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The Phosphatidylinositol 4-Kinase PI4KIII Is Required for the Recruitment of GBF1 to Golgi Membranes

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The Phosphatidylinositol 4-Kinase PI4KIII Is Required for the Recruitment of GBF1 to Golgi Membranes Research Article 2273 The phosphatidylinositol 4-kinase PI4KIII is required for the recruitment of GBF1 to Golgi membranes Karine Dumaresq-Doiron1, Marie-France Savard1, Salima Akam1, Santiago Costantino2 and Stephane Lefrancois1,* 1Centre de Recherche de l’Hôpital Maisonneuve-Rosemont et Département de Médecine, Université de Montréal, Montréal, Québec H1T 2M4, Canada 2Centre de Recherche de l’Hôpital Maisonneuve-Rosemont et Département d’Ophtalmologie et Institut de Génie Biomédicale, Université de Montréal, Montréal, Québec H1T 2M4, Canada *Author for correspondence ([email protected]) Accepted 7 April 2010 Journal of Cell Science 123, 2273-2280 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jcs.055798 Summary Sorting from the Golgi apparatus requires the recruitment of cytosolic coat proteins to package cargo into trafficking vesicles. An important early step in the formation of trafficking vesicles is the activation of Arf1 by the guanine nucleotide exchange factor GBF1. To activate Arf1, GBF1 must be recruited to and bound to Golgi membranes, a process that requires Rab1b. However, the mechanistic details of how Rab1 is implicated in GBF1 recruitment are not known. In this study, we demonstrate that the recruitment of GBF1 also requires phosphatidylinositol 4-phosphate [PtdIns(4)P]. Inhibitors of PtdIns(4)P synthesis or depletion of PI4KIII, a phosphatidylinositol 4-kinase localized to the endoplasmic reticulum and Golgi, prevents the recruitment of GBF1 to Golgi membranes. Interestingly, transfection of dominant-active Rab1 increased the amount of PtdIns(4)P at the Golgi, as detected by GFP-PH, a PtdIns(4)P-sensing probe. We propose that Rab1 contributes to the specificity and timing of GBF1 recruitment by activating PI4KIII. The PtdIns(4)P produced then allows GBF1 to bind to Golgi membranes and activate Arf1. Key words: GBF1, GGAs, Golgi apparatus, Phosphatidylinositol 4-phosphate, Rab1 Introduction in membrane trafficking (Lefrancois and McCormick, 2007; The intracellular sorting and trafficking of proteins is necessary to Manolea et al., 2008; Shinotsuka et al., 2002). Arf1 is involved in establish and maintain organelle function. The Golgi apparatus is the recruitment of COPI (Dascher and Balch, 1994), the Golgi- responsible for efficiently sorting and trafficking proteins to various localized -ear-containing, Arf-binding proteins (GGAs) and Journal of Cell Science cellular destinations, including the lysosomal compartment and adaptor protein (AP)-1 to Golgi membranes (Dell’Angelica et al., plasma membrane. The Golgi accomplishes this function by 2000a; Traub et al., 1993), and AP-3 to endosomal membranes packaging cargo destined for various destinations into trafficking (Ooi et al., 1998). The specificity of this recruitment depends on vesicles that are formed by the recruitment of various cytosolic the GEFs; recent evidence has shown that BIG2 is involved in the components, a process that cells tightly regulate (Bonifacino and recruitment of AP-1 (Shinotsuka et al., 2002), whereas GBF1 is Lippincott-Schwartz, 2003). ADP ribosylation factors (Arfs) are required for the recruitment of COPI (Alvarez et al., 2003; Deng required for the recruitment of proteins to various cellular et al., 2009) and the GGAs to Golgi membranes (Lefrancois and membranes, including the Golgi (Dell’Angelica et al., 2000b), McCormick, 2007). The interaction of GBF1 with membranes of endosomes (Ooi et al., 1998) and plasma membrane (Cohen et al., the Golgi is required for the conversion of Arf1-GDP to Arf1-GTP 2007). To initiate recruitment, Arfs must be converted from a (Niu et al., 2005). Although it has been shown that the small G GDP-bound to a GTP-bound state by a guanine nucleotide exchange protein Rab1 is involved in the recruitment of GBF1 (Monetta et factor (GEF) (Jackson and Casanova, 2000), whereas the GTPase- al., 2007), the mechanism of GBF1 interaction with Golgi activating proteins (GAPs) induce hydrolysis of GTP to GDP to membranes has not been elucidated, as efforts to identify a protein deactivate Arfs (Nie et al., 2003). The GEFs are a family of receptor for GBF1 on Golgi membranes have been unsuccessful. proteins that are thought to play a crucial role in regulating the Interestingly, the recruitment of ARNO to the plasma membrane specificity, location and timing of recruitment of proteins to requires Arf6 and the phosphoinositide phosphatidylinositol membranes. The ArfGEF family was originally identified in (4,5)-bisphosphate [PtdIns(4,5)P2] (Cohen et al., 2007). Saccharomyces cerevisiae as the Sec7 protein. Sec7 is common to Phosphoinositides are generated by various kinases that esterify a all members of this family and acts as the catalytic domain of the hydroxyl group on phosphatidylinositol with a phosphate at the 3, ArfGEFs, responsible for the activation of Arf (Jackson and 4 or 5 position (in any combination) of the inositol ring (Balla and Casanova, 2000). In mammalian cells, the smaller family members, Balla, 2006). We hypothesized that the recruitment of GBF1 to such as ARF nucleotide-binding site opener (ARNO), play a role Golgi membranes, which requires Rab1, might also require a in membrane signal transduction from the plasma membrane phosphoinositide. Because PtdIns(4)P is a predominant (Cohen et al., 2007), whereas the larger members of the family, phosphoinositide in the Golgi (Balla and Balla, 2006), we tested such as Golgi-specific brefeldin A resistance guanine nucleotide our hypothesis using inhibitors of PtdIns(4)P synthesis – exchange factor 1 (GBF1) and brefeldin-A-inhibited guanine wortmannin and phenylarsine oxide (PAO). To determine nucleotide exchange proteins 1 and 2 (BIG1 and BIG2), play a role specificity, we used small interfering RNA (siRNA) to deplete 2274 Journal of Cell Science 123 (13) PI4KIII, a phosphatidylinositol 4-kinase (PI4K) localized to the endoplasmic reticulum and early Golgi, which is sensitive to these inhibitors (Balla and Balla, 2006). In this paper, we report that the recruitment of GBF1 is mediated by PtdIns(4)P and identify PI4KIII as the enzyme required for this recruitment. Moreover, we provide evidence that the role of Rab1 in the recruitment of GBF1 is to activate the production of PtdIns(4)P. Results PtdIns(4)P is required for the recruitment of GBF1 to Golgi membranes To test whether PtdIns(4)P plays a role in the recruitment of GBF1 to Golgi membranes, we treated cells with DMSO, 5 M LY294002, 5 M wortmannin or 5 M PAO for 10 minutes. LY294002 is a potent inhibitor of phosphoinositide 3-kinases (PI3Ks), with an IC50 (half-maximal inhibitory concentration) of 1.4 M; wortmannin is an inhibitor of PI3Ks and type III PI4Ks, with an IC50 of 50-300 nM; PAO is an inhibitor of type III PI4Ks, with greater specificity for PI4KIII (IC50 of 1-5 M) than for PI4KIII (IC50 of 30 M). Following the inhibitor treatment, we performed immunofluorescence staining of the cells with the Golgi marker giantin to verify whether the inhibitors affected early Golgi morphology. This was necessary to ensure that any differences observed in GBF1 immunofluorescence staining were not due to disturbed Golgi morphology. Cells treated with DMSO and any of the three inhibitors showed strong perinuclear staining for giantin (Fig. 1A,E,I,M), suggesting that the Golgi was intact in the treated cells. To examine whether the inhibitors had an effect on the recruitment of GBF1, we stained cells for endogenous GBF1. Cells treated with either DMSO (Fig. 1B, arrow) or LY294002 (Fig. 1F, Fig. 1. PtdIns(4)P inhibitors block the recruitment of GBF1 to Golgi arrow) had a strong perinuclear staining pattern. However, in cells membranes. COS-7 cells grown on coverslips overnight were treated with treated with wortmannin (Fig. 1J, asterisk) and PAO (Fig. 1N, (A-D) DMSO, (E-H) LY294002, (I-L) wortmannin or (M-P) PAO for 10 asterisk), we found a diffuse staining pattern that lacked strong minutes and fixed in 4% paraformaldehyde. Immunofluorescence staining for perinuclear staining, suggesting that PtdIns(4)P was necessary for giantin (A,E,I,M), GBF1 (B,F,J,N), GGA3 (C,G,K,O) or AP-1 (D,H,L,P) was Journal of Cell Science the recruitment of GBF1 to Golgi membranes. Because we have then performed. Arrows in panels demonstrate the normal staining pattern of previously shown that the recruitment of the GGAs requires GBF1 GBF1 (B,F) and GGA3 (C,G) in mock- or LY294002-treated cells, whereas (Lefrancois and McCormick, 2007), we stained cells for asterisks show the diminished staining pattern of GBF1 (J,N) and GGA3 endogenous GGA3 to examine the effect of blocking the (K,O) in wortmannin- or PAO-treated cells. Scale bar: 10m. (Q)Western blot recruitment of GBF1 on GGA3 recruitment to Golgi membranes. (Wb) analysis of the absolute amount of GBF1, GGA3, AP-1 and actin in cells treated with the inhibitors as indicated. We found that wortmannin (Fig. 1K, asterisk) and PAO (Fig. 1O, asterisk) blocked the recruitment of GGA3, because we found a diffuse staining pattern compared with a strong perinuclear staining localized in DMSO-treated cells, but did not in cells treated with pattern in cells treated with DMSO (Fig. 1C, arrow) and LY294002 wortmannin (supplementary material Fig. S1D-F) or PAO (Fig. 1G, arrow). To verify that the inhibitors were not adversely (supplementary material Fig. S1G-I), because GBF1 was not affecting the recruitment of proteins to the Golgi in general, we recruited to Golgi membranes even though giantin staining seemed stained the cells for AP-1, a cytosolic protein complex recruited to normal. To ensure that the inhibitors were not causing the the trans-Golgi (Zhu et al., 1998). We found no difference in the degradation of GBF1, GGA3 and AP-1, we performed a western staining pattern of AP-1 at the Golgi in the cells treated with the blot analysis to compare the absolute amount of these proteins in inhibitors (Fig.
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