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

Satoshi Watanabe,Ilaria Sorrentino,Manami Harayama,Tiziana Tempio,Tiziana Anelli,Sara Sannino,Kenji Inaba,Roberto Sitia,Yuta Amagai,Shoji Masui,Momo Yamada

doi:10.1038/s41467-019-08429-1

Abstract

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.

Highlights

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)
When a mixture of ERp44 (60 μM) and ZnCl2 (120 μM) was loaded onto a size exclusion chromatography (SEC) column, the majority of ERp44 was eluted as non-covalent homodimers (Fig. 1c) with a molecular mass of ~80 kDa, as determined by SEC combined with multi-angle light scattering (SEC-MALS) analyses (Supplementary Fig. 1C)
The present studies reveal that Zn2+ regulates the structure, function and subcellular localization of ERp44, providing a Zn2 +-dependent mechanism regulating protein quality control in the early secretory pathway (ESP)

Summary

Introduction

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). Our findings reveal a role of Zn2+ as a key regulator of protein quality control at the ER-Golgi interface. The abundance and localization of ZnTs and ZIPs in the early secretory pathway (ESP) are consistent with the fundamental role of Zn2+ in regulating the structure and function of many secretory proteins. The high-resolution crystal structures of ERp44 at pH 6.5 and 7.234 revealed that protonation of key histidine and cysteine residues induces pHdependent conformational changes in ERp44, promoting the exposure of the positively charged client-binding site and allowing the essential cysteine (Cys29) to form intermolecular disulfide bonds with clients of thiol-mediated quality control[35,36]. Our findings identify a Zn2+-dependent mechanism of protein quality control at the ER–Golgi interface, revealing yet another physiological role for this essential metal ion in the secretory pathway

Methods

Results

Conclusion