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 mammals O-mannosylation is the only type of O-glycosylation. In an essential step toward the full understanding of protein O-mannosylation we mapped the O-mannose glycoproteome in baker's yeast. Taking advantage of an O-glycan elongation deficient yeast strain to simplify sample complexity, we identified over 500 O-glycoproteins from all subcellular compartments for which over 2300 O-mannosylation sites were mapped by electron-transfer dissociation (ETD)-based MS/MS. In this study, we focus on the 293 O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized protein O-mannosyltransferases. We find that O-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 that O-mannosylation is favored in unstructured regions and β-strands. Furthermore, O-mannosylation is impeded in the proximity of N-glycosylation sites suggesting the interplay of these types of post-translational modifications. The detailed knowledge of the target proteins and their O-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 of O-glycosylation from yeast to mammals

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 β-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

Franciszek Siedlecki's Way to the Theatre

Franciszek Wincenty Siedlecki was born in Krakow on 23rd July 1867. He was a graphic artist, painter, stage designer, and theatre critic. After his graduation from a secondary school, he became a law student at the Jagiellonian University. He also studied painting at the Academy of Arts in Munich and Academie Colarossi in Paris. Siedlecki met the theatre for the first time in his hometown Krakow. The year 1908 saw his debut as a decorator in the Town Theatre; he worked no Norwid's Krakus together with Józef Sosnowski. In the following artistic season he made the stage design projects to the Balladyna, Lilla Weneda, and Sen srebrny Salomei. In 1909 the director of the Great Theatre in Warsaw offered him a job of decorator, and he accepted the offer. He designed costumes for the Summer Night Dream. Two years later he was made head of the decorative department a the Unified Theatre. He made interesting decorations to Wesele, Taming of the Shrew, and Irydion. He was also famous for being a co-organiser of the Exhibition of Modern Scenic Painting. In the years of 1914-1919 he was in Switzerland, where he made penetrating analyses of Rudolf Steiner's anthroposophy, and designed stained-glass for Goetheanum. The final fourteen years of his life were devoted exclusively to painting and writing. In the years of 1921-1930 he collaborated with Warsaw journals as a theatrical reviewer. He published his reviews, sketches, and studies in “Sztuka i życie,” “Dzień Polski,” “Południe,”, “Sztuka i Praca,” “Muzyka,” “Rzeczpospolita,” “Życie i Teatr,” “Gazeta Administracji i Policji Państwowej,” and “Scena Polska.” He also participated in many international exhibitions, where he presented his oil paintings, drawings, prints, and water paintings. Having lived 67 years as an artist, he died after a long illness on 1st October 1934 in Warsaw

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

On the trails of ancient and modern Polish: A jubilee book dedicated to Professor Danuta Bieńkowska

Na publikację składają się teksty bardzo zróżnicowane pod względem podejmowanych tematów i stosowanej metodologii. Jest to specyfiką tomów jubileuszowych, które w pewien sposób scala odniesienie do osoby jubilata. W tym przypadku wszystkie teksty dobrze wkomponowują się w całość, tworzą zbiór niejednorodny, lecz ciekawy i oryginalny. Autorzy nawiązują do poruszanych przez jubilata tematów, twórczo je rozwijając lub z nimi polemizując. Pod tym względem osoba Jubilatki, Profesor Danuty Bieńkowskiej, jest gwarantem, że opisywane w publikacji zagadnienia nie mają marginalnego dla nauki charakteru. Tomem zainteresują się przede wszystkim badacze Biblii, jednak krąg potencjalnych odbiorców będzie oczywiście zdecydowanie szerszy ze względu na inne poruszane tu zagadnienia: kwestie normatywne, językoznawczo zorientowaną stylistykę, synchroniczne i diachroniczne badania nad polszczyzną. Z recenzji dr hab. Anetty Luto-Kamińskie