Modulation of mannosylphosphodolichol synthase and dolichol kinase activity in Trichoderma, related to protein secretion.

Abstract

It has been postulated that exoprotein secretion in Trichoderma is related to their O-glycosylation. In the present paper the involvement of phosphodolichol in this process is described and the key role of mannosylphosphodolichol (MPD) synthase in protein O-mannosylation is discussed. The effect of water soluble phospholipid precursors such as choline and Tween 80, known also to increase secretion of cellulases when added to the medium, on MPD-synthase activity is presented. This effect is positive in the Trichoderma reesei QM 9414 (a low producing strain) but has no influence on the enzyme activity from the RUT C-30 strain selected to overproduce secretion of exoproteins and known to contain an increased cellular amount of endoplasmic reticulum. The positive effect of addition of choline and Tween to the medium on the level of dolichol kinase activity is also demonstrated. The influence of cultivation temperature on the activity of the various enzymes involved in dolichol-dependent protein glycosylation i.e. MPD-synthase, dolichyl kinase and MPD/Protein mannosyl transferase was tested. For all enzymes cultivation at 35 degrees C led to the elevated activity, which was most striking for dolichol kinase, whereas for MPD-synthase and MPD/Protein mannosyl transferase the difference was only apparent in the assay when endogenous phosphodolichol was used as a substrate. Furthermore, lipid extract from the membranes cultivated at elevated temperature, when added to the enzyme obtained from Trichoderma grown at 25 degrees C, enhanced the dolichol kinase activity measured in the absence of exogenous dolichol.(ABSTRACT TRUNCATED AT 250 WORDS)