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.

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Many secretory enzymes acquire essential zinc ions (Zn2+) in the Golgi complex. ERp44, a chaperone operating in the early secretory pathway, also binds Zn2+ to regulate its client binding and release for the control of protein traffic and homeostasis. Notably, three membrane transporter complexes, ZnT4, ZnT5/ZnT6 and ZnT7, import Zn2+ into the Golgi lumen in exchange with protons. To identify their specific roles, we here perform quantitative Zn2+ imaging using super-resolution microscopy and Zn2+-probes targeted in specific Golgi subregions. Systematic ZnT-knockdowns reveal that ZnT4, ZnT5/ZnT6 and ZnT7 regulate labile Zn2+ concentration at the distal, medial, and proximal Golgi, respectively, consistent with their localization. Time-course imaging of cells undergoing synchronized secretory protein traffic and functional assays demonstrates that ZnT-mediated Zn2+ fluxes tune the localization, trafficking, and client-retrieval activity of ERp44. Altogether, this study provides deep mechanistic insights into how ZnTs control Zn2+ homeostasis and ERp44-mediated proteostasis along the early secretory pathway.

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