The Sec7 guanine nucleotide exchange factor GBF1 regulates membrane recruitment of BIG1 and BIG2 guanine nucleotide exchange factors to the trans-Golgi network (TGN) - PubMed (original) (raw)
The Sec7 guanine nucleotide exchange factor GBF1 regulates membrane recruitment of BIG1 and BIG2 guanine nucleotide exchange factors to the trans-Golgi network (TGN)
Jason Lowery et al. J Biol Chem. 2013.
Abstract
Three Sec7 guanine nucleotide exchange factors (GEFs) activate ADP-ribosylation factors (ARFs) to facilitate coating of transport vesicles within the secretory and endosomal pathways. GBF1 recruits COPI to pre-Golgi and Golgi compartments, whereas BIG1 and BIG2 recruit AP1 and GGA clathrin adaptors to the trans-Golgi network (TGN) and endosomes. Here, we report a functional cascade between these GEFs by showing that GBF1-activated ARFs (ARF4 and ARF5, but not ARF3) facilitate BIG1 and BIG2 recruitment to the TGN. We localize GBF1 ultrastructurally to the pre-Golgi, the Golgi, and also the TGN. Our findings suggest a model in which GBF1 localized within pre-Golgi and Golgi compartments mediates ARF activation to facilitate recruitment of COPI to membranes, whereas GBF1 localized at the TGN mediates ARF activation that leads to the recruitment of BIG1 and BIG2 to the TGN. Membrane-associated BIG1/2 then activates ARFs that recruit clathrin adaptors. In this cascade, an early acting GEF (GBF1) activates ARFs that mediate recruitment of late acting GEFs (BIG1/2) to coordinate coating events within the pre-Golgi/Golgi/TGN continuum. Such coordination may optimize the efficiency and/or selectivity of cargo trafficking through the compartments of the secretory pathway.
Figures
FIGURE 1.
GBF1 depletion or inactivation causes the dissociation of AP1, GGA2, and GGA3 from cellular membranes. A and B, HeLa cells were transfected with siRNA oligos directed against GBF1, incubated for 72 h, and stained with indicated antibodies. A, GBF1 depletion blocks recruitment of β-COP, and the GGA2 and AP1 adaptors. B, GBF1 depletion causes fragmentation of the TGN but does not block recruitment of Golgin-97 and Golgin-245. C, HeLa cells were transfected with GBF1/E794K, incubated for 24 h, and stained with indicated antibodies. Expression of GBF1/E794K blocks membrane recruitment of β-COP and GGA2 and causes fragmentation of the TGN but does not block ARL-dependent recruitment of golgin-97 and golgin-245. D, HeLa cells were treated with NO alone or with NO and GCA, fixed, and stained with indicated antibodies. NO causes fragmentation of the Golgi and the TGN without affecting membrane recruitment of β-COP, GGA3, AP1, and Golgin-245. GCA causes the release of β-COP, GGA3, and AP1 from membranes without affecting the recruitment of Golgin-245. GBF1-depleted cells are outlined. Bars are 10 mm.
FIGURE 2.
GBF1 depletion or inactivation causes dispersal of TGN46, MNK, and M6PR. A, HeLa cells were transfected with siRNA oligos directed against GBF1, incubated for 72 h, and stained with indicated antibodies. GBF1 depletion causes dispersion of MNK and TGN46. B–E, HeLa cells were mock-treated, treated with GCA alone, or treated with NO and GCA, fixed, and stained with the indicated antibodies. GCA causes complete dispersal of MNK, TGN46 and M6PR. Dispersal was limited in the presence of NO. GCA causes the dispersal of TGN46 while maintaining TGN elements containing golgin-245. Bars are 10 mm.
FIGURE 3.
GBF1 depletion or inactivation blocks recruitment of BIG1 and BIG2 to membranes. A–D, HeLa cells were transfected with either control siRNA or siRNA oligos directed against GBF1, BIG1, or BIG2 for 72 h. A, control and depleted cells were lysed, and the lysates were Western-blotted using the indicated antibodies. Western blots show specific depletion of GBF1, BIG1, and BIG2. Golgin-97 and calreticulin are shown as loading controls. B–D, control and depleted cells were fixed and stained with the indicated antibodies. Depletion of GBF1 blocks recruitment of BIG1 and BIG2 to membranes. Depletion of BIG1 or BIG2 has no effect on localization of GBF1. BIG2 depletion leads to partial nuclear relocation of BIG1. Simultaneous depletion of BIG1 and BIG2 has no effect on recruitment of GBF1 to membranes. E, HeLa cells were transfected with GFP-tagged wild-type GBF1 (GBF1) or GBF1/E794K (E794K). After 24 h, cells were fixed and stained with the indicated antibodies. Expression of wild-type GBF1 has no effect on BIG1 and BIG2 recruitment to perinuclear elements. In contrast, expression of GBF1/E794K caused dissociation and dispersal of BIG1 and BIG2. Bars are 10 mm.
FIGURE 4.
Effect of BFA and GCA on membrane association of BIG1 and BIG2. A–C, HeLa cells were treated with NO alone, with NO and BFA, or with NO and GCA, fixed, and stained with indicated antibodies. NO caused fragmentation of the Golgi and TGN. BIG1 and BIG2 remained on membranes in the presence of BFA but dissociated from membranes in the presence of GCA. In contrast, GBF1 remained on membranes in GCA-treated cells. D and E, HeLa cells were mock-treated with ethanol (ETOH) or with BFA or GCA and fractionated into post-nuclear supernatant (PNS), which was subsequently separated into supernatant containing cytosolic proteins (S) and membrane pellet (P). Equivalent amounts of cytosol and pellet fractions were analyzed by SDS-PAGE and blotted with indicated antibodies. In control cells, GBF1 and BIG2 are largely cytosolic. In the presence of BFA, both GBF1 and BIG2 translocated to membranes. In contrast, GCA caused translocation of GBF1 to membranes but caused dissociation of BIG2 from membranes.
FIGURE 5.
Active ARF4 and ARF5 mediate membrane recruitment of BIG1 and BIG2. A—C, HeLa cells were transfected with HA-tagged dominant active (Q71L) forms of ARF3 (A), ARF4 (B), and ARF5 (C) for 24 h. Cells were then treated with GCA, fixed, and stained with the indicated antibodies. Transfection with activated ARF4 and ARF5 cause association of BIG1 and BIG2 with membranes in GCA-treated cells. Expression of ARF3 does not rescue GCA-mediated dissociation of BIG1 and BIG2 from membranes. Bars are 10 mm. D, HeLa cells were transfected with HA-tagged inactive (T31N) or constitutively active (Q71L) forms of ARF1, ARF3, ARF4, and ARF5 for 24 h, and BIG2 was immunoprecipitated from cell lysates. The starting lysate (input) and the bound material were immunoblotted with anti-HA antibodies. Significant levels of active ARF4 and ARF5 but not ARF1 or ARF3 bind to BIG2. E, structural motifs within BIG2 and the BIG2/1–548 and the BIG2/1–250 constructs are shown. HDS, homology downstream of Sec7. HeLa cells were transfected with HA-tagged full-length BIG2 or BIG2/1–548 for 24 h, fixed, and stained with anti-HA antibodies. BIG2/1–548 targeted to the TGN. F, HeLa cells were transfected with HA-tagged constitutively active (Q71L) forms of ARF1, ARF3, ARF4, and ARF5 for 24 h and lysed. Lysates were incubated with purified GST or GST chimeras containing DCB+HUS (amino acids 1–548) or DCB (amino acids 1–250) domains of BIG2. The starting lysate (input) and the bound material were immunoblotted with anti-HA antibodies. Active ARF4 and ARF5, but not ARF1 or ARF3, bound to the DCB+HUS and the DCB only domain but not to GST.
FIGURE 6.
Localization of GBF1 within the secretory pathway. A–C, NRK cells were labeled with anti-GBF1 (small gold (G)) (A) and either anti-GM130 (B) or anti-clathrin (C) antibodies (large gold). GBF1 was detected throughout the Golgi stack and localized to early compartments as defined by the _cis_-Golgi marker GM130 and to late compartments defined by the TGN marker clathrin. Quantitation of GBF1 distribution is presented in Table 1. D, shown is a model for GBF1 function in the secretory pathway. In pre-Golgi, Golgi, and the TGN GBF1 catalyzes GDP/GTP exchange on ARF4/ARF5. Activation of these ARFs can be inhibited by GBF1 siRNA, expression of GBF1/E794K, BFA, or GCA. At the pre-Golgi and Golgi the activated ARFs regulate recruitment of COPI to pre-Golgi and Golgi membranes. At the TGN, the activated ARFs mediate recruitment of BIG1 and BIG2 to TGN membranes. Membrane-associated BIG1 and BIG2 then activate their substrates ARF1/ARF3, resulting in membrane recruitment of TGN coats, such as AP1 and the GGAs. We propose that GBF1 acts as a master regulator of COPI and clathrin-mediated coating events in the secretory pathway.
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