Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast Adnan Halima,1, Ida Signe Bohse Larsena, Patrick Neubertb, Hiren Jitendra Joshia, Bent Larsen Petersena,c, Sergey Y. Vakhrusheva, Sabine Strahlb, and Henrik Clausena,1 aCopenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark; bCentre for Organismal Studies (COS), University of Heidelberg, D-69120 Heidelberg, Germany; and cDepartment of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark Edited by Stuart A. Kornfeld, Washington University School of Medicine, St. Louis, MO, and approved October 12, 2015 (received for review June 16, 2015) Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and orthologous OGT/OGA genes is yeast, although OGA orthologs threonine residues (O-GlcNAcylation) is an essential process in all have not been identified in plants. Yeast uses primarily Ser and eukaryotic cells except yeast, including Saccharomyces cerevisiae Thr for phosphorylation, with tyrosine (Tyr) phosphorylation and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling being used to an extremely low extent (10). This is in contrast to and cellular processes in an intricate interplay with protein phosphor- other eukaryotic cells that phosphorylate all three residues for ylation and serves as a key sensor of nutrients by linking the hexos- extensive and vital signaling. It has been a longstanding puzzle amine biosynthetic pathway to cellular signaling. A longstanding why in yeast the coregulatory functions of O-GlcNAcylation conundrum has been how yeast survives without O-GlcNAcylation in are apparently not required or whether another type of protein light of its similar phosphorylation signaling system. We previously O-glycosylation, such as O-linked mannose (O-Man), takes on developed a sensitive lectin enrichment and mass spectrometry work- this role (6). flow for identification of the human O-linked mannose (O-Man) glyco- All eukaryotes except nematodes and plants have a well- proteome and used this to identify a pleothora of O-Man glycoproteins characterized O-Man glycosylation machinery for proteins traf- in human cell lines including the large family of cadherins and proto- ficking the secretory pathway, and the enzymes involved in this cadherins. Here, we applied the workflow to yeast with the aim to process are multitransmembrane-spanning dolichol phosphate characterize the yeast O-Man glycoproteome, and in doing so, we β-D-Man (Dol-P-Man):protein O-mannosyltransferases (PMTs) discovered hitherto unknown O-Man glycosites on nuclear, cytoplas- using the membrane-associated Dol-P-Man donor substrate and mic, and mitochondrial proteins in S. cerevisiae and S. pombe.Such transferring a single Man residue to selected Ser and Thr residues O-Man glycoproteins were not found in our analysis of human cell of proteins. These enzymes are located in the ER, with their cat- lines. However, the type of yeast O-Man nucleocytoplasmic proteins alytic domains oriented into the lumen (11, 12). Higher eukaryotic and the localization of identifiedO-Manresiduesmirrorthatofthe cells have two PMT isoenzymes (POMT1 and POMT2), and the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating initial O-Man glycans are elongated, branched, and capped by sialic that the two different types of O-glycosylations serve the same im- acids by a series of glycosyltransferases. Deficiencies in many of the portant biological functions. The discovery opens for exploration of enzymes involved in protein O-mannosylation in humans under- the enzymatic machinery that is predicted to regulate the nucleocy- lie a group of congenital muscular dystrophies (13). Yeast, in toplasmic O-Man glycosylations. It is likely that manipulation of this contrast, have a family of at least six PMTs, and the initial Man type of O-Man glycosylation will have wide applications for yeast bioprocessing. Significance glycoproteomics | O-glycosylation | yeast | mass spectrometry | signaling Nucleocytoplasmic dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) and phos- ll eukaryotic cells except yeast harbor a simple type of phorylation coregulate important cellular processes in all eukaryotic Aprotein O-glycosylation designated O-GlcNAcylation [dy- organisms except yeast, including Saccharomyces cerevisiae namic cycling of N-Acetylglucosamine (GlcNAc) on serine (Ser) and Schizosaccharomyces pombe. The lack of an equivalent and threonine (Thr) residues] in the cytosol and nucleus (1). nucleocytoplasmic O-glycosylation system in yeast has been O-GlcNAcylation is an essential process involving addition and difficult to explain given that O-GlcNAcylation is an essential removal of a single GlcNAc at Ser and Thr residues of nuclear, modification in higher organisms. Here, we reveal that yeast – cytoplasmic, and mitochondrial proteins (2 5). O-GlcNAcylation use O-linked mannose to modify nucleocytoplasmic proteins on is a widespread modification found on, for example, nucleo- evolutionary-conserved regions and sites normally occupied by porins, transcription factors, kinases, and cytoskeletal and O-GlcNAc in higher eukaryotes. The results presented in this chromatin proteins; it is involved in a plethora of biological study open new avenues for exploration of nutrient sensing processes and believed to play causal roles in diabetes, cancer, and signaling events based on nucleocytoplasmic O-glycosylation cardiovascular, and Alzheimer’s disease (6–8). Sites of O- in yeast. GlcNAcylation are often found at or in close proximity to protein phosphorylation sites, and the intricate interplay between both Author contributions: A.H., S.S., and H.C. designed research; A.H., I.S.B.L., P.N., and B.L.P. modifications is known to modulate many important processes in performed research; A.H., I.S.B.L., P.N., H.J.J., S.Y.V., S.S., and H.C. analyzed data; and A.H. cells (8, 9). The transfer of GlcNAc to proteins is carried out by the and H.C. wrote the paper. The authors declare no conflict of interest. O-GlcNAc transferase (OGT) using uridine diphosphate α-D- GlcNAc (UDP-GlcNAc) as a donor substrate, and a second hy- This article is a PNAS Direct Submission. drolytic enzyme, O-GlcNAcase (OGA), is available to remove the Data deposition: The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRoteomics IDEntifications (PRIDE) Partner Repos- GlcNAc monosaccharide in a regulated dynamic process (4, 5). itory (accession no. PXD002924). Discovered over 30 years ago, the OGT/OGA enzyme pair was 1 To whom correspondence may be addressed. Email: [email protected] or halim@sund. initially identified in mammals, but subsequent work has demon- ku.dk. strated O-GlcNAcylation in bacteria, filamentous fungi, plants, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. metazoans. The only eukaryotic cell type without identifiable 1073/pnas.1511743112/-/DCSupplemental. 15648–15653 | PNAS | December 22, 2015 | vol. 112 | no. 51 www.pnas.org/cgi/doi/10.1073/pnas.1511743112 Downloaded by guest on October 1, 2021 monosaccharide is extended only by additional mannose residues from total cell lysates. In striking contrast to our studies with through the actions of Golgi resident mannosyltransferases of the humancelllines,weinadditiontoproteinsenteringthese- KTR/MNT and MNN family that use GDP α-D-Man (GDP-Man) cretory pathway also identified a large number of O-Man as a donor substrate (11). The O-Man glycosylation and the action proteins annotated as classical nuclear, cytosolic, or mito- of multiple PMTs are essential for yeast (14), and O-Man plays chondrial proteins that are not expected to be exposed to the major roles in maintaining yeast cell wall integrity (15). known O-Man glycosylation machinery in the secretory path- Our knowledge of the proteins undergoing O-Man glycosylation way. The nucleocytoplasmic O-Man glycosites were located on in yeast and the specific sites of glycosylation is limited (11, 12), but proteins and in positions resembling that of the O-GlcNAcy- recently we developed a glycoproteomic strategy to probe the lation process in higher eukaryotes. Here, we describe this O- O-Man glycoproteome of human cells using genetic engineer- Man glycoproteome and suggest that the nucleocytoplasmic O- ing to simplify the O-glycan structures, the so-called “SimpleCell” Man modifications in yeast represent the missing equivalent to strategy, in combination with Concanavalin A (ConA) lectin chro- the O-GlcNAcylation process of higher eukaryotes, and future matography for enrichment of glycopeptides and mass spec- studies can now address the biosynthetic machinery and trometric sequencing (16). This resulted in identification of a biological functions. large number of O-Man glycoproteins and O-Man glycosites, demonstrating, for example, that cadherins and protocadherins Results and Discussion are major carriers of O-Man glycans. In the present study, we Probing the O-Man Glycoproteome of Saccharomyces cerevisiae and modified this strategy to probe the yeast O-Man glycoproteome Schizosaccharomyces pombe. Because yeast is known to produce A WT KRE2∆KTR1∆KTR3∆ N-linked O-linked Tryptic digests O-glycopeptides + PNGase F ConA + Trypsin LWAC MS Mannose N-Acetylglucosamine B C D CELL BIOLOGY WT Mannitol Sorbitol α-mannosidase 100 24 - + - + 146 91 Total 80