively in suppressing vesicle formation. We first confirmed this possibility, as incubation with an equal molar ratio of wild type and CTP led to significant inhibition in vesicle formation, with this inhibition being nearly complete when the CTP mutant was added at 10-fold excess (Supplementary Figure 4B). This inhibition was reversible, as adding the wild-type protein in excess of the CTP mutant reversed the effect (Supplementary Figure 4C). Consistent with these findings, the addition of CTP to the vesicle reconstitution system using Golgi membrane washed with 0.5 M KCl, which retained endogenous BARS, also inhibited vesicle formation (Figure 4A). Thus, we examined the Golgi membrane after this incubation to gain insight into how the CTP mutant inhibited vesicle formation. Strikingly, we detected an accumulation of COPI-positive buds (Figure 4B). Quantifying the immunogold labeling for both COPI and BARS, we found that the CTP mutant was localized mainly at the neck of the buds, while COPI was more randomly distributed throughout the arrested buds (Figure 4C). Quantitative EM also revealed that more COPI-positive buds were detected on Golgi membrane in the presence of the CTP mutant as compared to the control condition without CTP (Figure 4D). Parallel to this finding, significantly fewer COPI vesicles were formed in the presence of the CTP mutant as compared to the control condition, when only GAP was added during the second-stage incubation (Figure 4E). Thus, we concluded that BARS played a critical role at the fission step during COPI vesicle formation. Figure 4.The CTP mutant induces the accumulation of COPI-positive buds on Golgi membrane. (A) The CTP mutant inhibits the reconstitution of COPI vesicles using Golgi membrane washed with 0.5 M KCl. CHO Golgi membrane washed with 0.5 M KCl was used for the two-stage incubation system, with the second-stage incubation containing GAP and varying levels of different BARS as indicated. The level of β-COP released into the supernatant was quantified and then normalized to control, which is derived from the condition that used only GAP in the incubation. The mean of this normalized value derived from three independent experiments is shown with standard error. (B) The CTP mutant induces the accumulation of COPI-positive buds on Golgi membrane. CHO Golgi membrane washed with 0.5 M KCl was used for the two-stage incubation system, with GAP and CTP added at the second stage, followed by EM examination using anti-COPI subunit antibodies (upper panels) and anti-BARS antibody (lower panels); bar, 50 nm. (C) CTP localizes mostly to the neck of buds on Golgi membrane. Ten fields from the EM that resulted (B) were randomly selected for quantitation of the level of gold particles on either the neck region of the bud or the rest of the bud, and then expressed as fractional levels. The mean derived from three independent experiments is shown with standard error. (D) Quantitatio
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