Distinct but Redundant Roles of <scp>ER</scp> Cargo Receptors p24 and Erv29 in Facilitating Proper Secretion of Cellulases in <scp><i>Trichoderma reesei</i></scp>

Trichoderma reesei represents an important industrial workhorse for (hemi)cellulase production. However, relatively little is known about the details of its secretory pathway ensuring the extremely high-level enzyme secretion and how they might be leveraged for engineering improved protein production. Here, the functions of T. reesei ER cargo receptors p24 and Erv29 in trafficking cellulase were characterised. Whereas individual deletion of p24 or erv29 resulted in only a marginal effect on extracellular cellulase secretion, distinct intracellular trafficking pathways exist for individual hydrolytic enzyme in T. reesei. Notably, the simultaneous absence of p24 and Erv29 abolished the secreted production of cellulases but not xylanases. The secretion defect was accompanied by an apparent intracellular accumulation of cellulases. Mutations of residues on the cytosolic side of p24 and Erv29 supposed to mediate COPII coat recognition also compromised cellulase secretion although the overall ER exit sites (ERES) formation did not seem to be affected. We further revealed that a VPL motif following the signal peptide of CBH2 necessitates its efficient secretion mediated by Erv29. These results indicate that two specific ER cargo receptors complement each other to mediate the proper intracellular trafficking of cellulases and thus ensuring their extracellular secretion.