Functional dissection of the roles of COPII core components in Trichoderma reesei cellulase secretion

Trichoderma reesei is a major industrial producer of lignocellulolytic enzymes, yet the molecular mechanisms governing its high-yield protein secretion, particularly for the COPII-mediated early secretory pathways, remain inadequately explored. Here, we employed various genetic manipulations to dissect the functions of core COPII components in T. reesei cellulase secretion. Overexpression of the constitutively active form of the key COPII regulator GTPase, Sar1 H74L, induced severe mycelial growth defect and abolished the secretion of cellulases and xylanases, concurrently repressing the transcription of cellulase genes while elevating ER stress marker genes. Unlike Sar1, T. reesei COPII adaptor paralogs demonstrated functional specialization with differential subcellular localization and distinct impacts on enzyme secretion. Whereas Trsec24 knockdown abolished cellulase secretion, depletion of Trlst1 showed no detectable secretion defects. The Sec24 B-site was further confirmed to be necessary for cellulase secretion by mutagenesis and competitive inhibition. Of note, both Trlst1 and Trsec24 was found to be indispensable for the secretion of xylanase Xyn1, whereas knockdown of either gene exerted minor impact on Xyn2 secretion. These findings collectively advance our understanding of the early secretory pathway in T. reesei, shedding light on the cellular basis for its intrinsic high capacity of protein secretion.