Functional implications of MIR domains in protein O-mannosylation.

Protein O-mannosyltransferases (PMTs) represent a conserved family of multispanning endoplasmic reticulum membrane proteins involved in glycosylation of S/T-rich protein substrates and unfolded proteins. PMTs work as dimers and contain a luminal MIR domain with a ß-trefoil fold, which is susceptive for missense mutations causing α-dystroglycanopathies in humans. Here, we analyze PMT-MIR domains by an integrated structural biology approach using X-ray crystallography and NMR spectroscopy and evaluate their role in PMT function in vivo. We determine Pmt2- and Pmt3-MIR domain structures and identify two conserved mannose-binding sites, which are consistent with general ß-trefoil carbohydrate-binding sites (α, ß), and also a unique PMT2-subfamily exposed FKR motif. We show that conserved residues in site α influence enzyme processivity of the Pmt1-Pmt2 heterodimer in vivo. Integration of the data into the context of a Pmt1-Pmt2 structure and comparison with homologous ß-trefoil - carbohydrate complexes allows for a functional description of MIR domains in protein O-mannosylation.

Translational Regulation of Pmt1 and Pmt2 by Bfr1 Affects Unfolded Protein O-Mannosylation.

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.

Monitoring Protein Dynamics in Protein O-Mannosyltransferase Mutants In Vivo by Tandem Fluorescent Protein Timers.

For proteins entering the secretory pathway, a major factor contributing to maturation and homeostasis is glycosylation. One relevant type of protein glycosylation is O-mannosylation, which is essential and evolutionarily-conserved in fungi, animals, and humans. Our recent proteome-wide study in the eukaryotic model organism Saccharomyces cerevisiae revealed that more than 26% of all proteins entering the secretory pathway receive O-mannosyl glycans. In a first attempt to understand the impact of O-mannosylation on these proteins, we took advantage of a tandem fluorescent timer (tFT) reporter to monitor different aspects of protein dynamics. We analyzed tFT-reporter fusions of 137 unique O-mannosylated proteins, mainly of the secretory pathway and the plasma membrane, in mutants lacking the major protein O-mannosyltransferases Pmt1, Pmt2, or Pmt4. In these three pmtΔ mutants, a total of 39 individual proteins were clearly affected, and Pmt-specific substrate proteins could be identified. We observed that O-mannosylation may cause both enhanced and diminished protein abundance and/or stability when compromised, and verified our findings on the examples of Axl2-tFT and Kre6-tFT fusion proteins. The identified target proteins are a valuable resource towards unraveling the multiple functions of O-mannosylation at the molecular level.

Cellular Consequences of Diminished Protein O-Mannosyltransferase Activity in Baker's Yeast.

O-Mannosylation is a type of protein glycosylation initiated in the endoplasmic reticulum (ER) by the protein O-mannosyltransferase (PMT) family. Despite the vital role of O-mannosylation, its molecular functions and regulation are not fully characterized. To further explore the cellular impact of protein O-mannosylation, we performed a genome-wide screen to identify Saccharomyces cerevisiae mutants with increased sensitivity towards the PMT-specific inhibitor compound R3A-5a. We identified the cell wall and the ER as the cell compartments affected most upon PMT inhibition. Especially mutants with defects in N-glycosylation, biosynthesis of glycosylphosphatidylinositol-anchored proteins and cell wall ß-1,6-glucan showed impaired growth when O-mannosylation became limiting. Signaling pathways that counteract cell wall defects and unbalanced ER homeostasis, namely the cell wall integrity pathway and the unfolded protein response, were highly crucial for the cell growth. Moreover, among the most affected mutants, we identified Ost3, one of two homologous subunits of the oligosaccharyltransferase complexes involved in N-glycosylation, suggesting a functional link between the two pathways. Indeed, we identified Pmt2 as a substrate for Ost3 suggesting that the reduced function of Pmt2 in the absence of N-glycosylation promoted sensitivity to the drug. Interestingly, even though S. cerevisiae Pmt1 and Pmt2 proteins are highly similar on the sequence, as well as the structural level and act as a complex, we identified only Pmt2, but not Pmt1, as an Ost3-specific substrate protein.

Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae.

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 ß-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.

Defect in dolichol-dependent glycosylation increases sensitivity of Saccharomyces cerevisiae towards anti-fungal drugs.

Two temperature-sensitive Saccharomyces cerevisiae mutants, sec59-1 and dpm1-6, impaired, respectively, in dolichol kinase (Sec59p) and dolichyl phosphate mannose (DolPMan) synthase (Dpm1p), have an aberrant cell wall structure and composition. We tested their sensitivity to four classes of antifungal drugs used in clinical practice: 5-fluorocytosine, amphotericin B, caspofungin and itraconasole. The strains were resistant to itraconazole and sensitive to the other drugs used. The minimal inhibitory concentration (MIC) of caspofungin and amphotericin B was two-fold lower for sec59-1 and dpm1-6 than for the respective wild-type strains. The sensitivity of both mutants could be brought back to the wild-type level by a multicopy suppressor of the thermosensitive phenotype, the RER2 gene, encoding cis-prenyltransferase involved in dolichol biosynthesis. Biochemical analysis revealed slight changes of the cell wall composition, different in the mutants as compared to the wild-type in response to the drugs. Our data strongly support a relationship between dolichol phosphate level, protein glycosylation and antifungal sensitivity.