Figure 5. Golgi pH is elevated in Ube3a knockdown cells and in UBE3A^m−/p+ neurons.

(A–E) Golgi pH is elevated in Ube3a knockdown cells. (A) Schematic illustrating the method used to measure Golgi pH. YFP and CFP fluorophores are directed to the lumen of the GAusing the Golgi targeting domain ofβ-1,4-galactosyltransferase. YFP fluorescence increases as pH increases, while CFP fluorescence is pH stable. (B–C) Calibration of pGolgi-YFP:pGolgi-CFP ratios to absolute pH (see Methods for details). (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 (Ctrl) and Ube3a knockdown (KD) cells. Note the elevated pGolgi-YFP intensity in Ube3a KD cells compared to 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. AGolgi pH probe with tandem CFP and YFP fluorophores (pGolgi-CFP-YFP) was expressed in cortical neurons cultured from wildtype (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. Note the elevated YFP intensity in the UBE3A^m−/p+ neuron compared to the wildtype 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. Note the moderately elevated YFP intensity in the UBE3A^m−/p+ neuron compared to the wildtype 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.