In eukaryotic cells, N-glycosylation plays an essential role in regulating protein properties and activities. Transfer of N-glycans to selected asparagine residues in polypeptides is catalysed by oligosaccharyltransferase (OTase), which is a multimeric complex consisting of eight subunits in yeast. Ost3p and Ost6p are homologous mutually exclusive subunits in the yeast OTase, defining two OTase isoforms with distinct polypeptide substrate-specificities. The oxidoreductase activity of Ost3p/6p mediated by their N-terminal thioredoxin domain is important for efficient site-specific glycosylation. Absence of both Ost3p and Ost6p in yeast causes underglycosylation at many sites, and therefore results in a severe growth defect. To study the functions of Ost3p and Ost6p directly in glycosylation and more broadly, we constructed a tetracycline-off to conditionally knockdown OST3 in ∆ost6 yeast. This yeast strain therefore lacks both Ost3p and Ost6p when tetracycline is present.
Interestingly, it was found that abnormally fast-growing revertants could be easily obtained from the OST3/6 double-deficient yeast. Glycosylation occupancy analysis performed by mass spectrometry showed that most of the detectable glycosylation sites in the isolated growth rescued revertants were still severely underglycosylated. To identify possible suppressor mutations that might cause the growth rescue of the glycosylation defective yeast, we performed whole genome sequencing of the revertants. This sequencing data showed that in multiple independent revertants, mutations accumulated in the tetracycline-off system rather than in native yeast genes. Together, our data suggest that single mutational events that can suppress the important roles of OST3 and OST6 in yeast are very rare, and that yeast growth is very robust to severe defects in N-glycosylation.