Autophagy is a widely conserved intracellular degradation mechanism in eukaryotes that maintains cellular homeostasis by removing superfluous proteins, damaged organelles, protein aggregates, and intracellular pathogens. When macroautophagy is induced by starvation or rapamycin treatment, a cup-shaped intermediate structure called the phagophore forms at a single site adjacent to the vacuole. The phagophore expands through finger-like membrane protrusions and eventually seals to form an autophagosome, a closed double-membrane structure that sequesters autophagy cargos. The outer membrane of the autophagosome then fuses with the vacuole, releasing a structure surrounded by the inner membrane, called the autophagic body, into the vacuolar lumen. What happens next depends on the enzymatic machinery inside the vacuole.

In the budding yeast Saccharomyces cerevisiae, the autophagic body is degraded by Atg15, the sole vacuolar phospholipase. During its biosynthesis, Atg15 undergoes glycosylation and is transported to the vacuole via the multivesicular body pathway, where its precursor is processed into the mature form by the vacuolar serine protease Prb1. The active form of Atg15 exhibits phospholipase B-like activity and hydrolyzes the ester bonds of membrane phospholipids in the autophagic body, promoting its breakdown. The cargos released into the vacuolar lumen are then degraded by vacuolar proteases, many of which require maturation by the aspartic protease Pep4. Consequently, disruption of either ATG15, PEP4, or PRB1 causes the accumulation of autophagic bodies in the vacuole. This proteolytic cascade has long guided expectations for autophagic degradation in fungi.

The research team, comprising Hiroto Yamaguchi, Junka Fujita, and Manabu Arioka, had previously reported that in the filamentous fungus Aspergillus oryzae, entire nuclei are degraded by autophagy when cells are exposed to nutrient starvation. This process, termed nucleophagy, is a form of selective autophagy in which specific substrates such as organelles are selectively degraded rather than cytoplasmic components being non-selectively broken down as in bulk autophagy. In the nucleophagy of A. oryzae, entire nuclei are enclosed by autophagosomes and delivered to vacuoles for degradation. In strains disrupted for Aoatg1 and Aoatg8, the orthologs of the yeast autophagy initiation kinase and the ubiquitin-like protein required for autophagosome formation, both bulk autophagy and nucleophagy were significantly impaired. However, the role of vacuolar proteases in this fungal system remained unexplored.

To fill this gap, the researchers constructed A. oryzae mutants disrupted for pepE and Aoprb1, encoding the orthologs of the major vacuolar proteases Pep4 and Prb1 in Saccharomyces cerevisiae. A BLAST search using the Pep4 amino acid sequence identified two candidate orthologs in A. oryzae: PepE and Pep2. The amino acid sequence identity between Pep4 and PepE is 61 percent, and all four key catalytic residues known from yeast Pep4 are conserved in PepE. In contrast, Pep2 shares only 48 percent identity with Pep4 and lacks one of the conserved catalytic residues. The expression level of pep2 in hyphae is less than 1 percent of that of pepE. For the serine protease ortholog, a BLAST search identified AoPrb1, with an alignment score of 49 percent to yeast Prb1, and all main active residues were conserved. The researchers disrupted these genes by homologous recombination using adeA and sC selection markers, generating single and double deletion strains.

The first surprise came from phenotypic analysis. A decrease in the number of conidia is a phenotype commonly observed in A. oryzae autophagy-deficient strains, and the severely impaired conidiation in the Aoatg8 deletion strain again confirmed that autophagy is required for conidia formation in this fungus. Astonishingly, the pepE deletion strain showed no change in conidiation compared to the control strain, even when conidia formation was measured over time up to four days in culture. In marked contrast, the number of conidia in the Aoprb1 deletion strain significantly decreased, and this phenotype was further exacerbated in the pepE and Aoprb1 double deletion strain. These results immediately suggested that AoPrb1, but not PepE, plays a role in autophagy, contradicting the expectation based on the yeast model where Pep4 is the master activator of the vacuolar proteolytic system.

Fluorescence microscopy of strains expressing EGFP-AoAtg8, a marker for autophagic membranes, provided further evidence of this unexpected asymmetry. Under nutrient-rich conditions, the control strain displayed EGFP fluorescence in the cytoplasm but not in vacuoles stained with CMAC. Similar patterns were observed in the Aoatg1 and pepE deletion strains. Under nitrogen starvation, EGFP fluorescence was uniformly detected in the vacuoles of the control strain, indicating that autophagosomes containing EGFP-AoAtg8 formed upon nitrogen starvation were delivered to vacuoles where they were degraded, releasing free EGFP in the vacuolar lumen. In the Aoprb1 deletion strain, however, autophagic body-like clustered structures accumulated in the vacuoles, although some EGFP fluorescence appeared diffuse within the vacuoles, suggesting that EGFP-AoAtg8 was at least partially degraded. The pepE deletion strain showed no such accumulation, demonstrating that AoPrb1, but not PepE, is necessary for the degradation of autophagic bodies in A. oryzae.

The researchers then performed processing assays to quantify overall autophagy activity. When a target protein tagged with EGFP is transported to the vacuole and degraded via autophagy, only the EGFP moiety, which is resistant to degradation by vacuolar proteases, accumulates in the vacuole. The degradation ratio of EGFP-AoAtg8 in the control strain increased under nitrogen and carbon starvation conditions, while the Aoatg1 deletion strain showed significantly decreased degradation under all nutrient conditions. In the pepE deletion strain, no significant difference in degradation was observed, confirming that PepE is not essential for autophagy. Unexpectedly, the Aoprb1 deletion strain showed only a marginal decrease in degradation under starvation conditions, despite its characteristic phenotypes of severely impaired conidiation and autophagic body accumulation. However, the double deletion strain showed a significant reduction in degradation under all nutrient conditions, and the degradation was significantly reduced compared with the single deletion strains under carbon and nitrogen starvation conditions, suggesting that both PepE and AoPrb1 contribute to the degradation of EGFP-AoAtg8.

The enigma of the Aoprb1 single deletion strain, which accumulates autophagic bodies yet degrades marker proteins almost normally, may be explained by the fragility of accumulated autophagic body membranes during sample preparation. If these membranes quickly disrupted during processing, the proteins contained within would be readily exposed to and degraded by surrounding vacuolar proteases. It has been shown that processing by Pep4 alone can result in the generation of active vacuolar proteases. In the double deletion strain, even if autophagic body membranes were similarly disrupted, proteins would not be degraded due to greatly reduced vacuolar protease activity in cells lacking both PepE and AoPrb1. Previous studies in other systems support this leaky phenotype interpretation: in Saccharomyces cerevisiae, some degree of degradation was observed in prb1 deletion strains compared to the complete blockade seen in pep4 deletion strains, and in Fusarium graminearum, autophagic bodies accumulated in vacuoles of the Fgprb1 deletion strain yet GFP-FgAtg8 degradation was still observed at a reduced level.

For nucleophagy, the researchers used cells expressing Aspergillus nidulans histone H2B tagged with EGFP, which localizes to nuclei and serves as a hallmark for quantifying nucleophagy activity. The degradation ratio of AnH2B-EGFP increased under nitrogen and carbon starvation in the control strain, while the Aoatg8 deletion strain showed significantly decreased degradation under all conditions, confirming that nuclei were degraded via autophagy. In the pepE and Aoprb1 single deletion strains, no significant difference from the control was observed except for a slight reduction in the Aoprb1 strain under carbon starvation. In the double deletion strain, degradation was lower overall but significantly decreased only under carbon starvation. Fluorescence microscopy revealed that under nitrogen starvation, diffuse fluorescence was observed not only in the control strain but also in the pepE and Aoprb1 single deletion strains, suggesting nuclei had been degraded in the vacuole. In contrast, irregularly shaped fluorescent structures were detected in the double deletion strain, likely representing nuclei enclosed within accumulated autophagic bodies. These results suggest that while AoPrb1 partially contributes to nuclear degradation upon carbon starvation, vacuolar proteases other than PepE and AoPrb1 are involved in the degradation of nuclei during starvation-induced nucleophagy, pointing to an as-yet-unidentified proteolytic system that mediates the final breakdown of entire nuclei in this filamentous fungus.

Subject of Research: Roles of vacuolar proteases in autophagy and nucleophagy in the filamentous fungus Aspergillus oryzae

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

Article References: Yamaguchi, H., Fujita, J., & Arioka, M. (2026). Roles of vacuolar proteases in the autophagic degradation of nuclei and other substrates in Aspergillus oryzae. Molecular Biology Reports, 53(1), Article 1818. https://doi.org/10.1007/s11033-026-13002-3

Image Credits: AI Generated

DOI: 10.1007/s11033-026-13002-3

Keywords: autophagy, nucleophagy, vacuolar proteases, Aspergillus oryzae, filamentous fungi, PepE, AoPrb1, autophagic bodies, EGFP-AoAtg8, histone H2B, cell biology, molecular biology