title: Protein O-mannosyltransferases associate with the translocon to modify translocating polypeptide chains.
abstract: O-Mannosylation and N-glycosylation are essential protein modifications that are initiated in the endoplasmic reticulum (ER). Protein translocation across the ER membrane and N-glycosylation are highly coordinated processes that take place at the translocon-oligosaccharyltransferase (OST) complex. In analogy, it was assumed that protein O-mannosyltransferases (PMTs) also act at the translocon, however, in recent years it turned out that prolonged ER residence allows O-mannosylation of un-/misfolded proteins or slow folding intermediates by Pmt1-Pmt2 complexes. Here, we reinvestigate protein O-mannosylation in the context of protein translocation. We demonstrate the association of Pmt1-Pmt2 with the OST, the trimeric Sec61, and the tetrameric Sec63 complex in vivo by co-immunoprecipitation. The coordinated interplay between PMTs and OST in vivo is further shown by a comprehensive mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants. In addition, we established a microsomal translation/translocation/O-mannosylation system. Using the serine/threonine-rich cell wall protein Ccw5 as a model, we show that PMTs efficiently mannosylate proteins during their translocation into microsomes. This in vitro system will help to unravel mechanistic differences between co- and post-translocational O-mannosylation.
authors: From the Centre for Organismal Studies (COS), Cell Chemistry, Heidelberg University, 69120 Heidelberg, Germany and.
authors: Martin
authors: M
authors: Loibl
authors: Lina
authors: L
authors: Wunderle
authors: Johannes
authors: J
authors: Hutzler
authors: Benjamin L
authors: BL
authors: Schulz
authors: Markus
authors: M
authors: Aebi
authors: Sabine
authors: S
authors: Strahl
mesh_terms: D006031:Glycosylation; D008364:Mannosyltransferases / Q000032:analysis / Q000378:metabolism*; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase
keywords: Endoplasmic Reticulum (ER); Glycosylation; Glycosyltransferases; O-Mannosylation; OST; PMT; Protein Translocation; Yeast