Fig 1: Interaction of MGAT1 and giantin. (A) PLA in HeLa cells treated with DMSO, BFA, and BFA-WO. The proximity of MGAT1 and giantin was evaluated using mouse anti-MGAT1 and rabbit anti-giantin Abs. The closeness of GM130 and Man-I was examined using mouse anti-GM130 and rabbit anti-Man-I Abs. Red punctae indicate PLA signal, nucleus is in blue, DAPI; bars, 10 μm. (B) Quantitation of proximity ligation for indicated proteins is presented as the corrected total fluorescence intensity (a.u.). The results are measured as a mean ± SD; * p < 0.001. (C) Bottom panel: MGAT1 W-B of the complexes pulled down by anti-giantin Ab from the Golgi fractions isolated from HeLa cells: control, and recovered at 30 and 60 min of BFA-WO. The input of giantin is presented at the top panel. The IP using control rabbit IgG served as a control. (D) Giantin W-B of the lysate or the complex pulled down from the lysates of non-treated HeLa cells using biotinylated hMGAT1 N-terminal peptide and Dynabeads M-280 Streptavidin. Control samples are the Dynabeads incubated with cell lysate in the presence (+) or absence (−) of control peptide. (E) MGAT1 W-B of the complexes pulled down from the lysate of non-treated HeLa cells using giantin-GST N-terminal peptide immobilized by anti-GST Ab coupled epoxy beads. The anti-GST beads exposed to the cell lysate only and IP using control rabbit IgG are the control. (F) The proposed model of Golgi targeting. Enzymes that employ GM130-GRASP65 docking site are able to reach membranes during Golgi biogenesis, contrary to other Golgi resident proteins that use giantin. The targeting of the latter occurs after giantin dimerization and complete restoration of Golgi morphology.
Fig 2: Identification of VPS15 protein complexes.(a) The VPS15 protein complexes are similar in their organization and function in yeast and mammals. (b) Immunoprecipitations with VPS15 or VPS34 antibodies were done on cell lysates from control (ctrl) or patient (II.5) fibroblasts and known VPS15 and VPS34 interaction partners (VPS34, VPS15, UVRAG and ATG14) were detected by western blot. (c) Mass spectrometry data of VPS15 interaction partners were analysed, using the number of spectra found for VPS15 as a reference (100%). The number of spectra found for VPS34, GM130, UVRAG, Beclin1, Nrbf2 and Atg14L in the different samples (ctrl 1 and 2 and patient II.1, II.3 and II.5) was expressed relatively to the number of spectra found for VPS15 and percentages plotted on a histogram.
Fig 3: The overlap of giantin and Rab6a during Golgi biogenesis. (A) Confocal immunofluorescence images of Rab6a in HeLa cells after 60 min of BFA-WO, pretreated with scramble, giantin, GM130, or GRASP65 siRNAs. All confocal images acquired with the same imaging parameters; bars, 10 µm. (B) Quantification of cells with membranous Rab6a in cells presented in (A); n = 90 cells from three independent experiments, results are expressed as a mean ± SD; * p < 0.001. (C) Giantin immunostaining in DMSO- and BFA-treated HeLa cells. (D) Giantin immunostaining in HeLa cells after 60 min of BFA-WO, transfected with scramble, Rab6a siRNAs, and dominant-negative (GDP-bound) Rab6a(T27N). (E) Rab6a W-B of lysates of HeLa cells treated with corresponding siRNAs; ß-actin was a loading control. (F) Quantifications of cells with perinuclear Golgi in cells from (C,D); n = 90 cells from three independent experiments, results expressed as a mean ± SD; *, p < 0.001.
Fig 4: Giantin is necessary for the restoration of compact Golgi upon BFA washout. (A,B) Confocal immunofluorescence images of Golgi were collected in HeLa cells pretreated with different siRNAs for 72 h followed by exposure to 36 μM BFA for 60 min and then WO for 30 min. The combination of GRASP65 + giantin immunostaining was used for cells treated with scramble or giantin siRNAs, and giantin + GM130 and GRASP65 + GM130 were used for cells treated with GM130 and GRASP65 siRNAs, respectively. Images in the white boxes are enlarged and displayed as either green or red channels on the right side. Nuclei counterstained with DAPI (blue); bars, 10 μm. (C) Quantification of cells with perinuclear Golgi, as shown in (A,B); n = 90 cells from three independent experiments of giantin, GM130, and GRASP65 KD performed with two different combinations of target-specific siRNAs. Results are expressed as a mean ± SD; * p < 0.001. (D) Giantin, GM130, and GRASP65 W-B of lysates of HeLa cells treated with the corresponding siRNAs (I and II); β-actin was a loading control.
Fig 5: The disulfide bond in the luminal domain of giantin is critical for its dimerization and Golgi biogenesis. (A,B) Confocal immunofluorescence images of Golgi in HeLa cells transfected with WT giantin tagged with GFP at the C-terminus or with the same construct mutated at Cys3254 to Ser. Cells were stained with GM130 to validate localization in Golgi. (C,D) Immunostaining of MGAT1 (C) or GM130 (D) in HeLa cells overexpressing giantin-WT-GFP or giantin(-C3254S)-GFP and treated with 36 µM BFA for 60 min and then WO for 60 min. Control cells were exposed to the corresponding amount of DMSO. Images were captured using the EVOS M5000 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). Nuclei counterstained with DAPI (blue); bars, 10 µm. (E) Quantification of cells with perinuclear Golgi after BFA-WO for indicated Golgi markers; n = 90 cells from three independent experiments. Results are expressed as a mean ± SD; * p < 0.001. (F) GFP W-B of the GFP IP isolated from the lysates of HeLa cells transfected with giantin-WT-GFP or giantin(-C3254S)-GFP. The lysates were prepared in the presence or absence of 2 mM NEM followed by 5% ß-mercaptoethanol and resolved by 4–15% SDS-PAGE. Samples were normalized by the total protein concentration.
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