Recruitment of arfaptins to the trans-Golgi network by PI(4)P and their involvement in cargo export
2013; Springer Nature; Volume: 32; Issue: 12 Linguagem: Inglês
10.1038/emboj.2013.116
ISSN1460-2075
AutoresDavid Cruz-García, M. Bellido, Margherita Scarpa, Julien Villeneuve, Marko Jović, Marc Porzner, Tamás Balla, Thomas Seufferlein, Vivek Malhotra,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoArticle21 May 2013free access Source Data Recruitment of arfaptins to the trans-Golgi network by PI(4)P and their involvement in cargo export David Cruz-Garcia David Cruz-Garcia Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Maria Ortega-Bellido Maria Ortega-Bellido Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Margherita Scarpa Margherita Scarpa Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Julien Villeneuve Julien Villeneuve Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Marko Jovic Marko Jovic Section on Molecular Signal Transduction, Program for Developmental Neuroscience, NICHD, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Marc Porzner Marc Porzner Department of Internal Medicine I, University of Ulm, Ulm, Germany Search for more papers by this author Tamas Balla Tamas Balla Section on Molecular Signal Transduction, Program for Developmental Neuroscience, NICHD, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Thomas Seufferlein Thomas Seufferlein Department of Internal Medicine I, University of Ulm, Ulm, Germany Search for more papers by this author Vivek Malhotra Corresponding Author Vivek Malhotra Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author David Cruz-Garcia David Cruz-Garcia Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Maria Ortega-Bellido Maria Ortega-Bellido Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Margherita Scarpa Margherita Scarpa Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Julien Villeneuve Julien Villeneuve Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Marko Jovic Marko Jovic Section on Molecular Signal Transduction, Program for Developmental Neuroscience, NICHD, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Marc Porzner Marc Porzner Department of Internal Medicine I, University of Ulm, Ulm, Germany Search for more papers by this author Tamas Balla Tamas Balla Section on Molecular Signal Transduction, Program for Developmental Neuroscience, NICHD, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Thomas Seufferlein Thomas Seufferlein Department of Internal Medicine I, University of Ulm, Ulm, Germany Search for more papers by this author Vivek Malhotra Corresponding Author Vivek Malhotra Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author Author Information David Cruz-Garcia1,2, Maria Ortega-Bellido1,2, Margherita Scarpa1,2, Julien Villeneuve1,2, Marko Jovic3, Marc Porzner4, Tamas Balla3, Thomas Seufferlein4 and Vivek Malhotra 1,2,5 1Centre for Genomic Regulation (CRG), Barcelona, Spain 2Universitat Pompeu Fabra (UPF), Barcelona, Spain 3Section on Molecular Signal Transduction, Program for Developmental Neuroscience, NICHD, National Institutes of Health, Bethesda, MD, USA 4Department of Internal Medicine I, University of Ulm, Ulm, Germany 5Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain *Corresponding author. CRG—Centre de Regulació Genòmica, PRBB Building, Dr Aiguader, 88, Barcelona 08003, Spain. Tel.:+34 93 316 0235; Fax:+34 93 3969 983; E-mail: [email protected] The EMBO Journal (2013)32:1717-1729https://doi.org/10.1038/emboj.2013.116 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The BAR (Bin/Amphiphysin/Rvs) domain proteins arfaptin1 and arfaptin2 are localized to the trans-Golgi network (TGN) and, by virtue of their ability to sense and/or generate membrane curvature, could play an important role in the biogenesis of transport carriers. We report that arfaptins contain an amphipathic helix (AH) preceding the BAR domain, which is essential for their binding to phosphatidylinositol 4-phosphate (PI(4)P)-containing liposomes and the TGN of mammalian cells. The binding of arfaptin1, but not arfaptin2, to PI(4)P is regulated by protein kinase D (PKD) mediated phosphorylation at Ser100 within the AH. We also found that only arfaptin1 is required for the PKD-dependent trafficking of chromogranin A by the regulated secretory pathway. Altogether, these findings reveal the importance of PI(4)P and PKD in the recruitment of arfaptins at the TGN and their requirement in the events leading to the biogenesis of secretory storage granules. Introduction A large number of carriers form at the trans-Golgi network (TGN) that include clathrin-coated vesicles for trafficking to the endosomes, COPI for retrograde transport to the preceding Golgi cisternae and to the endoplasmic reticulum (ER), and carriers called CARTS for the trafficking of specific cargoes to the cell surface (De Matteis and Luini, 2008; Emr et al, 2009; Campelo and Malhotra, 2012; Valente et al, 2012; Wakana et al, 2012). The TGN is also the source of secretory storage granules in secretory cells and for the production of apical- and basolateral-specific carriers in polarized cells (Mellman and Nelson, 2008). How is the biogenesis of these different classes of transport carriers regulated at the TGN? For example, phosphatidylinositol 4-phosphate (PI(4)P) and the small GTPase Arf1 are required for the formation of clathrin-coated vesicles and CARTS but protein kinase D (PKD), which promotes the production of PI(4)P at the TGN, is required only for the biogenesis of the CARTS. In other words, there are components shared by processes that generate carriers destined for different membranes of the cell. How is this compartmentation or spatial segregation of transport machinery achieved at the TGN? It is also not known whether different carriers, mentioned above, form continuously or if there is a competition among different classes depending on the cargo. For example, does increase in secretory cargo affect the biogenesis of clathrin-coated or COPI vesicles and favour the formation of carriers destined for the cell surface? We are interested in membrane fission regulated by PKD, which is necessary for the generation of a specific class of transport carriers at the TGN. Two lipids diacylglycerol (DAG) and PI(4)P are essential for the PKD-dependent transport carrier biogenesis. DAG and Arf1 recruit PKD to the TGN, PKD is then activated by PKCη and the trimeric G protein subunits Gβγ, and promotes the production of PI(4)P by activating phosphatidylinositol 4-kinase IIIβ (PI(4)KIIIβ) (Baron and Malhotra, 2002; Diaz Anel and Malhotra, 2005; Hausser et al, 2005; Pusapati et al, 2010). As a result of the PI(4)P synthesis, the ceramide transfer protein (CERT), oxysterol binding protein (OSBP), and FAPP1 and FAPP2 are recruited to the TGN by virtue of their Pleckstrin homology (PH) domains (Levine and Munro, 1998, 2002; Godi et al, 2004). PKD phosphorylates CERT and OSBP and they detach from the TGN (Fugmann et al, 2007; Nhek et al, 2010). Based on these findings, it has been proposed that the PKD-dependent PI(4)P production, CERT and OSBP recruitment, and control of the binding of those proteins are essential for events leading to membrane fission to generate specific transport carriers (Graham and Burd, 2011; Bankaitis et al, 2012; Campelo and Malhotra, 2012). More recently, it has been shown that PKD and CtBP1-S/BARS are linked by 14-3-3γ to recruit and control a number of other proteins, which together regulate membrane fission (Valente et al, 2012). Strangely, thus far there is no evidence of a coat (like clathrin, COPI or COPII) or a BAR (Bin/Amphiphysin/Rvs) domain-containing protein in the PKD-dependent biogenesis of transport carriers. We have made a surprising finding that BAR domain-containing proteins arfaptin1 and arfaptin2 are recruited to the TGN by a PI(4)P-dependent reaction. Moreover, the binding of arfaptin1 to PI(4)P is regulated by PKD whereas the binding of arfaptin2 to PI(4)P is insensitive to PKD activity. We also report the specific requirement of arfaptin1 in the trafficking of chromogranin A (Cg A); the description of our findings follows. Results Arfaptins bind PI(4)P We have found that ceramide levels affect events leading to the biogenesis of transport carriers (Duran et al, 2012). The ceramide transport protein CERT binds PI(4)P at the TGN and thus controls the amount of ceramide imported into the TGN (Hanada et al, 2003). To study the connection between PI(4)P and CERT, we tested the effect of phenylarsine oxide (PAO), a chemical inhibitor of PI(4)P synthesis, on the localization of CERT at the TGN (Wiedemann et al, 1996). PAO treatment induced a partial dissociation of GFP-CERT from the TGN in HeLa cells (Figure 1A). Surprisingly, however, PAO treatment also dissociated arfaptin1 and arfaptin2 from the TGN used as controls in this experiment because they lack a typical PH domain for binding to PI(4)P (Figures 1B and C). The localization of β-COP and p230, two Golgi membrane peripheral proteins, was unaffected by PAO treatment (Figure 1B; Supplementary Figure S1). Arfaptins are metazoan-specific proteins that contain a BAR domain (Peter et al, 2004). Arfaptin1 was identified as a class I Arf-binding protein and reported to control Arf-dependent phospholipase D activation (Kanoh et al, 1997; Tsai et al, 1998; Williger et al, 1999). Subsequent studies revealed that arfaptin1 and the related protein arfaptin2 bind to both class I Arfs and Arl1 (Lu et al, 2001; Shin and Exton, 2001; Man et al, 2011). It has also been shown that the knockdown of Arl1 dissociates arfaptins from the TGN in HeLa cells (Man et al, 2011). Importantly, Arl1 is reported to recruit a guanidine nucleotide exchange factor (GEF) for Arf1 and thus activates Arf1 at the TGN (Christis and Munro, 2012). The question then arises whether arfaptins bind directly to Arf1 and Arl1, or to PI(4)P produced by the enzyme PI(4)KIIIβ, which is an effector of Arf1 at the TGN (Godi et al, 1999). Figure 1.Arfaptin1 and 2 bind to PI(4)P-containing membranes. (A) HeLa cells expressing GFP-CERT were treated for 5 min with 0.1% DMSO or 10 μM PAO and then visualized by fluorescence microscopy with anti-p230 antibody. (B) Non-transfected HeLa cells processed as in (A) were visualized with anti-arfaptin1, anti-arfaptin2 and anti-p230 antibodies, respectively. Scale bars, 10 μm. (C) Quantification of the immunofluorescence signal for arfaptin1 and arfaptin2 at the Golgi membranes is shown. Results are shown as the mean±s.e.m. of three experiments. The data were analysed using a paired Student's t-test (*, P<0.01 versus DMSO-treated cells). (D) Recombinant arfaptin1 and arfaptin2 were incubated with PC/PE/Ch liposomes containing or lacking PS or PI(4)P at 10 mol%. Liposomes (T, top fraction) were separated from the unbound proteins (B, bottom fraction) by flotation and analysed by SDS–PAGE followed by staining of the proteins by Coomassie blue.Source data for this figure is available on the online supplementary information page. Source Data for Figure 1D [embj2013116-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint We then tested the binding of arfaptins to phosphoinositides and other phospholipids by using a protein-lipid overlay assay and recombinant non-tagged arfaptin1 and 2. This analysis revealed that arfaptin1 binds to PI(3)P, PI(5)P and to less extent to PI(4)P, phosphatidylserine (PS) and PI(3,5)P2. Arfaptin2, on the other hand, showed a preference for binding to PI(3)P, PI(4)P, PI(5)P and PS (Supplementary Figure S2). It is known that PI(3)P is not contained in the TGN (van Meer et al, 2008). Moreover, little is known about the location and the function of PI(5)P (Grainger et al, 2012). PS is found at the cytoplasmic leaflets of the plasma membrane, TGN and endo-lysosomal compartments (Fairn et al, 2011). However, the arfaptins do not localize to compartments other than the TGN. Based on these reasons, we suggest that arfaptins bind anionic lipids and we provide further test of this binding with specific focus on PI(4)P. To test whether arfaptins can directly bind to PI(4)P-containing membranes, we carried out liposome flotation assays with recombinant arfaptin1 and 2 and liposomes containing or lacking PI(4)P. The amount of arfaptins found in the liposome fraction was barely detectable when the assay was performed with liposomes composed only of phosphatidylcholine (PC)/phosphatidylethanolamine (PE)/cholesterol (Ch) (Figure 1D). The Golgi membranes are known to contain the negatively charged phospholipid PS (van Meer et al, 2008; Fairn et al, 2011), we therefore assayed the binding of arfaptins to liposomes containing 10 mol% PS. Liposomes made of PC/PE/Ch/PS recruited 31 and 34% of arfaptin1 and 2, respectively, from the total amounts of these proteins included in the reaction mixture (Figure 1D). The binding increased to 58% for arfaptin1 and to 74% for arfaptin2 when the assay was performed with liposomes containing 10 mol% PI(4)P instead of PS (Figure 1D). These results indicate that although arfaptin1 and 2 can bind to membranes containing negatively charged phospholipids, such as PS, they bind more efficiently to membranes with PI(4)P. PI(4)P is required for the targeting of arfaptins to the Golgi complex We have taken advantage of a recently developed approach to acutely deplete PI(4)P by the rapamycin-induced recruitment of the Sac1 phosphatase to the Golgi membranes (Szentpetery et al, 2010). COS-7 cells were transfected with plasmids coding for the Golgi-associated TGN38-FRB-CFP recruiter together with the cytosolic mRFP-FKBP12-Sac1 and either GFP-arfaptin1 or GFP-arfaptin2. The localization of the PH domain of OSH1 fused to GFP was used as a control. The Golgi membrane-associated levels of Sac1 and arfaptins/OSH1 were monitored by live-cell confocal microscopy prior to and after adding rapamycin for up to 10 min (Figures 2A–C). The recruitment of Sac1 to the Golgi membranes rapidly dissociated OSH1, without any obvious effect on the localization of the arfaptins (Figures 2D–F). We then tested the requirement of PI(4)P in the recruitment of arfaptins by the following procedure. The cells were treated with brefeldin A (BFA) for 12 min, which is known to prevent Arf1-dependent binding of proteins to the Golgi membranes. Arfaptins dissociated from the TGN under these experimental conditions (Figure 2G). The cells were then washed to remove BFA and incubated in the absence or the presence of rapamycin for 1 h to allow the recruitment of Sac1 to the Golgi membranes. Quantification of the Golgi complex-associated levels of GFP-arfaptin1 and 2 after this procedure revealed that the Sac1 recruitment reduced the amount of both arfaptin1 and 2 that can re-associate with the Golgi membranes after BFA wash-out (Figures 2H and I). It is therefore clear that an Arf or Arf-like GTPase is required for the binding of arfaptins to the Golgi membranes. Importantly, these results reveal that PI(4)P is also necessary for the recruitment of arfaptins to the Golgi membranes. Figure 2.PI(4)P depletion at the Golgi reduces the rate of arfaptin1 and 2 association with the Golgi membranes. (A–F) COS-7 cells co-expressing mRFP-FKBP12-Sac1, TGN38-FRB-CFP and GFP-OSH1-PH (A, D), GFP-arfaptin1 (B, E) or GFP-arfaptin2 (C, F) were imaged by live-cell confocal microscopy and treated with 100 nM rapamycin to induce the recruitment of the cytosolic FKBP12-Sac1 to the Golgi complex. Time-lapse images of individual cells were recorded for 400 s and representative images are shown for GFP-OSH1-PH (A), GFP-arfaptin1 (B) and GFP-arfaptin2 (C) at 0 min (pre-recruitment) and at 1 and 2 or 5 min after recruitment. Graphs represent normalized Golgi fluorescence intensity for Sac1 and OSH1-PH (D) (n=6), arfaptin1 (E) (n=9) or arfaptin2 (F) (n=9) in cells with effective Sac1 recruitment. Values are shown as the mean±s.e.m. (G) COS-7 cells transfected with GFP-arfaptin1 were treated with BFA (5 μg/ml) for 12 min. Confocal microscopy images are shown at 0 min (pre-BFA) and at 5 and 12 min after treatment. (H) Confocal images of COS-7 cells co-expressing mRFP-FKBP12-Sac1, TGN38-FRB-CFP, and either GFP-arfaptin1 (upper panel) or GFP-arfaptin2 (lower panel), treated with BFA for 12 min, washed out and incubated with 100 nM rapamycin for 1 h. (I) Bar graphs depict normalized Golgi fluorescence intensities of arfaptin1 (upper graph) or arfaptin2 (lower graph) for untreated cells (arfaptin1 n=21, arfaptin2 n=18), or cells treated with BFA for 12 min (arfaptin1 n=26, arfaptin2 n=28), washed out and incubated in complete medium with DMSO (arfaptin1 n=23, arfaptin2 n=18) or 100 nM rapamycin (arfaptin1 n=41, arfaptin2 n=37) for 1 h. Values are shown as the mean±s.e.m. The data were analysed using the one-way ANOVA (*, P 85% compared with their levels in HeLa cells transfected with a control siRNA. Co-transfection with both siRNA for arfaptin1 and 2 induced similar knockdown levels as observed with the single transfection (Figure 6A). HeLa cells stably expressing horseradish peroxidase containing a signal sequence (HeLa-ss
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