Fig 1: The differential Golgi targeting mechanism for enzymes during Golgi biogenesis. (A,B) Immunostaining of GM130 and Man-I (A), and giantin and MGAT1 (B) in HeLa cells: control (DMSO-treated), BFA-treated, and BFA-WO for 30 min. White boxes indicate Golgi areas enlarged and shown in three channels on the right side. Nuclei were counterstained with DAPI (blue). All confocal images were acquired with the same imaging parameters; bars, 10 μm. (C,D) Representative 3D SIM imaging of HeLa cells after BFA-WO. Cells were co-stained with GM130 and Man-I (C), and giantin and MGAT1 (D). The Golgi area in the white boxes is enlarged and presented on the right side. The orthogonal views of each area are shown below; bars, 10 μm. (E) Immunostaining of GM130 and Man-I, and giantin and MGAT1 in HeLa cells recovered after BFA for 60 min. (F) Quantification of Pearson’s coefficient of colocalization for indicated proteins in cells presented in C and D; n = 15 cells for each series of SD SIM imaging; results expressed as a mean ± SD; * p < 0.001.
Fig 2: MALDI-IMS of N-glycans from Mgat1 cKO and control testis sections. (A) Top panels. H&E staining of 6 μm sections from paraffin-embedded testes of Mgat1 cKO and control (Mgat1[F/F]) males of 90 dpp (representative of n = 6; scale bar 100 μm). Lower panels: Testis sections treated with PNGase F and subjected to MALDI-IMS. The expression of a particular N-glycan is shown in each panel with its relative intensity scale (representative of two sections from each of three males). Gray sections are before treatment. (B) Profile of Mgat1[F/F] MALDI-IMS spectra (representative of two sections from three males). Inset shows expanded high m/z region. (C) Profile of Mgat1 cKO MALDI-IMS spectra. Inset shows expanded high m/z region. (D) Western blot for basigin (BSG) after Endo H treatment of testis extracts from 90 dpp control and Mgat1 cKO males. N-glycans are drawn with sugar symbols recommended by the Symbol Nomenclature for Glycans (Varki et al., 2015; Neelamegham et al., 2019); GlcNAc, blue square; Man, green circle; Gal, yellow circle; Fuc, red triangle; Sia, red diamond.
Fig 3: S12 phosphorylation impairs MAN1A1 interaction with MGAT1(A) MAN1A1 forms large protein complexes in interphase but not mitotic cells. Interphase (Int.) and mitotic (Mit.) cells expressing MAN1A1-myc were analyzed using western blot with or without denaturing. Note the high molecular weight bands of MAN1A1 in interphase cells without denaturing (lane 2).(B) Endogenous MAN1A1 interacts with MGAT1. HeLa cells were lysed and immunoprecipitated with rabbit IgG or a MGAT1 antibody as indicated followed by western blot of both proteins.(C) Endogenous MGAT1 interacts with MAN1A1-myc.(D) MAN1A1 interacts with MGAT1 in interphase but not mitotic cells. *IgG heavy chain.(E) MAN1A1 interacts with MAN2A1 in both interphase and mitotic cells.(F) MAN1A1 self-interacts in both interphase and mitotic cells.(G) CDK1-mediated mitotic S12 phosphorylation attenuates MAN1A1-MGAT1 interaction. RO-3306, 10 μM for 30 min.(H) S12 mutation of MAN1A1 does not affect MAN1A1-MAN2A1 interaction in interphase.(I) S12 mutation of MAN1A1 does not affect MAN1A1 self-interaction in interphase.(J) A schematic model showing the mitotic regulation of Golgi morphology and MAN1A1 activity by CDK1. Upon mitotic activation, CDK1 phosphorylates Golgi structural and fusion proteins, such as GRASP65, GM130, and VCIP135, leading to mitotic Golgi disassembly. Meanwhile, CDK1 phosphorylates MAN1A1 at S12, which inhibits MAN1A1 interaction with MGAT1 and reduces its α-mannosidase activity.
Fig 4: Rescue of Mgat1 cKO males by the Stra8-Mgat1-HA transgene. (A) H&E staining of testes and epididymi from Mgat1 cKO-Stra8-Mgat1-HA males of 150 dpp (one testis, duplicate sections, eight males). In Mgat1 cKO testis MNCs are apparent. In Mgat1 cKO-Stra8-Mgat1-HA testes MNCs were rare. In both Mgat1 cKO and Mgat1 cKO-Stra8-Mgat1-HA rescue mice, cell debris was present in the epididymal lumen. Scale bar 100 μm. (B) Lectin staining with L-PHA and GSA II in Mgat1 cKO compared to Mgat1 cKO-Stra8-Mgat1-HA testis shows that the transgene rescued lectin binding to complex N-glycans (n = 6, two sections per mouse). Scale bar 100 μm. (C) Western blot analysis for HA and ACTB in testes of non-Tg control (n = 3), six rescue mice from Mgat1 cKO-Stra8-Mgat1-HA and 6 from Mgat1 cKO-Ldhc-Mgat1-HA for expression of the transgene. (D) Western analysis for BSG in testicular germ cells from non-Tg (n = 3) and Mgat1 cKO-Stra8-Mgat1-HA males (n = 3) with and without digestion by Endo H. (E,F) MALDI-IMS of testis sections from Mgat1 cKO control and Mgat1 cKO-Stra8-Mgat1-HA rescue mice showing increased expression of complex N-glycans in the rescue mouse. Glycan symbols are described in the legend to Figure 1.
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% β-mercaptoethanol and resolved by 4–15% SDS-PAGE. Samples were normalized by the total protein concentration.
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