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Abstract

Protein O-mannosylation is an essential, conserved and abundant post-translational modification in the eukaryotic secretory pathway. Protein O-mannosylation is initiated at the endoplasmic reticulum (ER) by the covalent addition of single mannoses to serine and threonine residues of target polypeptides. This reaction is catalyzed by members of the evolutionarily conserved protein mannosyltransferase family (PMT). In the model organism Saccaromyces Cerevisiae, among its different biological roles, protein O-mannosylation has been described to target both unfolded and irreversibly misfolded proteins. The recently named unfolded protein O-mannosylation (UPOM) pathway is hypothesized to function as a fail-safe mechanism to prevent ineffective folding attempts for polypeptides that did not achieve the native conformation within certain time window. However, although UPOM has been described to target multiple misfolded model proteins, the mechanism by which the PMT machinery discriminates misfolded polypeptides is unknown. In fact, the question of whether UPOM is a physiologically relevant mechanism contributing to maintain homeostasis in the ER remains unanswered. In this current work, it is shown: I) The relevance of the Pmt1-Pmt2 complex as necessary to maintain ER homeostasis being integrated in the unfolded protein response (UPR). II) Aiming to unravel the UPOM machinery, the genes PGI1 (phosphoglucose isomerase) and BFR1 (Brefeldin A resistance) were found as necessary for efficient UPOM. On one hand, the characterization of PGI1 highlighted a direct link between O-mannosylation, cytosolic sugar metabolism, and ER stress. On the other hand, the RNA binding protein (RBP) Bfr1 was found to modulate the translational state of PMT mRNAs among many other ORFs, mainly coding for proteins localizing to the secretory pathway, suggesting a role as a local translational control factor. III) Aiming to address the physiological relevance of UPOM in the ER protein quality control system, the impact of O-mannosylation on the stability of a subset of PMT target proteins was investigated. Both, protein stabilization and de-stabilization effects were presented. The prevalence of either effect was largely dependent on the nature of the O mannosylation substrate. In line with this, Pmt1 is shown to be necessary for the degradation of the beta-1,6 synthase Kre6 when mislocalized in the vacuole.