Fig. 1

Zn²⁺ binds to ERp44, affecting its structure and localization. a Updated crystal structure of ERp44 at 2.0 Å resolution (PDB ID: 5GU6). The three Trx-like domains (a, b, and b′) and C-tail are shown in green and magenta, respectively. The inset shows a close-up view of the His-cluster composed of His299, His328, and His332. b ITC raw data (upper) and binding isotherm data (lower) for titration of ZnCl₂ (500 µM) into ERp44 (30 µM) at pH 7.2. Bars represent the errors in the peak integration for each injection estimated by NITPIC⁷². The global analysis was performed to estimate the K_d and ΔH values with SEDPHAT⁷³ assuming 1:1 binding. The apparent K_d is shown with 68.3% confidence interval in brackets. c SEC analysis of ERp44 (60 µM) in the presence (red line) or absence (blue line) of Zn²⁺ ions (120 µM). Peak fractions of the ERp44-ZnCl₂ mixture eluted at 12.5–14.0 mL were analyzed by non-reducing SDS-PAGE (right), suggesting that major portion of ERp44 forms non-covalent homodimers in a Zn²⁺-dependent manner. d ANS fluorescence spectra were measured for ERp44 at pH 7.2 or 6.2 in the absence (left) or presence (right) of ZnCl₂. The higher and blue-shifted fluorescence peaks observed at lower pH and those in the presence of Zn²⁺ suggest the exposure of hydrophobic surfaces in ERp44. Subsequent addition of EDTA to the ERp44-ZnCl₂ mixture returned the fluorescence to the initial level. e Confocal immunofluorescence images showing the intercellular localization of endogenous ERp44 (in green) in HeLa cells. Cells were co-stained with an antibody to GM130 (in red) and with DAPI (in blue) to highlight the Golgi and nucleus, respectively. Note that ERp44 accumulates in the Golgi upon Zn²⁺ deprivation and returns to the ER upon Zn²⁺ replenishment. Scale bars, 10 µm. f Quantitative analyses of the Pearson’s correlation coefficients for the co-localization of endogenous ERp44 with GM130 based on the immunofluorescence images shown in e. Dots indicate individual data points (≥70 cells for each condition). Bars indicate the means ± SD. ****p < 0.0001. g Time-lapse fluorescence imaging of YFP-ERp44 in living cells. HeLa cells transfected with YFP-ERp44 were treated with TPEN for 30 min and then with ZPT or DMSO (vehicle) for 30 min. As described for endogenous ERp44, Zn²⁺ depletion and replenishment alters intracellular localization of YFP-ERp44 in a reversible manner