Abstract
O-mannosylation is implicated in protein quality control in Saccharomyces cerevisiae due to the attachment of mannose to serine and threonine residues of un- or misfolded proteins in the endoplasmic reticulum (ER). This process also designated as unfolded protein O-mannosylation (UPOM) that ends futile folding cycles and saves cellular resources is mainly mediated by protein O-mannosyltransferases Pmt1 and Pmt2. Here we describe a genetic screen for factors that influence O-mannosylation in yeast, using slow-folding green fluorescent protein (GFP) as a reporter. Our screening identifies the RNA binding protein brefeldin A resistance factor 1 (Bfr1) that has not been linked to O-mannosylation and ER protein quality control before. We find that Bfr1 affects O-mannosylation through changes in Pmt1 and Pmt2 protein abundance but has no effect on PMT1 and PMT2 transcript levels, mRNA localization to the ER membrane or protein stability. Ribosome profiling reveals that Bfr1 is a crucial factor for Pmt1 and Pmt2 translation thereby affecting unfolded protein O-mannosylation. Our results uncover a new level of regulation of protein quality control in the secretory pathway.
Highlights
Glycosylation is a major protein modification that includes the addition of a sugar moiety onto a protein [1]
We stably introduced endoplasmic reticulum (ER)-green fluorescent protein (GFP) into the innocuous HO locus of pmt1∆, pmt2∆, and pmt4∆ cells
In pmt1∆ and pmt2∆ cells reporter fluorescence is considerably enhanced compared to wild type whereas the GFP signal in pmt4∆ is not affected (Figure 1B,C)
Summary
Glycosylation is a major protein modification that includes the addition of a sugar moiety onto a protein [1]. While most studies of O-mannosylation focus on the role of this modification during normal protein maturation along the secretory pathway, recently it has been demonstrated that there exists non-canonical O-mannosylation of proteins due to un- or misfolding [18] This so-called unfolded protein O-mannosylation (UPOM) has been proposed as a molecular timer that is active in the early stages of ER protein quality control to abrogate futile folding cycles and save valuable cellular resources [19]. The consequences of UPOM strongly depend on the substrate proteins which have been shown to be later eliminated by the cell either by ERAD [20], vacuolar degradation [21] or cellular exclusion [22] To date this modification has been observed for several mutated proteins, not for their wild type counterparts [20,21,22,23,24,25]. To this end we took advantage of ER-targeted slow-folding GFP as a UPOM-reporter and identified brefeldin A resistance factor 1 (Bfr1) as an enhancer of Pmt and Pmt translation
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