The Angelman Syndrome Protein Ube3a/E6AP Is Required for Golgi Acidification and Surface Protein Sialylation

Gross alteration of organellar ultrastructure is limited to the GA and to tissues lacking Ube3a. A, Comparable morphologies of the peripheral ER (e) and nuclear envelope (n) in visual cortical neurons from WT, UBE3A^m−/p+, and UBE3A^−/− mice. Scale bars, 500 nm. B, Immunoblot analysis of Ube3a expression in brain and kidney of WT, UBE3A^m−/p+, and UBE3A^−/− mice. C, Electron micrographs of GA in WT, UBE3A^m−/p+, and UBE3A^−/− kidney. There is GA swelling in UBE3A^−/− kidney (asterisk) and unaltered GA morphology in UBE3A^m−/p+ kidney. Scale bars, 500 nm.

Ube3a KD disrupts Golgi morphology. A, Knockdown of endogenous Ube3a in clone 9 cells stably expressing Ube3a shRNA. NT, Nontransfected cells; KD, KD cells stably expressing shRNA against Ube3a; Ctrl, cells stably expressing a nontargeting scrambled shRNA. B, Quantification of Ube3a levels in scrambled control (Ctrl) and Ube3a KD cells, clones KD and KD2, relative to nontransfected (NT) clone 9 cells. **p ≤ 0.001. ns, Not significant (p = 0.30). C, D, Electron micrographs of GA in (C) control cells stably expressing scrambled shRNA and (D) stable Ube3a KD cells clones KD and KD2. The GA in control cells have narrow, elongated cisternae arranged in a characteristic stacked structure (white arrows). In contrast, Golgi cisternae in Ube3a KD cells are swollen (asterisks) and disorganized. Scale bars, 500 nm.

Secretory trafficking of VSVGts-GFP and VAMP2-SEP is not disrupted by loss of Ube3a. A–C, VSVGts-GFP progression from the ER to the plasma membrane through the GA was monitored after temperature-induced ER release (see Materials and Methods). VSVGts-GFP distribution and immunoreactivity at the plasma membrane (Surface VSVG) in (A) control and (B) Ube3a KD cells at 0, 30, and 60 min after release from ER-exit blockade. Scale bars, 10 μm. C, Quantification of surface VSVGts-GFP, measured by surface antibody staining, relative to total VSVGts-GFP, measured by intrinsic GFP fluorescence. No significant differences were evident in VSVG surface accumulation between control cells and Ube3a KD cells. Scale bars, 10 μm. p > 0.2 at all time points. D–H, Cargo transport from the GA to the plasma membrane was measured in UBE3A^m−/p+ and WT cortical neurons by monitoring VAMP2-SEP exocytic events in dendrites. D, Schematic of VAMP2-SEP trafficking. Upon leaving the GA, newly synthesized VAMP2-SEP is trafficked into dendrites (1), where it is exocytosed at the plasma membrane (2). VAMP2-SEP is then rapidly endocytosed (3) and retrafficked to the axon, where it accumulates at axonal terminals (4). VAMP2-SEP fluoresces at neutral pH levels and is quenched at the acidic pH levels of intracellular organelles. E, Time-lapse projections (6.5 min) of WT and UBE3A^m−/p+ neuronal dendrites expressing VAMP2-SEP. Arrowheads indicate exocytic events that occurred during the time-lapse. Scale bar, 10 μm. F, Time-lapse montage of selected exocytic events (arrowheads) from dendrites in E, one image per 5 s, showing the appearance and disappearance of VAMP2-SEP on the dendritic surface over time. Scale bar, 5 μm. G, Quantification of exocytic event rate (N_events × min⁻¹ × μm⁻¹ × 100) in WT and UBE3A^m−/p+ neurons; p = 0.28. H, Demonstration of the pH-sensitive properties of VAMP2-SEP in WT and UBE3A^m−/p+ dendrites. There is weak diffuse VAMP2-SEP fluorescence under basal conditions and the much brighter, punctate VAMP2-SEP fluorescence after NH₄Cl neutralizes pH in intracellular compartments. Scale bar, 10 μm.

Golgi pH is elevated in Ube3a KD cells and in UBE3A^m−/p+ neurons. A–E, Golgi pH is elevated in Ube3a KD cells. A, Schematic illustrating the method used to measure Golgi pH. YFP and CFP fluorophores are directed to the lumen of the GA using the Golgi targeting domain of β-1,4-galactosyltransferase. YFP fluorescence increases as pH increases, whereas CFP fluorescence is pH stable. B, C, Calibration of pGolgi-YFP:pGolgi-CFP ratios to absolute pH (see Materials and Methods). B, Quantification of pGolgi-YFP and pGolgi-CFP fluorescence (arbitrary units) at various defined pH values documenting their distinct pH sensitivities. C, Calibration curve (pGolgi-YFP:pGolgi-CFP fluorescence) used to calculate pH values. pH = 0.53 × YFP:CFP + 4.14, R² = 0.9. D, pGolgi-YFP and pGolgi-CFP fluorescence in control and Ube3a KD cells. There is elevated pGolgi-YFP intensity in Ube3a KD cells compared with control cells. Scale bar, 10 μm. E, Quantification of Golgi pH in control (Ctrl) and Ube3a KD cells. **p < 0.001. F–H, Golgi pH is elevated in UBE3A^m−/p+ neurons. F, Schematic of the probe used to measure Golgi pH in cultured cortical neurons. A Golgi pH probe with tandem CFP and YFP fluorophores (pGolgi-CFP-YFP) was expressed in cortical neurons cultured from WT and UBE3A^m−/p+ mice. G, pGolgi-CFP-YFP fluorescence in WT and UBE3A^m−/p+ neurons. White circles outline the nuclear region for orientation. There is elevated YFP intensity in the UBE3A^m−/p+ neuron compared with the WT neuron. Scale bar, 10 μm. H, Average YFP:CFP fluorescence ratios of pGolgi-CFP-YFP in UBE3A^m−/p+ neurons normalized to values in WT. **p = 0.001. I–K, ER pH is modestly elevated in UBE3A^m−/p+ neurons. I, Schematic of the probe used to measure intralumenal ER pH. pER-CFP-YFP is a soluble protein targeted to the ER lumen by its N-terminal calreticulin signal sequence and C-terminal KDEL retrieval sequence. J, pER-CFP-YFP fluorescence in WT and UBE3A^m−/p+ neurons. There is moderately elevated YFP intensity in the UBE3A^m−/p+ neuron compared with the WT neuron. Scale bar, 10 μm. K, Average YFP:CFP fluorescence ratios of pER-CFP-YFP in UBE3A^m−/p+ neurons after normalization to values in WT. *p < 0.01.

Loss of surface protein sialylation in cells lacking Ube3a. A–E, Loss of α-2,6-sialic acid conjugates in Ube3a KD cells. A, Schematic illustrating labeling of α-2,6 linked sialic acid conjugates with SNA-fluorescein. GlcNAc, N-acetylglucosamine; Fluor, fluorescein. B, Surface labeling of α-2,6-sialic acid conjugates in control cells (cell membranes indicated with white arrowheads) and Ube3a KD cells using SNA-fluorescein. Pretreatment with neuraminidase abolished SNA labeling. Scale bar, 10 μm. C, Quantification of surface bound SNA-fluorescein on scrambled shRNA control (Ctrl) and Ube3a KD cells. **p < 0.001. D, Total cellular lysates from control and Ube3a KD cells after SDS-PAGE and blotting with SNA-HRP. Molecular mass markers are shown. E, Corresponding average integrated SNA-HRP signal demonstrating reduced sialylation in Ube3a KD cells. **p < 0.001. F–H, Reduction in total protein sialylation in Ube3a KD cells revealed by metabolic labeling. F, Schematic illustrating metabolic labeling and isolation of sialylated proteins using covalent FLAG-phosphine chemistry (see Materials and Methods). ManNAz, a synthetic azide derivative precursor to sialic acid (SA), is incorporated into endogenous glycans (F1), allowing reaction and covalent tagging with FLAG-phosphine (F2), and immunoprecipitation with an anti-FLAG antibody (F3). G, FLAG immunoreactivity after SDS-PAGE and immunoblotting of immunoprecipitates from control (Ctrl) and Ube3a KD cells with (+) or without (−) exposure to ManNAz. Molecular mass markers are shown. There are reduced levels of high molecular mass sialylconjugates in Ube3a KD cells and an absence of corresponding molecular species without prior metabolic labeling. The dark band at ∼55 kDa is the IgG band from the immunoprecipitation. Species <50 kDa are nonspecific as they are present in the absence of ManNAz. H, Average FLAG immunoreactivity (>60 kDa) in immunoblots, such as shown in G, and from samples exposed to ManNAz and treated with neuraminidase before FLAG-phosphine, defining background levels. There is reduction of protein sialylation in Ube3a KD cells. p > 0.3 for Ube3a KD versus negative controls (ANOVA). *p < 0.05 for Ube3a KD versus control.