EXPRESSION AND SECRETION OF BARLEY CYSTEINE ENDOPEPTIDASE-B AND CELLOBIOHYDROLASE-I IN TRICHODERMA-REESEI

Localization of expression and secretion of a heterologous barley cyst eine endopeptidase (EPB) and the homologous main cellobiohydrolase I ( CBHI) in a Trichoderma reesei transformant expressing both proteins we re studied. The transformant was grown on solid medium with Avicel cel lulose and lactose to induce the cbh1 promoter for the synthesis of th e native CBHI and the recombinant barley protein linked to a cbh1 expr ession cassette. Differences in localization of expression between the two proteins were clearly indicated by in situ hybridization, indirec t immunofluorescence, and immunoelectron microscopy. In young hyphae, native-size recombinant epb mRNA was localized to apical compartments. In older cultures, it was also seen in subapical compartments but not in hyphae from the colony center. The recombinant EPB had a higher mo lecular weight than the native barley protein, probably due to glycosy lation and differential processing in the fungal host. As was found wi th its transcripts, recombinant EPB was localized in apical and subapi cal compartments of hyphae. The cbh1 mRNA and CBHI were both localized to all hyphae of a colony, which suggests that the endogenous CBHI wa s also secreted from these. In immunoelectron microscopy, the endoplas mic reticulum and spherical vesicles assumed to contribute to secretio n were labeled by both CBHI and EPB antibodies while only CBHI was loc alized in elongated vesicles close to the plasma membrane and in hypha l walls. The results indicate that in addition to young apical cells, more mature hyphae in a colony may secrete proteins.