Cloning and functional analysis of the dpm2 and dpm3 genes from Trichoderma reesei expressed in a Saccharomyces cerevisiae dpm1Δ mutant strain

Abstract

AbstractInTrichoderma reesei, dolichyl phosphate mannose (dpm) synthase, a key enzyme in the O-glycosylation process, requires three proteins for full activity. In this study, thedpm2anddpm3genes coding for the DPMII and DPMIII subunits ofT. reeseiDPM synthase were cloned and functionally analyzed after expression in theSaccharomyces cerevisiae dpm1Δ[genotype (BY4743;his3Δ1; /leu2Δ0; lys2Δ0; /ura3Δ0; YPR183w::kanMX4] mutant. It was found that apart from the catalytic subunit DPMI, the DPMIII subunit is also essential to form an active DPM synthase in yeast. Additional expression of the DPMII protein, considered to be a regulatory subunit of DPM synthase, decreased the enzymatic activity. We also characterizedS. cerevisiaestrains expressing thedpm1,2,3ordpm1, 3genes and analyzed the consequences ofdpmexpression on protein O-glycosylationin vivoand on the cell wall composition.