Subcellular fractionation of a hypercellulolytic mutant, Trichoderma reesei Rut-C30: localization of endoglucanase in microsomal fraction

Abstract

The growing mycelia of Trichoderma reesei Rut-C30 are richly endowed with endoplasmic reticula and a variety of pleomorphic subcellular bodies. Mycelia of the culture growing in presence of avicel pHlOl was fractionated in sucrose density gradients, and several morphologically and biochemically distinct fractions were isolated. Mycelia were homogenized in a Bead Beater, and the homogenate was freed of nucleus and wall fragments by low-speed centrifugation before fractionation. Organelle-free cytosol, which did not penetrate the gradient, contained (of the total) 72% of the vanadate-sensitive ATPase, 26% of carboxymethyl cellulase (CMCase), 2% of cytochrome c reductase, and 13% of the protein. Significant fractions separated on a gradient were light vesicles containing heavily stained material inside and ribosomes attached to the outside surface, intact vesicles resembling condensing vacuoles, large vesicles derived from the plasma membrane, and heavy vesicles containing crystalline material. The light-vesicle fraction contained a large portion of the cell-bound CMCase activity. The particle-bound ATPase and cytochrome c reductase activities were concentrated in heavy fractions. The fractionation in the presence of MgCl2 improved the preservation of subcellular bodies derived from the endoplasmic reticula. Although the CMCase activity of the light-vesicle fraction was 4 times higher than the activity in the heavy-vesicle fraction, the CMCase antibody-binding capacities of both fractions were about the same. This discrepancy between the catalytic activity and the antibody-binding capacity suggests that the heavy vesicles might have contained considerable amount of inactive CMCase compared with that present in the light vesicles.

Overexpression of the Saccharomyces cerevisiae Mannosylphosphodolichol Synthase-Encoding Gene in Trichoderma reesei Results in an Increased Level of Protein Secretion and Abnormal Cell Ultrastructure

“…Despite the progress that has been made in studies of the enzymology and molecular biology of Trichoderma hydrolytic proteins (37), the pathway of protein secretion and its regulation are still poorly understood (1,34). The presence of cleavage sites for the signal peptidase and a Kex2-like dipeptidyl peptidase in many cellulase prepropeptides (2,7), the results of immunoelectron microscopy (6,22), and the recent cloning of a sar1 homologue (39) support the hypothesis that the T. reesei secretory pathway involves transport from the endoplasmic reticulum through the Golgi complex to the plasma membrane.…”

Extracellular vesicles carry cellulases in the industrial fungus Trichoderma reesei

“…Since cellulases are secreted enzymes, the control of their biosynthesis may require various steps of the secretory pathway [68]. In T. reesei , studies have shown that cellulases are resident in the endoplasmic reticulum-derived vesicles attached to the outside surface of the membrane [69]. First of all, the cellulase expression and secretion requires an induction that might include the formation of new proteins for constructing secretory pathways [70].…”

Glucose-induced secretion of Trichoderma reesei xylanases

“…While we cannot completely rule out the possibility that these findings are due to mutation in strain ATX2-12 as a result of random integration of plasmid pAT5, we favor the interpretation that these differences are glucose specific. Ghosh and coworkers (9)(10)(11)(12) documented that in the hypersecretory mutant T. reesei RUT C-30, an increased content of the endoplasmic reticulum is developed during growth on cellulose, and they interpreted this finding as explaining the increased rate of secretion of cellulases by this mutant. These authors also showed that this proliferation of the endoplasmic reticulum could be inhibited by the addition of glycerol, hence pointing to a possible repression of endoplasmid reticulum biosynthesis by some kind of carbon catabolite control in T. reesei (11).…”

“…The biochemical understanding of the secretory pathway in filamentous fungi is still in its infancy (for a review, see reference 24). Previous evidence pointed to the existence in T. reesei of two secretory pathways, one constitutive and one inducible (12). Organelles involved in the latter have been shown to accumulate in the hypersecretory mutant strain T. reesei RUT C-30 (9)(10)(11)(12).…”