Zinc regulates ERp44-dependent protein quality control in the early secretory pathway

Zinc ions (Zn2+) are imported into the early secretory pathway by Golgi-resident transporters, but their handling and functions are not fully understood. Here, we show that Zn2+ binds with high affinity to the pH-sensitive chaperone ERp44, modulating its localization and ability to retrieve clients like Ero1α and ERAP1 to the endoplasmic reticulum (ER). Silencing the Zn2+ transporters that uptake Zn2+ into the Golgi led to ERp44 dysfunction and increased secretion of Ero1α and ERAP1. High-resolution crystal structures of Zn2+-bound ERp44 reveal that Zn2+ binds to a conserved histidine-cluster. The consequent large displacements of the regulatory C-terminal tail expose the substrate-binding surface and RDEL motif, ensuring client capture and retrieval. ERp44 also forms Zn2+-bridged homodimers, which dissociate upon client binding. Histidine mutations in the Zn2+-binding sites compromise ERp44 activity and localization. Our findings reveal a role of Zn2+ as a key regulator of protein quality control at the ER-Golgi interface.

. ZnT5, 6 and 7 knockdown altered subcellular localization of ERp44, but not Ero1α (A) Confocal immunofluorescence images showing the intercellular localization of endogenous ERp44 (in green) in HepG2 cells with ZnT5/6/7 triple knockdown, with respect to untreated cells (siControl). Cells were co-stained for GM130 (in red) and DAPI (in blue). Note that ERp44 accumulates in the Golgi upon ZnT5/6/7 triple knockdown. Scale bar, 10 µm. (B) Quantitative analyses of Pearson's correlation coefficients for the co-localization of endogenous ERp44 with GM130 based on the immunofluorescence images shown in A. Dots indicate individual data points (> 30 cells for each conditions). Bars indicate the means ± SD. ***, p < 0.0001 (C) Subcellular localization of YFP-ERp44 in ZnT5/6/7 triple knockdown HeLa cells. Scale bar, 10 µm. (D) Quantitative analyses of Pearson's correlation coefficients for the co-localization of YFP-ERp44 with GM130 based on the immunofluorescence images shown in C. Dots indicate individual data points (> 30 cells for each conditions). Bars indicate the means ± SD. ****, p < 0.00001 (E-G) RT-PCR analyses confirm the efficiency of ZnT5, ZnT6 and ZnT7 silencing in HeLa cells subline1 (E), subline2 (F) and HepG2 cells (G). Average of 3 independent experiments ± SD. (H) Cells expressing Ero1-Myc and YFP-ERp44 were treated with or without TPEN and ZPT and analyzed by immunofluorescence. Note that most Ero1-Myc remains in the ER whilst YFP-ERp44 accumulates in the Golgi upon Zn 2+ depletion. This is likely due to retention of Ero1-Myc by PDI (Otsu et al., 2006) as well as rapid secretion of Ero1-Myc molecules from the Golgi. Scale bar, 10 µm. (I) ITC raw data (upper) and binding isotherm data (lower) for titration of ZnCl2 (500 µM) into Ero1 (30 µM) at pH 7.0

Supplementary Figure 6. Structure determination of Zn 2+ -bound ERp44
(A) An asymmetric unit contains four ERp44 molecules and a total of ten strong anomalous peaks from the bound Zn 2+ ions. Anomalous difference Fourier map at 10  in the crystallographic asymmetric units are shown in magenta. (B) Initial SAD-phased electron density map in the neighborhood of the His-cluster at 1 . Anomalous difference Fourier map at 15  is also shown in magenta. (C) The overall structure of the Zn 2+ -bound ERp44 homodimer is shown in a putty representation, with the tube radius increasing from low to high B-factor. (D) Comparison of the C-tail conformation. The b' domain (white) and C-tail (magenta) of the four independent ERp44 molecules in the asymmetric unit are shown in a putty representation, with the tube radius increasing from low to high B-factor. (E, F) Close-up views of the conformation of the C-terminus of Mol A (E) and Mol B (F). Dashed lines represent disordered residues. (G) Close-up view of the interactions formed at the Zn 2+ -bound His-cluster (site 1). Coordination to Zn 2+ and van der Waals contacts are represented by yellow and green dashed lines, respectively.

Supplementary
where Xbound(i) is the concentration of Zn 2+ bound to ERp44 after the i-th injection, Mt(i) is the total concentration of ERp44 after the i-th injection, and Xfree(i) is the concentration of free Zn 2+ after the i-th injection. Note that the observed concave downward curves indicate positive cooperativity in Zn 2+ binding to sites 1 and 2. (B) Saturation analysis based on the observed ITC data, where Xfree is plotted against fractional saturation (R(i) =Xbound(i) / Mt(i)). Calculated saturation curves with Hill coefficients are represented by solid lines. (C) SEC analysis of the Zn 2+ -dependent dimerization of ERp44 (60 µM) in the presence of indicated concentrations of Zn 2+ at pH 6.7. (D) ITC raw data (upper) and binding isotherm data (lower) for 100-fold dilution of the mixture of (left) 100µM ERp44 and 200 µM ZnCl2 or (right) 10 µM ERp44 and 20 µM ZnCl2 into buffer (20 mM BisTris pH6.2, 150 mM NaCl).  Figure 11. Zn +2 -bound dimer of ERp44 can dissociate to form the heterodimer with Ero1 SEC-MALS analysis for the mixture of ERp44 (60 µM), ZnCl2 (120 µM) and lastly Ero1 (60 µM) was performed at room temperature. A SEC profile of the mixture is shown by a blue line. Averaged molecular masses calculated by MALS are indicated by red lines for the peak fractions, P2 and P3. Figure 12. Zn 2+ affects the formation of ERp44-Ero1 complexes in cells (A) Pull-down assay for Zn 2+ -dependent complex formation between overexpressed ERp44 and Ero1. HeLa cells co-transfected with Ero1-Myc and YFP-Mock or YFP-ERp44 were treated with 10 µM TPEN for 30 min and then subsequently with 10-50 µM ZPT for 15 min. Cell lysates were subjected to immunoprecipitation with GST-GFP-Nanobody. The right panel shows the level of ERp44-bound Ero1-Myc normalized against the level of input Ero1-Myc. Data are the means ± SEM (N=3, one-way ANOVA followed by Tukey's test). ***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., not significant (p > 0.05). (B) Co-immunoprecipitation of endogenous ERp44-Ero1 complexes. Lysates from HeLa cells were subjected to immunoprecipitation with anti-ERp44 (36C9), and the immunoprecipitates (IP), leftovers (LO) and input lysates (Lys) analyzed by immunoblotting under non-reducing conditions on 4-12% polyacrylamyde gradient gels. After transfer, the nitrocellulose filter was sequentially decorated with a rabbit anti-ERp44 polyclonal antibody followed by and anti-rabbit Alexa 700 secondary antibody, and subsequently with a mouse monoclonal anti-Ero1 antibody (2G4) followed by anti-mouse Alexa 488 antibodies. Clearly, Ero1 is associated with ERp44 non-covalently (see the ~65 kDa band) as well as covalently (see the higher molecular weight bands, stabilized by addition of ZnCl2). The panel on the right shows an overlay of the two signals, that highlights the ERp44-Ero1 covalent complexes (yellow bands). Note that Zn 2+ depletion by TPEN treatment impaired the formation of both covalent and non-covalent ERp44-Ero1 complexes. Note also the slower mobility of intracellular ERp44 in TPEN treated cells (lanes 2 in the right panels), reflecting O-glycosylation in the Golgi. (C) Pull-down assay for detection of the ERp44 homodimer or the ERp44-Ero1 binary complex formed in cells. HeLa cells co-expressing FLAG-tagged ERp44, YFP-ERp44 and Ero1-Myc were treated with DMSO or 2.5 µM ZPT for 15 min. Cell lysates were subjected to pull-down assay using GST-GFP-Nanobody and immunoblotted with indicated antibodies. The signal intensity of coprecipitated FLAG-ERp44 relative to that of whole FLAG-ERp44 in cell lysates was quantified, and the data are shown in the right graph. Data are the means ± SEM (N=3).

Supplementary Figure 13. Existence of Zn 2+ -bridged homodimers of ERp44 in cells (A) Flowcharts of sequential elution assays shown in panels B and C. (B) Presence of Zn 2+ -dependent and covalent homodimers (oligomers) in living cells. HepG2 cells
were transfected with Halo-ERp44 WT or 3HA, or an ER resident Halo alone (-) as a control, and lysed and handled in solutions supplemented with ZnCl2. Aliquots of post-nuclear supernatants corresponding to 1 mg of total protein were incubated with immobilized Halo ligands. Beads were washed several times before the first elution with TPEN, washed again and then eluted with DTT and SDS (lane 4-15). Aliquots of total lysates from Halo or WT Halo-ERp44 expressing cells (40 or 10 µg) were analyzed to obtain an indicative quantification of endogenous ERp44 (lane 1-2). Aliquots (25 µg) of the lysates before and after precipitation with the Halo-ligand beads were decorated with anti-Halo to validate precipitation (lanes 16-21). (C) HepG2 cells co-transfected with Halo-ERp44 and HA-ERp44 (both WT or both 3HA) were handled as above, using buffers without (left panel) or with (right panel) added ZnCl2. Clearly, the presence of Zn 2+ stabilizes the non-covalent complexes involving Halo-ERp44 WT. Importantly, however, significant portion of Halo-and HA-ERp44 was co-precipitated also even without added ZnCl2. In contrast, little if any Halo-or HA-ERp44 3HA is eluted by TPEN from beads covered with the 3HA mutants.
(D) Flowchart of the SEC assay shown in panel E. (E) SEC analysis for the microsomal lysate of HEK293T cells. Each fraction was analyzed by nonreducing immunoblotting with D17A6 anti-ERp44 antibodies. Arrows indicate a possible noncovalent homodimer of endogenous ERp44. The lysate was treated with TPEN (middle) and TCEP (right), respectively.

Supplementary Figure 14. ERp44 dimerization is not essential for exit from the ER to the Golgi.
HeLa cells transfected with YFP-ERp44 (WT or H277/281A) or YFP-Mock were treated with 10 µM TPEN/Opti-MEM for 2 h and fixed with 4% PFA/PBS. Nuclei were stained with DAPI. Fluorescent images were obtained with laser scanning confocal microscopy. Scale bars, 10 µm. Like ERp44 (WT), the dimerization-defective mutant (H277/281A) accumulates in the Golgi area after TPEN treatment. Figure 15. Structural rationalization for the sequential Zn 2+ binding to ERp44. Superposition of metal-free monomer and Zn 2+ -bound dimer of ERp44. The b' domains of these two states are superimposed to each other. The structure of Zn 2+ -free ERp44 monomer is represented by a ribbon diagram and colored in green (a domain), yellow (b domain), blue (b' domain) and magenta (C-tail). For the Zn 2+ -bound dimer, one protomer is shown in white, while the other is represented by surface model and colored in the same way as indicated above.