DUAL REGULATION OF ERP44 ACTIVITY IN THE EARLY SECRETORYCOMPARTMENT

Secretory proteins start their journey from the endoplasmic reticulum to the Golgi undergoing stringent quality control to guarantee the integrity of the secretory proteome. This fundamental process is mediated by the coordinated action of numerous resident chaperones and enzymes. Amongst these, ERp44 is rather unique in that it is a multifunctional chaperone involved in maintaining redox homeostasis and in controlling thiol-mediated quality control and calcium and KDEL-Rs signalling. The crystal structure of ERp44 reveals 3 domains, a, b and b’, and a C terminal tail that connects domain a with b. Here we demonstrate that ERp44 activity is regulated by the pH gradient existing between the ER and Golgi. The more acidic Golgi pH (about 6.5) induces conformational changes in the ERp44 C-terminal tail that simultaneously expose the substrate-binding site and RDEL motif. In this way, ERp44 and its client proteins can be retrieved to the ER, where the higher pH (about 7.1) likely induces complex dissociation. The region at the interface between the C-tail and the substrate binding site is crucial to sense the changes of pH from the ER to the Golgi, acting like a sensor, intrinsic in the amino acidic composition of the protein. Alteration of Golgi pH induces increased secretion of ERp44 clients, due to a lower retentive activity of the chaperone. Moreover, we found that the conserved histidines located at the border between the b’ domain and the flexible C-tail are important for regulating ERp44 localization and function. Unlike wild type ERp44, mutants lacking key histidines are O-glycosylated in the Golgi and in part secreted. Interestingly, co-expression of client proteins restores retention of mutant ERp44, suggesting that conserved histidines regulate RDEL exposure in the absence of clients. Surprisingly, not all the clients bind ERp44 in the same compartment of the secretory pathway. Our data indicate that, depending on their affinity for ERp44 binding site and on the surrounding environment (different pH and/or variable cofactor concentration), ERp44-substrate complexes form before or downstream the site where O-linked glycosylation takes place. The client-induced retrieval mechanisms may allow the distinct and sequential localization of ERp44 interactors along the early secretory pathway. Remarkably, endogenous ERp44 can also undergo O-glycosylation in physiological conditions. A cyclic oscillation of the amount of O-glycosylated ERp44 is observed in stromal endometrial cells during the menstrual cycle, suggesting a pathophysiological role of this chaperone in these processes.