Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae

Abstract

O-Mannosylation is a vital protein modification conserved from fungi to humans. Yeast is a perfect model to study this post-translational modification, because in contrast to mammalsO-mannosylation is the only type ofO-glycosylation. In an essential step toward the full understanding of proteinO-mannosylation we mapped theO-mannose glycoproteome in baker's yeast. Taking advantage of anO-glycan elongation deficient yeast strain to simplify sample complexity, we identified over 500O-glycoproteins from all subcellular compartments for which over 2300O-mannosylation sites were mapped by electron-transfer dissociation (ETD)-based MS/MS. In this study, we focus on the 293O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized proteinO-mannosyltransferases. We find thatO-mannosylation is not only a prominent modification of cell wall and plasma membrane proteins, but also of a large number of proteins from the secretory pathway with crucial functions in protein glycosylation, folding, quality control, and trafficking. The analysis of glycosylation sites revealed thatO-mannosylation is favored in unstructured regions and β-strands. Furthermore,O-mannosylation is impeded in the proximity ofN-glycosylation sites suggesting the interplay of these types of post-translational modifications. The detailed knowledge of the target proteins and theirO-mannosylation sites opens for discovery of new roles of this essential modification in eukaryotes, and for a first glance on the evolution of different types ofO-glycosylation from yeast to mammals.

Fig. 1.

The O-Man glycoproteomics strategy on yeast mutant KTRΔ. A, Illustration of the O-Man glycosylation pathway and the workflow for enrichment and analysis of O-Man glycopeptides by Con A lectin chromatography and HCD/ETD-MS/MS. B, C, Simplified O-Man glycans in the mutant strain KTRΔ. B, Western blot analysis of the O-mannosylated targets Hsp150 and Kex2. 30 μg of total cell lysates were separated by SDS-PAGE and analyzed by Western blot using protein-specific antibodies as indicated (see Experimental Procedures). Block of glycan elongation is monitored by electrophoretic mobility shift in WT and mutant strains as indicated. C, Relative quantitation of peptide-bound single mannose in WT and indicated strains by HPEAC-PAD. Mean values of three biological replicates ± standard deviation are shown.

Fig. 2.

The yeast O-Man glycoproteome. A, 511 O-Man glycoproteins from all subcellular compartments were identified by HCD/ETD-MS/MS (left pie chart). Among 1144 proteins targeted to the ER (right pie chart; extracted from Ast et al. (38) and referred herein as secretome), 293 O-Man glycoproteins (left pie chart) could been assigned. Within this fraction, SRP-independently translocating proteins are specifically enriched (middle pie chart) (also see supplemental Table S2) B, Proteins of the cell wall, the plasma membrane (PM) and the secretory pathway are enriched in the O-Man glycoproteome. The percentage of proteins from the corresponding subcellular compartments in the total yeast proteome (light gray) and the O-Man glycoproteome (dark gray) are shown. Higher values in the O-Man glycoproteome indicate an overrepresentation of proteins from a specific compartment in relation to the yeast proteome and vice versa. C, Coverage of proteins from distinct subcellular compartments in the O-Man glycoproteome. The percentage of proteins from different subcellular compartments identified within the O-Man glycoproteome (dark gray) are shown. For comparison, the distribution of recently described N-linked glycoproteins of S. cerevisiae is depicted (light gray) (39). B, C, Annotations are based on manual curation from SGD and selected high-confidence cellular compartment GO terms from the Compartments database. Proteins without any annotation according to these criteria are summarized as not annotated (NA). The corresponding listings of proteins including classification of subcellular localization can be found in supplemental Table S2. D, GO term enrichment analysis using DAVID (v6.7). GO term enrichment was performed on all identified O-Man glycoproteome Uniprot accessions (extracted from supplemental Table S3) using GO FAT terms, an EASE score of 0.1, and the S. cerevisiae reference list. Enrichment scores were extracted for the respective GO term clusters and clusters were named by representative terms. Biological process (GO_BP), molecular function (GO_MF) and cellular compartment (GO_CC) are shown.

Fig. 3.

Organisation of O-Man sites in protein domains in comparison with O-GalNAc. O-Man and O-GalNAc sites were compared between yeast and human to understand the overall distribution of glycosylation on proteins. Transmembrane information was retrieved from UniProt (Oktober, 2015), and domain information retrieved from Interpro 53.0. Using the “R” package Rgator, sites were classified into different regions on the protein (glycodomains), and summaries of the numbers produced.

Fig. 4.

O-Mannosylation of Pdi1 and PDI-like proteins. A, Schematic illustration of structural domains from Pdi1 and the PDI-like ER proteins Eug1, Mpd1 and Eps1 from baker's yeast. Active sites (stars) and thioredoxin domains (green) are highlighted. Putative N-linked glycosylation sites (Y shapes) are indicated as well as all O-Man sites (green circles) identified. O-Man sites are generally clustered in specific regions of the thioredoxin domains, flanking the active sites and the hydrophobic core region. B, Ribbon diagram and transparent surface representation of Pdi1 (PDB: 2b5e) with indicated a, b, b', and a' domains. Reactive sites of the a and a' domain, Cys⁶¹ and Cys⁴⁰⁶, are indicated in red. Mapped O-Man sites are indicated in blue. Highlighted excerpt is featuring the active site Cys⁴⁰⁶ next to the O-Man site Thr⁴⁵² as indicated. For representation, a mannose residue was modeled in the diagram and manually fitted for attachment and orientation using Pymol. C, Multiple sequence alignment of the corresponding regions in Pdi1 homologs from fungi and mammalian origin.

Fig. 5.

Analysis of sequential and structural features of O-Man sites. A, O-Man sites were analyzed using an in-house prepared “R” script performing sliding window analyses on protein sequences. The default sequence window size was 21 amino acids, ten amino acids N- and C-terminal of the respective site of interest. An example of a sliding window analysis result measuring the Ser/Thr content in percent is shown for Hsp150. Values calculated this way include the general Ser/Thr content in percent, the hydropathy, and the FoldIndex(c) as a measure for structural disorder. Additionally, values for the probability for α-helices and β-strand secondary structures were calculated externally using NetSurfP 1.1. Positions of O-Man modifications were extracted from supplemental Table S3. The corresponding values were plotted in histograms and bar plots, and the corresponding sequence windows were used for generation of Logo plots. In each case, only proteins and site information from proteins entering the secretory pathway (according to Ast et al. (38)) were taken into account. B, C, Bar plot summarizing the results of B, α-helix and C, β-strand prediction using NetSurfP 1.1. Bars represent the number of O-Man sites (dark gray) in percent that are located in protein regions with a minimum secondary structure probability as indicated. O-Man sites were compared with the distribution of all Ser/Thr sites from proteins entering the secretory pathway (light gray). O-Man sites were found underrepresented in regions of high α-helices, but overrepresented in regions of high β-strand probability. D–F, Histograms show the results of sliding window calculations of D, the general Ser/Thr content, E, the hydropathy, and F, the FoldIndex(c) surrounding the O-Man sites. O-Man sites (black) were compared with the distribution of all Ser/Thr positions in the secretome (gray). O-Man sites were found to be overrepresented in regions of 30–50% Ser/Thr content. In addition to that, O-Man sites are situated in hydrophilic and intrinsically disordered protein regions. G, One-sided Logo plot of analyzed sequence windows (using WebLogo 3.4). Size of the characters indicates the occurrence of amino acids within the sequence windows surrounding the O-Man site. Amino acids are colored according to their hydrophobicity: hydrophilic amino acids are blue, neutral amino acids are green, and hydrophobic amino acids are black. H, Two-sided Logo-plot of analyzed sequence windows (using Two Sample Logo). Sequence windows of O-Man sites were compared against the sequence windows of all Ser/Thr positions in the secretome. A threshold for enrichment and depletion of p > 0.01 was used. Amino acids are colored according to their chemical properties: polar amino acids are depicted in green, neutral amino acids are purple, basic amino acids are blue, acidic amino acids are red, and hydrophobic amino acids are black.

Fig. 6.

Context dependent O-mannosylation of a model acceptor substrate in in vitro translation/translocation/glycosylation assays. A, B, O- and N-linked glycosylation was monitored in vitro using a cell free translation/translocation/glycosylation assay described by Loibl et al. (8). A, Translated proteins and translocation products in the presence of yeast microsomes (yM) of the FLAG-tagged Ccw5-based model substrate TSTQATSS (schematically represented in supplemental Fig. S4) and B, the indicated derivatives thereof are shown. Details are outlined under Experimental Procedures. Translocation products were confirmed in the presence of Proteinase K (ProtK) and the detergent Triton X-100 (TX-100). Proteins of purified microsomes were treated with Endo H to remove N-linked glycans. Proteins were separated by SDS-PAGE and analyzed by Western blot using anti-FLAG antibodies.