Discovery of a Nucleocytoplasmic O-Mannose Glycoproteome in Yeast

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 14 46 17 Mut (11%)

31 60 Mitochondrial 215 177 83 (51%) 160 (38%) 77 40 66 S3 4 Relative Abundance Yeast 30 63 20 S3 20 40 60 80 Extracellular Cytoplasmic/Nuclear Nuclear/Cytoplasmic/Mitochondria Not Assignable 0 0 1 29 30 31 Time (min) Mut WT

Fig. 1. Identification of a novel type of nucleocytoplasmic protein O-mannosylation. (A) Graphic depiction of the glycoproteomic approach for identification of yeast O-mannosylation. WT or KRE2ΔKTR1ΔKTR3Δ Mut yeast total cell lysates treated with trypsin and PNGase F are enriched by ConA LWAC and glycopeptides identified by MS. (B) Pie chart showing the cellular localization of identified proteins with O-Man glycosites from total cell lysates of WT and Mut S. cerevisiae and a cytoplasmic preparation (S3) from WT S. cerevisiae. The composite results (Total) illustrate the total nonredundant number of O-Man glycoproteins identified. The identified O-Man glycoproteins assigned to the secretory pathway (Extracellular) will be described elsewhere. (C) GC-MS analysis of mannitol and sorbitol standards (gray trace). GC-MS profile of yeast reducing-end sugars released by reductive β-elimination (black trace). (D) Analysis of O-Man anomericity using jack bean α-mannosidase. Bar chart shows total (nuclear, cytoplasmic, and mitochondrial) number of identified O-Man glycoproteins for WT and Mut S. cerevisiae strains before (–) and after (+) α-mannosidase treatment.

Halim et al. PNAS | December 22, 2015 | vol. 112 | no. 51 | 15649 Downloaded by guest on October 1, 2021 heterogeneous elongated polymannose structures, we initially used The Nucleocytoplasmic O-Glycans Are Based on Man-α-O-Ser/Thr. Our the KRE2ΔKTR1ΔKTR3Δ mutant strain (Mut), lacking the α1,2- O-glycoproteomics strategy largely hinges on the α-Mannose mannosyltransferase (KRE2) involved in the second biosynthetic specificity of the lectin ConA used for the LWAC as well as the step of O-Man glycans and additional gene family members (KTR1 MS identification of the mass increment for hexose residues. Thus, and KTR3), to enable analysis of the O-Man glycoproteome with the approach does not reveal the absolute stereochemistry of the more simplified O-Man glycan structures (Fig. 1A) resembling our identified modifications. We therefore performed a series of ex- SimpleCell strategy (16). periments to verify the anomeric and epimeric configuration of the We used total cell lysates obtained by vortexing with glass identified peptide-linked hexose residue. First, we released O-linked beads in Rapigest detergent for trypsin digestion. N-linked glycans glycans by reductive β-elimination and analyzed the O-glycan were removed by PNGase F digestion, and the digests were sub- profiles by MALDI-TOF mass spectrometry and found Hex2–5 jected to ConA lectin weak affinity chromatography (LWAC) for oligosaccharides to be the dominating species (Fig. S2). This gly- enrichment of O-Man glycopeptides. Enriched O-Man glycopep- coprofiling does not enable us to selectively quantify the compo- tides were further fractionation by isoelectric focusing (IEF) and sition of hexose structures on the nucleocytoplasmic glycoproteins, analyzed by nanoflow liquid chromatography-mass spectrometry but as shown in Dataset S1, we identified nucleocytoplasmic pro- (nLC-MS/MS) (Fig. 1A). Through this approach using the teins with both a single and a disaccharide hexose structure, showing KRE2ΔKTR1ΔKTR3Δ Mut, we identified a considerable number of that at least some elongation occurs. Mammalian nucleocytoplasmic O-glycoproteins and O-glycosites with one or more hexoses attached O-GlcNAc is not elongated by other glycans, but in plants this using both higher energy collision dissociation (HCD) and electron type of glycosylation is elongated (17). The glycans released transfer dissociation (ETD) fragmentation modes. A summary of the by reductive β-elimination were subsequently subjected to acid identified glycoproteins from S. cerevisiae ispresentedinFig.1B. hydrolysis and trimethylsilyl derivatization before GC-MS anal- In total we mapped 291 unique O-Man glycoproteins and ∼1,000 ysis. This approach enables the reducing-end hexose to be dif- O-Man glycosites in Mut total cell lysates. ferentiated from internal or terminal hexose residues through its distinct retention time in GC-MS, and for the β-eliminated yeast Discovery of a Nucleocytoplasmic O-Man Glycoproteome. Analysis of O-glycans, the reducing-end hexose displayed a similar retention the identified proteins revealed that a large proportion (n = 83, time and fragmentation pattern as the mannitol standard, thus 29%) were known or predicted nuclear and/or cytosolic proteins confirming that the peptide-linked hexose is a mannose residue without recognizable signal peptides. This finding was in striking (Fig. 1C and Fig. S3). Having resolved the epimeric configuration contrast to our previous analysis of the O-Man glycoproteome of of the glycan, we then turned our attention to the anomeric con- human cells, where all identified glycoproteins were known or figuration of the mannose–peptidelinkage.Weperformedjack predicted to traffic the secretory pathway (16). We therefore also bean α-mannosidase digestion on the ConA-enriched and IEF- inspected all identified glycoproteins with signal peptides for the fractionated samples from both WT and KRE2ΔKTR1ΔKTR3Δ specific localization of O-glycosites with respect to known or yeast and found that essentially all mannose residues were hydro- predicted membrane orientation and found sites predicted to be lyzed by the α-mannosidase treatment (Fig. 1D and Dataset S1). located in the cytosolic compartment (Dataset S1). Thus, more Approximately 25% of the identified O-Man glycopeptides were than a third of the O-Man glycoproteins identified in Mut readily identified as the corresponding peptides without the Man S. cerevisiae were classical nuclear, cytosolic, or mitochondrial residues after digestion (Dataset S1). This suggests that the proteins that are not exposed to the known O-Man glycosylation anomericity of the mannose linkage is in an α-configuration. machinery in the secretory pathway (Fig. 1B). Thus, we conclude that all identified O-Man glycosites involved To exclude the possibility that the unexpected finding was α-linked Man residues hitherto known only on proteins trafficking related to the KRE2ΔKTR1ΔKTR3Δ Mut, we further explored the secretory pathway. this by analyzing total cell lysates of wild-type (WT) S. cerevisiae, which resulted in identification of 261 O-Man glycoproteins of Nucleocytoplasmic O-Man Proximity to Phosphorylation Sites. The which a similar fraction of 91 (35%) glycoproteins were from 160 identified O-Man nucleocytoplasmic glycoproteins identified nuclear, cytoplasmic, or mitochondrial compartments. Also, we in S. cerevisiae included transcription factors, nucleoporins, his- sought to enrich for cytoplasmic proteins by analyzing a crude tones, and kinases, which are all classical proteins undergoing cytoplasmic fraction (S3) of fractionated WT yeast, and this O-GlcNAcylation as well as phosphorylation in higher eukary- resulted in identification of a number of unique nucleocyto- otes (Dataset S1). For the identified nucleocytoplasmic yeast plasmic glycoproteins (Fig. 1B and Dataset S1). In total, we glycoproteins, 80% are known to be phosphorylated (18–21), and identified 162 unique glycoproteins from Mut and WT S. cerevisiae as much as 19% (221 in total) of the identified O-Man glycosites strains with O-glycosites that were either only found in cytosolic, were found to be identical to previously identified phosphory- nuclear, or mitochondrial compartments (n = 160) or where the lation sites (Dataset S3), a relatively high number considering sites identified were located in the cytosolic part of transmembrane that neither the current yeast phosphoproteome nor the O-Man proteins (n = 2). glycoproteome presented here is complete. In a preliminary study, we also explored the O-Man glycoproteome An illustrative example was the glycogen phosphorylase (GP) en- of the fission yeast S. pombe (WT) using the same experimental zyme, which mobilizes cellular energy by breaking down glycogen into approach as above (Fig. 1A) with total cell lysates. We identified 178 glucose-1-phosphate. The mechanism of GP activation is conserved O-Man glycoproteins, of which 87 (49%) are known to have cyto- among eukaryotes and involves, in addition to allosteric elements, a solic, nuclear, or mitochondrial localization (Fig. S1 and Dataset S2). single phosphorylation at Thr31 in yeast and Ser15 in mammals, both The identified O-Man proteins classified as nucleocytoplasmic greatly locatedintheN-terminalregulatorydomain(Fig.2A) (22, 23). We expanded the total nucleocytoplasmic glycoproteome, as there was identifiedanO-ManglycanontheregulatoryThr31ofyeastGP, little overlap among the datasets from S. cerevisiae and S. pombe,with demonstrting that nucleocytoplasmic O-Man in yeast may compete only a few identified in both. directly with phosphorylation for functionally important sites. Clearly In the following, we focus on the deepest nucleocytoplasmic O-Man glycosylation of Thr31 in GP will block phosphorylation, and O-Man glycoproteome data obtained from S. cerevisiae (Dataset it is expected that this will severely affect activation of the enzyme. S1). The O-Man glycoproteome subset comprising glycoproteins Thus, the nucleocytoplasmic O-Man glycosylation may therefore and domains known to be exposed to the secretory pathway will function as an important control switch for utilization of reserve be presented in a separate publication. carbohydrates in yeast.

15650 | www.pnas.org/cgi/doi/10.1073/pnas.1511743112 Halim et al. Downloaded by guest on October 1, 2021 A Glycogen phosphorylase eukaryotes, and the discovery suggests that yeast possesses a 100 200 300 400 500 600 700 800 hitherto unidentified O-glycosylation machinery that operates in cytoplasmic, nuclear, and mitochondrial compartments. Human Yeast O-Man Glycosites and Comparison with Mammalian O-GlcNAcylation Sites. We selected examples of orthologous proteins with known Ser15 O-GlcNAcylation sites and identified O-Man glycosites for more Human 1 MAKPLTDQEK------RRQISIRGIV 20 Yeast 26 LTRRLTGFLPQEIKSIDTMIPLKSRALWNKHQVK 59 detailed analysis. Although the existence of O-GlcNAc on histones was questioned in a recent study (28), O-GlcNAcylation is Thr31 believed to be one of several posttranslational modifications (PTMs) that constitute the histone code and regulate histone B Histone H2B interactions with DNA and effector proteins, and this PTM has 20 40 60 80 100 120 been found on histones H2A, H2B, and H4 (29). It has further been demonstrated that O-GlcNAcylation of human histone Human Yeast H2B in response to glucose levels modulates the transcriptional response by promoting monoubiquitination of lysine residue 120 (30). A glucose-dependent response is also observed in yeast where Human STITSREIQTAVRLLLPGELAKHAVSEGTKAVTKYTSSK 125 the orthologous histone H2B.1 undergoes monoubiquitination at STISAREIQTAVRLILPGELAKHAVSEGTRAVTKYSSSTQA 131 Yeast the conserved lysine residue, although the preceding step promoting this monoubiquitination has not been identified (31). We found C Plasma membrane ATPase O-Man glycosites on the highly conserved yeast histone ortholog 200 400 600 800 H2B.1 positioned virtually in the same region as O-GlcNAcylation on the human histone H2B (Fig. 2B). We found O-Man residues on 129 Mouse the peptide TKYSSST but could not assign the exact positions of Yeast the glycosites; however, an O-GlcNAc site has been identified in the highly conserved TSS sequon. Although we in this first limited study did not identify O-Man glycosites on other histones, the results Mouse CRSAGIKVIMVTGDHPITAKAIA 626 FIYDEVRKLILRRYPGGWVEKETYY 1020 suggest that O-Man glycosylation is part of the histone code in Yeast ARHLGLRVKMLTGDAVGIAKETC 569 STRSVEDFMAAMQRVSTQHEKET-- 918 yeast, similar to O-GlcNAcylation in higher eukaryotes. + Thr558 The plasma membrane H -ATPase (PMA1) is O-GlcNAcylated O-Mannosylation O-GlcNAcylation Phosphorylation Extracellular region in the cytosolic region in mammals (24), and we identified O-Man glycosites on the yeast PMA1 ortholog that were conserved and Fig. 2. Graphic illustration of cross-talk between O-Man glycosylation and overlapping with the known O-GlcNAcylation sites as well as phosphorylation with analogy to O-GlcNAcylation in other eukaryotes. (A) Sequence alignment (grayscale proportional to conservation) of human phosphorylation sites (Fig. 2C). In particular, we found O-Man at and yeast GP with expansion of the N-terminal domain showing the co- Thr558, a conserved residue within the ATP binding motif of CELL BIOLOGY occupancy at the regulatory Thr31 residue. (B) Alignment of human and PMA1 (32), which is also O-GlcNAcylated in mammals (24). The yeast histone H2B proteins demonstrating C-terminal O-GlcNAc and O-Man consequence of glycosylation at Thr558 is still unclear, but given modifications, respectively. Human H2B lysine 120 is indicated in yellow. that Thr558 is in immediate proximity to the ATP molecule, it is (C) Alignment of mouse and yeast plasma membrane ATPase proteins. Left reasonable to predict that the O-glycan will have an impact on ATP expansion shows part of the ATP binding motif (yellow) with overlapping binding (Fig. S6). PMA1 was further identified with O-Man O-GlcNAc/O-Man occupancy at Thr558. Right expansion shows the C-terminal modifications on the C-terminal regulatory domain where one site, regulatory domain modified by O-Man and phosphorylations. Ser899, has also been identified as being phosphorylated (Fig. 2C). The C-terminal region is a critical region that undergoes glucose- We also compared the proximity of all O-Man glycosites to dependent phosphorylation, leading to rapid PMA1 activation (33, known phosphorylation sites, which further demonstrated that 34). Upon glucose depletion, PMA1 is reversibly deactivated within the O-Man glycosites assigned to nucleocytoplasmic proteins in minutes, suggesting a negative regulation driven by PTMs, although this mechanism is not fully understood. general tended to have phosphorylation sites closer than the O-Man In conclusion, we describe the existence of a hitherto unknown glycosites assigned to proteins and protein domains exposed to the nucleocytoplasmic type of protein O-mannosylation in S. cerevisiae secretory pathway (Fig. S4). Thus, the nucleocytoplasmic O-Man and S. pombe. We previously used the same strategy to character- glycoproteins clearly resemble the mammalian O-GlcNAcylated ize the human O-Man glycoproteome and found no evidence of proteins. O-mannosylation of nucleocytoplasmic proteins (16). Because yeast We further analyzed the amino acid sequences surrounding is also the only known eukaryote to lack the OGT/OGA enzymes the nucleocytoplasmic O-Man glycosites, but this approach did responsible for O-GlcNAcylation, it is likely that nucleocytoplas- not reveal any apparent sequence motifs for glycosylation (Fig. mic O-mannosylation is unique to yeast. Although other fungi in- S5) similar to what has been reported for O-GlcNAcylation (24). cluding molds like Aspergillus niger have OGT/OGA homologous However, a higher proportion of flanking hydrophobic residues genes, further studies are needed to determine if these also have was observed for nucleocytoplasmic O-Man glycosites compared nucleocytoplasmic O-Man glycoproteins and ultimately if the two with O-Man glycosites found on extracellular proteins. nuclecytoplasmic types of glycosylation can coexist. The O-Man Analysis of the subset of proteins for which mammalian glycosites on nucleocytoplasmic proteins share the characteristic orthologs could be reliably established (79/160) revealed that feature of overlap with phosphorylation sites and are positioned 24% of these have also been reported to be O-GlcNAcylated in similarly to O-GlcNAc sites on evolutionary conserved proteins rodents or humans (Table S1) (24–27). This analysis is likely and sites. We therefore propose that O-mannosylation of nucleo- biased by the limited depth of the O-glycoproteome data avail- cytoplasmic proteins in yeast serves the equivalent functions as the able currently, and we expect the overlap to increase with deeper O-GlcNAcylation process in higher eukaryotes. The nutrient-sensing characterization of the two glycoproteomes. Thus, the identified role of O-GlcNAcylation is likely to be mirrored by O-mannosylation O-Man glycosylation of nucleocytoplasmic proteins is likely to in yeast, with the difference being that GDP-Man, and not UDP- represent the equivalent to O-GlcNAcylation found in higher GlcNAc, will likely serve the role of donor substrate and key nutrient

Halim et al. PNAS | December 22, 2015 | vol. 112 | no. 51 | 15651 Downloaded by guest on October 1, 2021 sensor in yeast. The biosynthesis of GDP-Man would thus link nLC-MS/MS and Data Analysis. Mass spectrometric analyses were performed glucose and nucleotide metabolism in a network node capable of essentially as previously described (16). Samples were analyzed on a setup integrating nutrient signals that are ultimately relayed as nucleo- composed of an EASY-nLC 1000 (Thermo Fisher Scientific) interfaced via a nanoSpray Flex ion source to an LTQ-Orbitrap Velos Pro hybrid spectrometer cytoplasmic O-Man glycosylation. The apparent shift to nutrient (Thermo Fisher Scientific). The EASY-nLC 1000 was equipped with a polar end- sensing by UDP-GlcNAc in higher eukaryotes from GDP-Man capped C18-silica column 21 cm in length, 75 μm in inner diameter, and 1.9 μm may indicate a need to evolve a more complex nutrient sensor in particle size. A data-dependent mass spectral acquisition routine, HCD, and capable of incorporating glucose, nucleotide, amino acid, and fatty subsequent ETD scan were used for all runs. Briefly, a precursor MS1 scan (m/z acid metabolism by flux through the hexosamine biosynthetic path- 355–1,700) of intact peptides was acquired in the Orbitrap at a resolution set- way. Yeast is the only eukaryotic cell type without identifiable ting of 30,000, followed by Orbitrap HCD-MS2 and ETD-MS2 of the five most orthologous OGT/OGA genes involved in O-GlcNAcylation, and abundant multiply-charged precursors in the MS1 spectrum; a minimum MS1 signal threshold of 50,000 ions was used for triggering data-dependent frag- our findings here provide an explanation, as the enzymes required α mentation events; and MS2 spectra were acquired at a resolution of 15,000. fortransferandremovalof -Mannose residues are likely to have Data processing was carried out using Proteome Discoverer 1.4 software entirely different structures and be encoded by nonhomologous (Thermo Fisher Scientific) as previously described (16) with minor modifications, genes. Identifying the enzymes responsible for the nucleocytoplas- as outlined below. Raw data files (.raw) were processed using the Sequest HT mic O-Man will provide unique opportunities to regulate a broad node and searched against the canonical S. cerevisiae proteome (7,225 entries) spectrum of cellular processes in yeast with huge potential for yeast- downloaded from the Uniprot database (October 2013) or the canonical based bioproduction. S. pombe proteome (5,092 entries; September 2015). Spectral assignments at the medium confidence level (P > 0.01) and below were resubmitted to a Materials and Methods second Sequest HT node using semispecific tryptic cleavage. Final results were filtered for high-confidence (P < 0.01) identifications only. Spectra matched to Yeast Strains and Culture Conditions. The following S. cerevisiae strains were peptides with nucleocytoplasmic O-Man modifications were inspected manually a Δ Δ Δ Δ used: WT (BY4741, MAT his3 1 leu2 0 met15 0 ura3 0), the KTR/MNT to verify the accuracy of the assignments. The mass spectrometry proteomics Δ Δ Δ triple mutant (BY4741 except kre2 ::His3-GFP, ktr1 ::SAT, ktr3 ::KanMX4;a data have been deposited in the ProteomeXchange Consortium (36) via the gift of Howard Bussey, McGill University, Montreal), and BY5457 loxp:: PRoteomics IDEntifications (PRIDE) partner repository with the dataset identi- Δ α pep4 ::loxp [WT BY5457 (MAT ura3-52::p1785 leu2-3,112 his4-519 trp1 fier PXD002924. Signal peptides were retrieved manually from Uniprot or r Δ pho3-1 pho5-1 can ) with the additional loxp::pep4 ::loxp modification; a through the SignalP tool (server version 4.1), and transmembrane domains gift from Rosa Laura Lopez Marques, University of Copenhagen, Copenhagen). were predicted with Trans Membrane Hidden Markov Model (TMHMM) (server WT cells (BY4741) were grown in rich medium containing 2% (wt/vol) version 2.0); both tools are available at www.cbs.dtu.dk/services/. glucose, 2% (wt/vol) peptone, and 1% (wt/vol) yeast extract (YPD) under Cytoplasmic proteins (S3 fraction) were prepared as follows: Cells (BY5457 aerobic conditions at 30 °C with constant shaking at 170 rpm in a rotary loxp::pep4Δ::loxp) were grown as above in YPAD medium (YPD supple- shaker incubator (I Series 26, New Brunswick Scientific Co., Inc.). Cell mented with 0.01% Adenine hemisulfate wt/vol). Cells were pelleted by growth was determined by measuring the optical density at a wavelength centrifugation at 800 × g for 15 min at 4 °C and suspended in a 1:5 ratio in of 600 nm (OD600). S. cerevisiae cultures were grown to midlog phase 50 mL lysis buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl) supplemented with – × (OD600 0.8 1.1) and harvested by centrifugation at 3,000 g for 5 min. one tablet of cOmplete protease inhibitor (Roche) and lysed by French press Growth conditions and harvesting of BY5457 cells are described below. at 4 °C at 40 Kpsi (S1 fraction). The S1 fraction was cleared by centrifugation The S. pombe WT (no-marker h–) strain, a derivative of the WT hetero- at 5,000 × g at 4 °C for 15 min. The supernatant (S2) was subjected to ul- thallic strains 972h– and 975h+, was used. The fission yeast cells were tracentrifugation at 45,000 × g at 4 °C for 30 min, followed by 100,000 × g at propagated at 30 °C in YES media (5 g/L yeast extract, 30 g/L glucose, 4 °C for 3 h, resulting in the cytoplasmic S3 (supernatant) fraction. The S3 225 mg/L adenine, 225 mg/L leucine, and 225 mg/L uracil) and harvested in fraction was trypsin-digested, ConA-enriched, and purified by Stage tips as the late exponential phase by centrifugation (3,000 × g for 2 min) and described above. For each LWAC elution fraction, 5% of the total sample lysed using glass beads as described below. was injected and analyzed by HCD fragmentation only.

LWAC Isolation of O-Man Glycopeptides. Cell extracts were prepared from a MALDI and GC-MS Analysis. The S3 fraction (13 mL) was dialyzed against 6 L

total of 100 OD600 units of packed yeast cells with acid-washed 5-mm glass 50 mM NH4HCO3, pH 7.8, using 8,000 Da MWCO membranes, and concentrated

beads in ice-cold 0.1% Rapigest (Waters Corp.) in 50 mM NH4HCO3 by vor- by evaporation. Following reduction and alkylation (described above), the texing in reciprocal shaker (Hybaid RiboLyser) for 4 × 25 s with 1-min in- cytosolic proteins were trypsin-digested ON and purified by Sep-Pak C18 tervals at 4 °C. The bottom of the tube was punctured, and the lysate was (Waters) columns. The tryptic peptides were dried, resolubilized in 100 mM β collected. Unbroken cells and larger cell debris were removed by a low- NaOH and 1 M NaBH4, and incubated ON at 50 °C. The -elimination reaction μ speed centrifugation step at 1,500 × g for 5 min at 4 °C. The cleared lysates was terminated by the addition of 8 L glacial acetic acid, and the released were heated at 80 °C for 10 min, followed by reduction in 5 mM dithio- O-glycan alditols were separated from proteins by a second Sep-Pak C18 threitol at 60 °C for 30 min and alkylation in 10 mM iodoacetamide at room (Waters) purification. Reduced O-glycans were desalted by Dowex AG 50W ×8 cation exchange resin (Bio-Rad) followed by repeated (×5) addition of temperature (RT) in darkness for 30 min. Samples were digested with 25 μg 500 μL 1% (vol/vol) acetic acid in methanol and evaporation over a stream of trypsin (Roche) overnight (ON), heat-inactivated by incubation at 95 °C for N gas. For MALDI analysis, released oligosaccharides were permethylated 20 min, and treated with 8 U PNGase F ON at 37 °C. An additional 4 U 2 essentially as previously described (37). Briefly, released oligosaccharides PNGase F was added and incubated at 37 °C for 4 h. The digests were were dried in glass vials to which 18 mg NaOH powder, 150 μL dimethyl acidified with 12 μL TFA, incubated at 37 °C for 20 min, cleared by centri- sulfoxide with 0.1% H O (vol/vol), and 30 μL methyl iodide were added; the fugation at 10,000 × g for 10 min, and purified by Sep-Pak C18 (Waters) 2 mixture was incubated at RT for 1 h, and the reaction was terminated by columns. The LWAC protocol for isolation of O-Man glycopeptides was as addition of 150 μL ice-cold H2O followed by 200 μL chloroform. The organic previously described (16). Sep-Pak–purified peptides were concentrated by phase was washed five times with 1 mL H2O and finally dried with a stream evaporation, and the reduced solution was diluted with an equal volume of of N2 gas. Permethylated oligosaccharides were reconstituted in 30 μL 50% 2 × ConA buffer A (40 mM Tris·HCl, pH 7.4, 300 mM NaCl, 2 mM CaCl2/MgCl2/ methanol in H2O (vol/vol), and 1 μL was cocrystalized with an equal amount MnCl2/ZnCl2, 1 M urea) before loading in a 2.8-m-long ConA lectin agarose of matrix [10 mg/mL 2,5-dihydroxybenzoic acid and 2.5 mM sodium acetate column. The column was washed with 10 column volumes (CVs) of ConA in 70% acetonitrile in H O (vol/vol)]. MALDI-TOF (Autoflex Speed, Bruker μ 2 buffer A at 100 L/min before elution with five CVs of ConA buffer B (20 mM Daltonics) was operated in the reflector mode using positive polarity and · Tris HCl, pH 7.4, 150 mM NaCl, 1 mM CaCl2/MgCl2/MnCl2/ZnCl2, 0.5 M methyl- 2,000 laser shots per spot. For GC-MS, reduced O-glycans were spiked with α-D-glucopyranoside/methyl-α-D-mannopyranoside) at 50 μL/min. Fractions 1 μg myo-inositol (internal standard) and depolymerized by incubation in 0.5 containing glycopeptides were purified by in-house packed Stage tips N methanolic HCL (Supelco) at 80 °C for 16 h. Monosaccharides were per- (Empore disk-C18, 3M) and further fractionated by IEF as previously de- O–trimethylsilylated with Tri-Sil reagent (Thermo Scientific) at 80 °C for 30 min. scribed (35). For each IEF fraction, 50% was analyzed by nLC-MS/MS as de- GC-MS analysis was performed using a TRACE GC Ultra gas chromatograph scribed below. The remaining 50% of each IEF fraction was digested at 37 °C coupled to a PolarisQ ion trap mass spectrometer. Samples were injected ON with 30 U/mL jack bean α-mannosidase (Prozyme) in 100 mM sodium (splitless mode) at 40 °C (1 min), and the oven temperature was ramped to acetate, 2 mM Zn2+, pH 5 before analysis by nLC-MS/MS. 150 °C (25 °C/min) followed by an increase to 200 °C (1 °C/min) before a final

15652 | www.pnas.org/cgi/doi/10.1073/pnas.1511743112 Halim et al. Downloaded by guest on October 1, 2021 ramp to 260 °C (10 °C/min), where it was held for 5 min. Monosaccharides Marques are acknowledged for providing S. cerevisiae strains. Rasmus were identified by comparison of retention times and mass spectra to hexose Hartmann-Petersen is acknowledged for providing the S. pombe strain. and hexitol standards. All retention times were relative to the myo-inositol This work was supported by Kirsten og Freddy Johansen Fonden, A.P. Møller og Hustru Chastine McKinney Møllers Fond til Almene Formaal, internal standard. the Novo Nordisk Foundation, Danish Council for Strategic Research (APC- GlyVac, 12-131859), a program of excellence from the University of Copen- ACKNOWLEDGMENTS. The authors thank Gerald W. Hart, James C. hagen (CDO2016), the Danish National Research Foundation (DNRF107), Paulson, Hudson Freeze, and Bernhard Henrissat for helpful discussions and the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 1036, and comments on the manuscript. Howard Bussey and Rosa Laura Lopez Project 11.

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