TY - JOUR T1 - Functional analysis of Ost3p and Ost6p containing yeast oligosaccharyltransferase<em>s</em> JF - bioRxiv DO - 10.1101/2021.05.17.443621 SP - 2021.05.17.443621 AU - Julia D. Neuhaus AU - Rebekka Wild AU - Jillianne Eyring AU - Rossitza N. Irobalieva AU - Julia Kowal AU - Chia-wei Lin AU - Kaspar P. Locher AU - Markus Aebi Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/05/17/2021.05.17.443621.abstract N2 - The oligosaccharyltransferase (OST) is the central enzyme in the N-glycosylation pathway. It transfers a defined oligosaccharide from a lipid-linker onto the asparagine side chain of proteins. The yeast OST consists of eight subunits and exists in two catalytically distinct isoforms that differ in one subunit, Ost3p or Ost6p. The cryo-electron microscopy structure of the Ost6p containing complex was found to be highly similar to the Ost3p containing OST. OST enzymes with altered Ost3p/Ost6p subunits were generated and functionally analysed. The three C-terminal transmembrane helices were responsible for the higher turnover-rate of the Ost3p vs. the Ost6p containing enzyme in vitro and the more severe hypoglycosylation in Ost3p lacking strains in vivo. Glycosylation of specific OST target sites required the N-terminal thioredoxin domain of Ost3p or Ost6p. This Ost3p/Ost6p dependence was glycosylation site but not protein specific. We concluded that the Ost3p/Ost6p subunits modulate the catalytic activity of OST and provide additional specificity for OST substrate recognition.Competing Interest StatementThe authors have declared no competing interest.ALGasparagine linked glycosylationCHScholesteryl hemisuccinateCryo-EMcryo-electron microscopyDab2,4-diaminobutanoic acidDDMN-dodecyl-beta-D-maltopyranosideDKOdouble knock-outDol-Pdolichyl-phosphateDol-PPdolichyl-pyrophosphateEDTAethylenediaminetetraacetic acidEGTAethyleneglycoltetraacetic acidERendoplasmic reticulumGlcglucoseGlcNAcN-acetylglucoseamineLLOlipid-linked oligosaccharideManmannoseOSToligosaccharyltransferasessOSTsingle-subunit oligosaccharyltransferaseTAMRAtetramethylrhodamineTMtransmembrane span ER -