Roles of vacuolar proteases in the autophagic degradation of nuclei and other substrates in Aspergillus oryzae

We previously reported that in the filamentous fungus Aspergillus oryzae entire nuclei were degraded by autophagy when cells are exposed to nutrient starvation. In this study we investigated the roles of vacuolar proteases in autophagic degradation of nuclei (nucleophagy) and bulk autophagy in A. oryzae . We constructed A. oryzae mutants disrupted for pepE and Aoprb1 encoding the orthologs of major vacuolar proteases Pep4 and Prb1, respectively, in Saccharomyces cerevisiae . Surprisingly, disruption of pepE did not result in any autophagy-related phenotypic alterations such as reduced conidiation and decrease in starvation-induced substrate degradation. In contrast, Aoprb1 disruptant exhibited controversial phenotypes; while conidiation was severely impaired and accumulation of autophagic bodies in the vacuoles was observed, degradation of AoAtg8 and histone H2B, markers for autophagic flux and nucleophagy, respectively, exhibited slight or no significant reduction. Double disruption of both pepE and Aoprb1 markedly impaired AoAtg8 degradation, whereas nucleophagy was only partially reduced. These results suggest that in A. oryzae PepE does not play a major role in the autophagic processes, and nuclear degradation requires additional protease(s) other than PepE and AoPrb1.