Updated publication reference for PubMed record(s): 26644575. Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine (Ser) and threonine (Thr) residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, e.g Saccharomyces cerevisiae. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation, and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway (HBP) to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-Mannose (O-Man) glycoproteome, and used this to identify a pleothora of O-linked mannose glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and doing so we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic and mitochondrial proteins in Saccharomyces cerevisiae. The type of nucleocytoplasmic proteins and the localization of identified O-Man residues mirrors that of the O-GlcNAc glycoproteome in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzyme machinery that is predicted to regulate the discovered nucleocytoplasmic O-Man glycosylation. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing.