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2 Acta Biochimica Polonica

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Solubilization and one-step purification of mannosylphosphodolichol synthase from Trichoderma reesei

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Kruszewska J. S. , Perlinska-Lenart U. , Palamarczyk G.

tom 43

nr 2

Mannosylphosphodolichol synthase (MPD-synthase) (EC 2.4.1.830) catalyzing formation of MPD from GDPMan and dolichylphosphate (PD) has been purified from T. reesei cellular membranes almost to homogeneity. Selective solubilization of the enzyme was followed by one step purification on Phenyl-Sepharose column. SDS/ PAGE of the purified enzyme fraction revealed the presence of a protein band of 31 kDa corresponding to the apparent molecular mass of the MPD-synthase purified from S. cerevisiae [Babczinski, P. et al. (1980) Eur. J. Biochem. 105,509-515; Haselbeck A. (1989) Eur. /. Biochem. 181, 663-6681. During solubilization, the enzyme was stabilized by the presence of a lipophilic substrate dolichylphosphate and phospholipids as well as by protease inhibitors. The Phenyl-Sepharose purified enzyme had an absolute requirement for dolichylphosphate and was activated by cAMP dependent protein kinase.

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

100%

Kruszewska J. , Kubicek C. P. , Palamarczyk G.

1994

tom 41

nr 3

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-man- nosylation 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-dependcnt protein glycosylation i.e. MPD-synthase, dolichyl kinase and MPD/ Protein mannosyl transferase was tested. For all enzymes cultivation at 35°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°C, enhanced the dolichol kinase activity measured in the absence of exogenous dolichol. All these results suggest that the amount of endogenous dolichol as well as phosphodolichol in Trichoderma might be increased upon cultivation of the fungus at elevated temperature.