Functional implications of MIR domains in protein -mannosylation.

Protein -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 β-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.

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

O-mannosylation is implicated in protein quality control in 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.

Monitoring Protein Dynamics in Protein -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 -mannosylation, which is essential and evolutionarily-conserved in fungi, animals, and humans. Our recent proteome-wide study in the eukaryotic model organism revealed that more than 26% of all proteins entering the secretory pathway receive -mannosyl glycans.

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

-Mannosylation is a type of protein glycosylation initiated in the endoplasmic reticulum (ER) by the protein -mannosyltransferase (PMT) family. Despite the vital role of -mannosylation, its molecular functions and regulation are not fully characterized. To further explore the cellular impact of protein -mannosylation, we performed a genome-wide screen to identify mutants with increased sensitivity towards the PMT-specific inhibitor compound R3A-5a.

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.

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.