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

Abstract

In Trichoderma reesei, dolichyl phosphate mannose (dpm) synthase, a key enzyme in the O-glycosylation process, requires three proteins for full activity. In this study, the dpm2 and dpm3 genes coding for the DPMII and DPMIII subunits of T. reesei DPM synthase were cloned and functionally analyzed after expression in the Saccharomyces 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 characterized S. cerevisiae strains expressing the dpm1, 2, 3 or dpm1, 3 genes and analyzed the consequences of dpm expression on protein O-glycosylation in vivo and on the cell wall composition.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

“…Furthermore, a chimeric T. reesei DPMI protein with the yeast transmembrane domain at the C terminus was also not functional in a yeast dpm1⌬ mutant (581). Expression of different combinations of the dpm1, dpm2, and dpm3 genes from Trichoderma in yeast revealed that besides the DPMI catalytic subunit, DPMIII subunit is indispensable for rescuing the dpm1⌬ mutation (582). In contrast, the Dpm1 protein from S. cerevisiae itself is functional in Trichoderma.…”

“…In Trichoderma, DPM synthase forms an enzymatic complex of three proteins, DPMI, DPMII, and DPMIII (581,582), which contrasts with the situation in yeast, where DPM synthase consists of a single Dpm1 protein (583,584). The yeast Dpm1 protein is an equivalent of the DPMI catalytic subunit of Trichoderma DPM synthase (582).…”

“…In Trichoderma, DPM synthase forms an enzymatic complex of three proteins, DPMI, DPMII, and DPMIII (581,582), which contrasts with the situation in yeast, where DPM synthase consists of a single Dpm1 protein (583,584). The yeast Dpm1 protein is an equivalent of the DPMI catalytic subunit of Trichoderma DPM synthase (582). This difference in DPM synthase structures was a reason for grouping the enzymes into two groups: the S. cerevisiae group, having a single-protein enzyme, and the human group, with an enzymatic complex of DPM synthases (585).…”

Dolichol phosphate mannose synthase from the pathogenic yeast Candida albicans is a multimeric enzyme

“…Moreover, we prove that the expression of CaDPM1 alone was not sufficient to rescue the lethal deletion of S. cerevisiae DPM1 (Fig. 2), similar to human and S. pombe DPM1 [12] or T. reseei dpm1 [14]. When expressed in S. cerevisiae CaDPM1 together with CaDPM3 were essential for the formation of functional DPMS complex which suppressed ScΔdpm1 phenotype, whereas additional presence of CaDpm2 increased the enzymatic activity and improved cell fitness.…”

“…The enzymes belonging to the second class are composed of three subunits. To this class belong human [11], Schizosaccharomyces pombe [12], Trichoderma reesei [13,14] or Arabidopsis thaliana DPMS [15]. The group of T. Kinoshita in a series of papers described functions and interactions of three subunits of human DPMS.…”