The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Abstract

Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics, or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase complex, the key enzyme of N-glycosylation. Deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity towards compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas deletion of OST6, a functional homolog of OST3, has no effect. Oligosaccharyltransferase activity in vitro determined in membranes from rot1-1ost3∆ cells was found to be decreased to 45% compared to wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase CPY, Gas1 or DPAP B, displayed an underglycosylation pattern. By affinity chromatography a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the Omannosylation process, as glycosylation of distinct glycoproteins of this type were affected as well. Altogether the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation.

Protein O-mannosylation: What we have learned from baker's yeast

“…Both types of glycosylation are initiated in the ER, O-mannosylation by distinct PMT complexes and N-glycosylation by the membrane protein complex OST [129]. Activity of both enzyme complexes depends on the essential ER chaperone Rot1 [130], and they can act on the same acceptor protein (e.g., [94][95][96]131]). It was shown that PMTs and OST compete for acceptor protein substrates, and that O-mannosylation can precede N-glycosylation, suggesting that PMTs have the potential to alter the number of N-linked glycans [96] (M.L.…”

Impact of Yeast Glycosylation Pathway on Cell Integrity and Morphology

“…Moreover, Rot1 was also found to be involved in the O-mannosylation process, as glycosylation of distinct glycoproteins of this type was affected as well. Altogether, it can be assumed that Rot1 acts also as a chaperone required to ensure proper glycosylation [38].…”

BIONIC: Biological Network Integration using Convolutions

“…6). ROT1 encodes an essential chaperone required for N-and O-glycosylation in yeast 55 , and is required for normal levels of β-1,6-glucan 56 . Both KRE5 and BIG1 are also required for proper β-1,6 glucan synthesis 57,58 .…”