N-glycosylation plays an essential role in protein folding and function in eukaryotic cells. Transfer of glycan to selected asparagine residues in polypeptides is catalysed by oligosaccharyltransferase (OTase), which is a multimeric complex consisting of eight subunits. Ost3p and Ost6p are mutually exclusive subunits in the yeast OTase, defining two OTase isoforms with distinct substrate-specificities. The oxidoreductase activity of Ost3p/6p mediated by the N-terminal thioredoxin domain is important for efficient site-specific glycosylation. Absence of both Ost3p and Ost6p in yeast causes underglycosylation at many glycosylation sites and therefore results in a severe growth defect. To study the functions of Ost3p and Ost6p in and out of glycosylation, a tetracycline-off system was recruited for conditional knock-down of OST3, while OST6 was genomically deleted by replacing the whole gene with a HisMx cassette. This double-deficient strain has a normal growth rate when tetracycline is absent, as the expression of OST3 is stimulated by the attachment of the tetracycline-controlled transactivator to the tetracycline responsive element in the Tet promoter. Interestingly, we found that the double-deficient yeast had a more severe growth defect than the double-knockout yeast, suggesting that additional mutations had accumulated in the double-knockout yeast. To identify possible suppressor mutations, we isolated fast-growing colonies of the double-deficient yeast and sequenced their genomes. This sequencing data showed that in multiple independent colonies mutations accumulated in the tetracycline-off system rather than in native yeast genes, suggesting that single mutational events that can suppress the important role of OST3 and OST6 in yeast are very rare.