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

Roles of vacuolar proteases in the autophagic degradation of nuclei and other substrates in Aspergillus oryzae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Roles of vacuolar proteases in the autophagic degradation of nuclei and other substrates in Aspergillus oryzae Hiroto Yamaguchi, Manabu Arioka This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8802980/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Aspergillus oryzae autophagy autophagic body nucleophagy Pep4 Prb1 vacuolar protease Figures Figure 1 Figure 2 Figure 3 Introduction Autophagy is a widely conserved intracellular degradation mechanism in eukaryotes [ 1 ]. It plays important roles in maintaining cellular homeostasis by removing superfluous proteins, damaged organelles, protein aggregates, and intracellular pathogens [ 2 ]. When macroautophagy, a general form of autophagy, is induced by starvation or rapamycin treatment, a cup-shaped intermediate structure called phagophore is formed at phagophore assembly site, a single site around the vacuole. Phagophore grows by extending finger-like membrane protrusions and eventually seals to form an autophagosome, a closed double-membrane structure in which autophagy cargoes are sequestered. Then the outer membrane of autophagosome fuses with vacuole (lysosome in mammals), releasing a structure surrounded by the inner membrane, autophagic body, into the lumen of vacuole [ 3 ]. In the budding yeast Saccharomyces cerevisiae , the autophagic body is degraded by Atg15, the sole vacuolar phospholipase [ 4 – 6 ]. During synthesis, Atg15 undergoes glycosylation, and after the Atg15 precursor is transported to the vacuole via the multivesicular body pathway, it is matured through the processing by vacuolar serine protease Prb1 [ 7 , 8 ]. The active form of Atg15 exhibits phospholipase B-like activity and hydrolyzes the ester bonds of the membrane phospholipids in the autophagic body, thereby contributing its breakdown. Then, substrates released into the vacuolar lumen are degraded by vacuolar proteases, many of which, including Prb1, require maturation by an aspartic protease Pep4 [ 9 ]. Therefore, disruption of either ATG15 , PEP4 , or PRB1 causes the accumulation of autophagic bodies in the vacuole [ 4 , 10 ]. Autophagy is classified into two types: bulk autophagy and selective autophagy. In bulk autophagy, substrates are non-selectively degraded, whereas in selective autophagy, specific substrates such as organelles, protein aggregates, and invading microbes are selectively degraded. Among selective autophagy, the process that specifically degrades nuclei is called nucleophagy [ 11 ]. We previously reported that in the nucleophagy of the filamentous fungus Aspergillus oryzae , the entire nuclei are enclosed by autophagosomes and degraded in the vacuoles [ 12 ]. In the strains disrupted for Aoatg1 and Aoatg8 , the orthologs for S. cerevisiae ATG1 and ATG8 , respectively, bulk autophagy activity as well as nucleophagy activity was significantly suppressed [ 13 , 14 ]. In S. cerevisiae , Atg1 is a kinase essential for the initiation of autophagy, and Atg8 is a ubiquitin-like protein required for membrane fusion during autophagosome formation and for the expansion of phagophores [ 15 ]. In addition, in the strain disrupted for Aoatg15 , an ortholog for S. cerevisiae ATG15 , autophagic bodies containing nuclei accumulated in the vacuole, while the suppression of nuclear degradation was only partial [ 14 ]. In this study, in order to investigate the roles of vacuolar proteases in nucleophagy and bulk autophagy in A. oryzae , we constructed and analyzed the strains disrupted for pepE and/or Aoprb1 , the orthologs for S. cerevisiae PEP4 and PRB1 , respectively. We found that the disruption of pepE did not affect the autophagic processes. In contrast, Aoprb1 disruptant exhibited controversial phenotypes; while conidiation defect and accumulation of autophagic bodies in the vacuoles suggested a role of AoPrb1 in autophagy, no significant decrease in the degradation of autophagic substrates was observed. Double disruption of pepE and Aoprb1 impaired bulk autophagy, but only partially affected nucleophagy. These results suggest that in A. oryzae PepE does not play a major role in the autophagic processes, and nuclear degradation is mediated by protease(s) other than PepE and AoPrb1. Material and methods Strains and growth media A. oryzae strains used in this study are listed in Table S1 . To disrupt pepE , 1.0 kb upstream and downstream flanking regions of pepE and adeA marker were amplified by PCR, and fused by fusion PCR. The amplified fragment was cloned into pUC19, and the DNA fragment containing the deletion cassette was amplified by PCR and transformed into A. oryzae NSRku70-1-1. Disruption of pepE was confirmed by PCR. Disruption of Aoprb1 was conducted in a similar way using sC as a selection marker. The plasmid pgEGA8 containing A. oryzae niaD and egfp linked to Aoatg8 [ 16 ] and the plasmid pNH2BG containing niaD and egfp linked to Aspergillus nidulans histone h2b [ 14 ] were used to transform NSRku70-1-1A (control), Δ Aoatg1 , Δ pepE , Δ Aoprb1 , and Δ pepE Δ Aoprb1 strains. M, M + met, Czapek-Dox (CD), and CD + met media were prepared as described [ 14 ]. CD medium containing 1% casamino acids (CD + A) and CD lacking NaNO 3 (CD-N) or glucose (CD-C) were used for induction of autophagy. Processing assay Mycelia were inoculated in 50 mL of DPY medium and cultured at 30℃ for 24 h. Then medium was replaced with 20 mL of CD + A, CD-N, or CD-C medium, and the mycelia were further cultured at 30℃ for 6 h. After cultivation, the mycelia were frozen in liquid nitrogen and disrupted using ShakeMan6 (Bio Medical Science). The mycelial powder was suspended in 400 µL of extraction buffer [50 mM Tris-HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, Protease Inhibitor Cocktail (Promega)], and the suspension was centrifuged at 4℃ at 13,000 rpm for 5 min. The supernatant was recovered, mixed with 5× Laemmli sample buffer, and boiled at 100℃ for 3 min. SDS-PAGE, immunoblotting, and band quantification were conducted as described [ 14 ]. Fluorescence microscopy observation Conidia or hyphae were cultured in a glass-bottom dish in 200 µL DPY medium at 30℃ for 24 h. Then the medium was replaced with CD + A or CD-N medium, and the mycelia were further cultivated at 30℃ for 6 h. When vacuoles were stained, 7-amino-4-chloromethylcoumain (CMAC) was added 30 min before observation to achieve a final concentration of 10 µM. When nuclei were stained, cells were fixed in 4% paraformaldehyde, stained by 4’,6-diamidino-2-phenylimdole (DAPI) at 2.5 µg/mL, and observed using a fluorescence microscope BX53-33-PH (Olympus, Tokyo, Japan) equipped with a 100× objective lens. Images were captured by DP73 CCD camera and analyzed by cellSens software (Olympus, Tokyo, Japan). Results and Dscussion Identification of pepE and Aoprb1 in A. oryzae To identify Pep4 ortholog in A. oryzae , a BLAST search was performed using Pep4 amino acid sequence as the query on FungiDB ( https://fungidb.org/fungidb/app ). Two proteins, PepE (AO090003000693) and Pep2 (AO090038000229), were identified as orthologs in A. oryzae (Fig. S1 A and S1B). In Pep4 of S. cerevisiae , D109 and D294 (Fig. S1 A and S1B, squares in red) are known as the main catalytic residues, and W116 and Y152 (Fig. S1 A and S1B, squares in blue) are known as residues involved in the catalytic site hydrogen bonding [ 17 ]. The amino acid sequence identity between Pep4 and PepE is 61%, and D109, D294, W116, and Y152 in Pep4 are all conserved in PepE. On the other hand, the amino acid sequence identity between Pep4 and Pep2 is 48% and D294 is not conserved in Pep2. In addition, the expression level of pep2 is less than 1% of that of pepE when grown in the dextrin peptone medium according to the data in the Comprehensive Aspergillus oryzae genome database across the clades (CAoGDX) ( https://nribf21.nrib.go.jp/CAoGDX/ ) [ 18 ]. Therefore, we focused only on PepE in this study and pepE gene was disrupted by homologous recombination using the adeA selection marker (Fig. S2 A). Similarly, to identify Prb1 ortholog in A. oryzae , a BLAST search was performed using Prb1 amino acid sequence as the query on FungiDB, and AO090020000517, named AoPrb1, was identified as an ortholog in A. oryzae (Fig. S1 C). In Prb1 of S. cerevisiae , D325, H357, and S519 (Fig. S1 C, squares in red) are thought to be the main active residues. The alignment score between Prb1 and AoPrb1 was 49%, and the main active residues in Prb1 were all conserved in AoPrb1. To analyze the function of AoPrb1 in autophagy, Aoprb1 gene was disrupted by homologous recombination using the sC selection marker (Fig. S2 B). Furthermore, Aoprb1 gene was disrupted using Δ pepE as a host, generating Δ pepE Δ Aoprb1 double deletion strain. Phenotypic analysis of Δ pepE , Δ Aoprb1 , and Δ pepE Δ Aoprb1 strains A decrease in the number of conidia is a phenotype commonly observed in A. oryzae autophagy-deficient strains [ 14 , 16 , 19 ]. Therefore, the numbers of conidia in the control, Δ Aoatg8 , Δ pepE , Δ Aoprb1 , and Δ pepE Δ Aoprb1 strains were measured and compared. When conidia were inoculated at the center of PD medium and cultured at 30℃ for 4 days, radial growth was almost similar among the strains tested (Fig. 1 A). In contrast, the conidiation in Δ Aoatg8 was severely impaired, again indicating that autophagy is required for conidia formation in A. oryzae (Fig. 1 B). Surprisingly, Δ pepE did not show any change in conidiation compared to the control strain. Conidia formation over time up to 4 days in culture was measured, but there was no difference between the control and Δ pepE strains (Fig. S3 ). In contrast, the number of conidia in Δ Aoprb1 significantly decreased, which further decreased in Δ pepE Δ Aoprb1 . These results suggest that AoPrb1, but not Pep4, plays a role in autophagy. Deletion of Aoprb1 , but not pepE , causes accumulation of autophagic bodies In S. cerevisiae , it is known that when PEP4 or PRB1 is disrupted, autophagic bodies accumulate in the vacuole [ 10 ]. This is because Pep4 proteolytically activates Prb1, and Prb1 directly activates Atg15, the sole phospholipase in the vacuole that is responsible for the degradation of membranes of autophagic bodies [ 5 , 7 , 8 ]. To investigate whether Δ pepE , Δ Aoprb1 , and Δ pepE Δ Aoprb1 also accumulate autophagic bodies in the vacuoles, we observed EGFP-AoAtg8-expressing strains by fluorescence microscopy. Atg8 localizes to autophagosomes and fluorescent protein-labeled Atg8 has been used as a marker for autophagic membranes [ 20 ]. Cells pre-grown in the nutrient-rich medium for 24 h were shifted to and grown for 6 h in the CD + A (synthetic CD medium containing casamino acids) or CD-N (CD without nitrogen source) medium. When grown in CD + A medium, the control strain displayed EGFP fluorescence in the cytoplasm, but not in the vacuoles labeled by CMAC, a dye commonly used for vacuole staining (Fig. 2 A). Similar cytoplasmic EGFP fluorescence was observed in the Δ Aoatg1 and Δ pepE strains. In some cells EGFP puncta which were thought to be aggregates of AoAtg8 oligomers [ 19 ] were also observed (Fig. 2 A, white arrowheads). In contrast, irregularly-shaped fluorescent clusters, some of which colocalized with vacuoles, were present in the Δ Aoprb1 and Δ pepE Δ Aoprb1 strains (Fig. 2 A, yellow arrowheads). Although the identity of these structures is unclear, they were likely to be autophagic bodies, as autophagic bodies involved in the homeostatic recycling of cellular components might accumulate in the vacuole in the absence of vacuolar protease activity. In the nitrogen starvation condition, EGFP fluorescence was uniformly detected in the vacuoles in the control strain (Fig. 2 B, arrows), indicating that the autophagosomes containing EGFP-AoAtg8 formed upon nitrogen starvation were delivered to vacuoles where they were degraded, releasing free EGFP in the vacuolar lumen. In Δ Aoatg1 , the fluorescence of EGFP-AoAtg8 remained in the cytoplasm, indicating that the autophagic delivery of EGFP-AoAtg8 to the vacuole did not occur. In Δ pepE , EGFP fluorescence was detected in the vacuoles as observed in the control strain. In marked contrast, in Δ Aoprb1 and Δ pepE Δ Aoprb1 , autophagic body-like clustered structures accumulated in the vacuoles (Fig. 2 B, yellow arrowheads). It should be noted, however, that some EGFP fluorescence appeared diffuse within the vacuoles, suggesting that EGFP-AoAtg8 was at least partially degraded. Collectively, these results indicate that AoPrb1, but not PepE, is necessary for the degradation of autophagic bodies in A. oryzae . Another ortholog of Pep4, Pep2, might substitute the function of PepE, but Pep2 lacks one of the putative catalytic residues corresponding to D294 in Pep4 and the expression level of pep2 is far lower than that of pepE . We thus speculate that Pep2 is not involved in the activation AoPrb1 and the activation mechanism of AoPrb1 differs from that of Prb1. It is of note, however, that the orthologs of PepE and Pep2 are conserved in other filamentous fungi such as Aspergillus fumigatus , Aspergillus nidulans , and Neurospora crassa , and the four residues related to the catalytic function of Pep4 are all conserved in these orthologs. Since it seems that the expression level of PepE orthologs is higher than that of Pep2 orthologs, as observed in A. oryzae , Pep2 orthologs might have overlapping and compensatory functions with PepE orthologs in these species. Degradation of marker proteins in the processing assay To investigate the effect of disrupting pepE and/or Aoprb1 on the overall autophagy activity, the processing assay using EGFP-AoAtg8 as a marker was performed. When the target protein tagged with EGFP is transported to vacuole and degraded via autophagy, only the EGFP moiety, which is resistant to degradation by vacuolar proteases, accumulates in the vacuole. Since Atg8 is transported to vacuole along with substrates via autophagy, GFP-Atg8 has been used in the processing assay to quantify the overall activity of autophagy [ 14 , 21 , 22 ]. The degradation ratio of EGFP-AoAtg8 in the control strain increased under nitrogen and carbon starvation conditions (Fig. 3 A). In Δ Aoatg1 , the degradation ratio significantly decreased compared to the control strain under all nutrient conditions, indicating that AoAtg8 is degraded through autophagy. In Δ pepE , although the degradation ratio under nutrient-rich condition partially decreased, in the nitrogen and carbon starvation conditions no decrease in the degradation was observed; rather, the degradation ratio was slightly increased. These results again indicate that PepE is not essential for autophagy. In Δ Aoprb1 , however, unexpected from its characteristic phenotypes of autophagy deficiency such as severely impaired conidiation and accumulation of autophagic bodies, only a marginal decrease in the degradation ratio was observed compared to the control strain in the carbon starvation condition, and in the nitrogen starvation condition no significant difference was detected. These results are apparently inconsistent with poor conidiation phenotype and accumulation of autophagic bodies observed in Δ Aoprb1 . The reason for this discrepancy is not clear, but previous studies have shown that the deletion of PRB1 or its ortholog alone leads to a leaky phenotype in terms of degradation of substrate proteins. First, in contrast to the complete blockade of degradation of Pex11-GFP in pep4 Δ strain of S. cerevisiae , some degree of degradation was observed in prb1 Δ [ 23 ]. Second, while the degradation of proteasomal subunits was abolished in pep4 Δ prb1 Δ, degradation was evident in prb1 Δ [ 24 ]. Third, in Fusarium graminearum , even though autophagic bodies accumulated in the vacuoles of Δ Fgprb1 strain, degradation of GFP-FgAtg8 was still observed, albeit at a reduced level compared to the wild type [ 25 ]. In this study, some EGFP fluorescence appeared diffuse within the vacuoles, suggesting that EGFP-AoAtg8 was at least partially degraded in Δ Aoprb1 . Thus, it is likely that loss of PRB1 or its orthologs reduces, but does not completely abolish, autophagy activity, thereby affecting conidiation. In contrast, in Δ pepE Δ Aoprb1 , the degradation ratio partially but significantly decreased under all nutrient conditions. Nucleophagy in the stains deficient for vacuolar proteases Finally we investigated the roles of pepE and Aoprb1 on nucleophagy using the cells expressing Aspergillus nidulans histone H2B (AnH2B)-EGFP. When AnH2B-EGFP is expressed in A. oryzae , it localizes to nuclei and therefore the degradation of AnH2B-EGFP has been used as a hallmark to quantify nucleophagy activity [ 13 , 14 ]. As reported in the previous study [ 14 ], the degradation ratio increased under nitrogen and carbon starvation conditions in the control strain, while in Δ Aoatg8 the degradation ratio significantly decreased under all nutrient conditions, indicating that nuclei were degraded via autophagy (Fig. 3 B). In Δ pepE and Δ Aoprb1 , no significant difference from the control strain was observed, except for a slight decrease when Δ pepE was starved for nitrogen source. In contrast, the degradation ratio significantly decreased in Δ pepE Δ Aoprb1 , although the decrease was not as evident as that observed for EGFP-AoAtg8. These results suggest that vacuolar protease(s) other than PepE and AoPrb1 were involved in the degradation of nuclei. It is enigmatic that almost no decrease was detected in the degradation of EGFP-AoAtg8 and AnH2B-EGFP in the Δ Aoprb1 strain compared to the wild type strain. One possible explanation for this is that if the membranes of autophagic bodies accumulated in the vacuoles of Δ Aoprb1 were fragile and quickly disrupted during sample preparation for processing assay, proteins contained in the autophagic bodies were readily exposed to and degraded by surrounding vacuolar proteases; it has been shown that processing by Pep4p alone could result in the generation of active vacuolar proteases [ 26 ]. In contrast, in Δ pepE Δ Aoprb1 , the degradation of both EGFP-AoAtg8 and AnH2B-EGFP was significantly decreased. In this case, even if autophagic body membranes were similarly disrupted, proteins were not degraded due to greatly reduced vacuolar protease activity in the cells lacking both PepE and AoPrb1. 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Fungal Genet Biol 144:103449. 10.1016/j.fgb.2020.103449 Mechler B, Müller H, Wolf DH (1987) Maturation of vacuolar (lysosomal) enzymes in yeast: proteinase yscA and proteinase yscB are catalysts of the processing and activation event of carboxypeptidase yscY. EMBO J 6:2157–2163. 10.1002/j.1460-2075.1987.tb02483.x Additional Declarations No competing interests reported. Supplementary Files TableS1.docx TableS2.docx Supplementalfigures.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8802980","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":605324105,"identity":"17af7433-2a63-486b-9d12-1ff2a2a4a16a","order_by":0,"name":"Hiroto Yamaguchi","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Hiroto","middleName":"","lastName":"Yamaguchi","suffix":""},{"id":605324106,"identity":"adfeb7ae-d507-405f-9adb-cf2537dfa69b","order_by":1,"name":"Manabu Arioka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABJElEQVRIie3QsUrEMBjA8S8E6lLtel16r9CQtfRFXDwK3QKC4NQhcpBbDucegr7C9Q1SCnUpvVW4wSv3Ak7ioGLSU6lcy+Emkj+0hJBfkhbAZPqTIY44BAAY887c58AfJrEi3+vwIdLuWXS37pC+nJviqkmT1akzU+uekhB86eTNQgTgzCRcnO+TUT2ZkmW5ZmmhbpiWkSIYk0zEMKrOgKY9x1RIuBtrzbgi+JhL8B+3ltsIddUHAGrvi3FL3mt2p8mbJhLvyHiA+JpkQrKlJuiLZIr4A4RUaEoW1xHLFMnnZWS7ElOS1rFNqgnv+xavOmqa+XPIbldFvnlJQu9EIjVzGXjefVHSnj/2I6me3U2QpQdI0AOi22v7xttfEJPJZPq3fQDLTmorZuq/6QAAAABJRU5ErkJggg==","orcid":"","institution":"The University of Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Manabu","middleName":"","lastName":"Arioka","suffix":""}],"badges":[],"createdAt":"2026-02-06 05:39:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8802980/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8802980/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104744782,"identity":"8c525238-0ddc-4843-b6fa-8a8a3c220138","added_by":"auto","created_at":"2026-03-16 17:17:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":135150,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic analysis of D\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epepE\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, D\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAoprb1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and D\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epepE\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAoprb1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The phenotype of \u003cem\u003epepE\u003c/em\u003e or/and \u003cem\u003eAoprb1\u003c/em\u003e deletion strains were compared. Conidia (1.0×10\u003csup\u003e4\u003c/sup\u003e) of control (NSRku70-1-1AsC), D\u003cem\u003eAoatg8\u003c/em\u003e (DAoatg8sC), D\u003cem\u003epepE\u003c/em\u003e (DpepEsC), D\u003cem\u003eAoprb1\u003c/em\u003e, and D\u003cem\u003epepE\u003c/em\u003eD\u003cem\u003eAoprb1\u003c/em\u003e strains were inoculated on the center of PD medium and incubated for 4 days at 30℃. (B) The numbers of conidia were counted by collecting the conidia from the plates that had been cultured under the condition in (A), using a hemocytometer.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8802980/v1/84db581745974560d9024fff.png"},{"id":104744783,"identity":"60ff9c31-1090-49cb-bb54-86bf0d00d963","added_by":"auto","created_at":"2026-03-16 17:17:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":214362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroscopy observation of EGFP-AoAtg8\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe conidia or hyphae of EGFP-AoAtg8-expressing control, D\u003cem\u003eAoatg1\u003c/em\u003e, D\u003cem\u003epepE\u003c/em\u003e, D\u003cem\u003eAoprb1\u003c/em\u003e, and D\u003cem\u003epepE\u003c/em\u003eD\u003cem\u003eAoprb1\u003c/em\u003e strains were cultured in a glass-bottom dish in 200 mL DPY medium for 24 h at 30℃. The medium was then replaced with CD+AA (A) or CD-N (B) medium, and the mycelia were further cultivated for 6 h at 30℃. Vacuoles were stained by CMAC. Scale bars, 5 mm. White arrowheads, AoAtg8 oligomers; yellow arrowheads, autophagic body-like structures in the vacuoles; arrows, EGFP fluorescence diffused in the vacuoles.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8802980/v1/4b1fb646a907a9ffb6c5a321.png"},{"id":104744784,"identity":"b3507eb7-b013-49a3-848b-fcc40b0eb766","added_by":"auto","created_at":"2026-03-16 17:17:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":100632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of vacuolar degradation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epepE\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e or/and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAoprb1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion strains by processing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mycelia were inoculated in 50 mL of DPY medium and cultured at 30℃ for 24 h. The medium was replaced with 20 mL of CD+A, CD-N, or CD-C medium, and the mycelia were further cultured for 6 h at 30℃. The mycelia were then frozen in liquid nitrogen, disrupted, and proteins were extracted. The degradation ratio (%) was calculated by dividing the band intensity of free EGFP with the sum of intensities of free EGFP and EGFP-fused full-length protein. Data are means ± standard deviation. Statistical analysis was performed by Student’s \u003cem\u003et\u003c/em\u003e-test in which pairwise comparison of the degradation ratios was performed between the control and each gene disruption strain grown in the same condition. (A) Degradation of EGFP-AoAtg8. EGFP-AoAtg8-expressing control, D\u003cem\u003eAoatg1\u003c/em\u003e, D\u003cem\u003epepE\u003c/em\u003e, D\u003cem\u003eAoprb1\u003c/em\u003e, and D\u003cem\u003epepE\u003c/em\u003eD\u003cem\u003eAoprb1\u003c/em\u003e strains were used for the processing assay. *, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Control and D\u003cem\u003eAoprb1\u003c/em\u003e, n=9; D\u003cem\u003eAoatg1\u003c/em\u003e, D\u003cem\u003eAoatg15\u003c/em\u003e, and D\u003cem\u003epepE\u003c/em\u003e, n=3; D\u003cem\u003epepE\u003c/em\u003eD\u003cem\u003eAoprb1\u003c/em\u003e, n=6. (B) Degradation of AnH2B-EGFP. AnH2B-EGFP-expressing control, D\u003cem\u003eAoatg8\u003c/em\u003e, D\u003cem\u003epepE\u003c/em\u003e, D\u003cem\u003eAoprb1\u003c/em\u003e, and D\u003cem\u003epepE\u003c/em\u003eD\u003cem\u003eAoprb1\u003c/em\u003e strains were used for the processing assay. *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001. D\u003cem\u003epepE\u003c/em\u003eD\u003cem\u003eAoprb1\u003c/em\u003e, n=9; others, n=3.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8802980/v1/708fe0abdc78b18c8775ac8d.png"},{"id":108803732,"identity":"1f259334-7b8f-4b7a-ac02-15ca19fed13b","added_by":"auto","created_at":"2026-05-08 15:05:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":767298,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8802980/v1/6d6eafbe-c51b-49ba-868c-21699d5dc9aa.pdf"},{"id":104783274,"identity":"795bc845-6a98-4e90-9a01-adae54e24afc","added_by":"auto","created_at":"2026-03-17 07:58:30","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":23348,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8802980/v1/19256ae3f1aadb2f5fd6de52.docx"},{"id":104744786,"identity":"b630d9f2-a094-48e8-8b69-44bc995fa13f","added_by":"auto","created_at":"2026-03-16 17:17:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20266,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8802980/v1/ae0c5916a062c3dfa215b65a.docx"},{"id":104744787,"identity":"78e400a6-c92c-474a-be1c-2212edbec649","added_by":"auto","created_at":"2026-03-16 17:17:28","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5818246,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigures.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8802980/v1/86aa18828b365910287e33e1.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Roles of vacuolar proteases in the autophagic degradation of nuclei and other substrates in Aspergillus oryzae","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAutophagy is a widely conserved intracellular degradation mechanism in eukaryotes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It plays important roles in maintaining cellular homeostasis by removing superfluous proteins, damaged organelles, protein aggregates, and intracellular pathogens [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. When macroautophagy, a general form of autophagy, is induced by starvation or rapamycin treatment, a cup-shaped intermediate structure called phagophore is formed at phagophore assembly site, a single site around the vacuole. Phagophore grows by extending finger-like membrane protrusions and eventually seals to form an autophagosome, a closed double-membrane structure in which autophagy cargoes are sequestered. Then the outer membrane of autophagosome fuses with vacuole (lysosome in mammals), releasing a structure surrounded by the inner membrane, autophagic body, into the lumen of vacuole [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the budding yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, the autophagic body is degraded by Atg15, the sole vacuolar phospholipase [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. During synthesis, Atg15 undergoes glycosylation, and after the Atg15 precursor is transported to the vacuole via the multivesicular body pathway, it is matured through the processing by vacuolar serine protease Prb1 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The active form of Atg15 exhibits phospholipase B-like activity and hydrolyzes the ester bonds of the membrane phospholipids in the autophagic body, thereby contributing its breakdown. Then, substrates released into the vacuolar lumen are degraded by vacuolar proteases, many of which, including Prb1, require maturation by an aspartic protease Pep4 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, disruption of either \u003cem\u003eATG15\u003c/em\u003e, \u003cem\u003ePEP4\u003c/em\u003e, or \u003cem\u003ePRB1\u003c/em\u003e causes the accumulation of autophagic bodies in the vacuole [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAutophagy is classified into two types: bulk autophagy and selective autophagy. In bulk autophagy, substrates are non-selectively degraded, whereas in selective autophagy, specific substrates such as organelles, protein aggregates, and invading microbes are selectively degraded. Among selective autophagy, the process that specifically degrades nuclei is called nucleophagy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We previously reported that in the nucleophagy of the filamentous fungus \u003cem\u003eAspergillus oryzae\u003c/em\u003e, the entire nuclei are enclosed by autophagosomes and degraded in the vacuoles [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the strains disrupted for \u003cem\u003eAoatg1\u003c/em\u003e and \u003cem\u003eAoatg8\u003c/em\u003e, the orthologs for \u003cem\u003eS. cerevisiae ATG1\u003c/em\u003e and \u003cem\u003eATG8\u003c/em\u003e, respectively, bulk autophagy activity as well as nucleophagy activity was significantly suppressed [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In \u003cem\u003eS. cerevisiae\u003c/em\u003e, Atg1 is a kinase essential for the initiation of autophagy, and Atg8 is a ubiquitin-like protein required for membrane fusion during autophagosome formation and for the expansion of phagophores [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, in the strain disrupted for \u003cem\u003eAoatg15\u003c/em\u003e, an ortholog for \u003cem\u003eS. cerevisiae ATG15\u003c/em\u003e, autophagic bodies containing nuclei accumulated in the vacuole, while the suppression of nuclear degradation was only partial [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, in order to investigate the roles of vacuolar proteases in nucleophagy and bulk autophagy in \u003cem\u003eA. oryzae\u003c/em\u003e, we constructed and analyzed the strains disrupted for \u003cem\u003epepE\u003c/em\u003e and/or \u003cem\u003eAoprb1\u003c/em\u003e, the orthologs for \u003cem\u003eS. cerevisiae PEP4\u003c/em\u003e and \u003cem\u003ePRB1\u003c/em\u003e, respectively. We found that the disruption of \u003cem\u003epepE\u003c/em\u003e did not affect the autophagic processes. In contrast, \u003cem\u003eAoprb1\u003c/em\u003e disruptant exhibited controversial phenotypes; while conidiation defect and accumulation of autophagic bodies in the vacuoles suggested a role of AoPrb1 in autophagy, no significant decrease in the degradation of autophagic substrates was observed. Double disruption of \u003cem\u003epepE\u003c/em\u003e and \u003cem\u003eAoprb1\u003c/em\u003e impaired bulk autophagy, but only partially affected nucleophagy. These results suggest that in \u003cem\u003eA. oryzae\u003c/em\u003e PepE does not play a major role in the autophagic processes, and nuclear degradation is mediated by protease(s) other than PepE and AoPrb1.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e \u003cb\u003eStrains and growth media\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. oryzae\u003c/em\u003e strains used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. To disrupt \u003cem\u003epepE\u003c/em\u003e, 1.0 kb upstream and downstream flanking regions of \u003cem\u003epepE\u003c/em\u003e and \u003cem\u003eadeA\u003c/em\u003e marker were amplified by PCR, and fused by fusion PCR. The amplified fragment was cloned into pUC19, and the DNA fragment containing the deletion cassette was amplified by PCR and transformed into \u003cem\u003eA. oryzae\u003c/em\u003e NSRku70-1-1. Disruption of \u003cem\u003epepE\u003c/em\u003e was confirmed by PCR. Disruption of \u003cem\u003eAoprb1\u003c/em\u003e was conducted in a similar way using \u003cem\u003esC\u003c/em\u003e as a selection marker. The plasmid pgEGA8 containing \u003cem\u003eA. oryzae niaD\u003c/em\u003e and \u003cem\u003eegfp\u003c/em\u003e linked to \u003cem\u003eAoatg8\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and the plasmid pNH2BG containing \u003cem\u003eniaD\u003c/em\u003e and \u003cem\u003eegfp\u003c/em\u003e linked to \u003cem\u003eAspergillus nidulans histone h2b\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] were used to transform NSRku70-1-1A (control), Δ\u003cem\u003eAoatg1\u003c/em\u003e, Δ\u003cem\u003epepE\u003c/em\u003e, Δ\u003cem\u003eAoprb1\u003c/em\u003e, and Δ\u003cem\u003epepE\u003c/em\u003eΔ\u003cem\u003eAoprb1\u003c/em\u003e strains. M, M\u0026thinsp;+\u0026thinsp;met, Czapek-Dox (CD), and CD\u0026thinsp;+\u0026thinsp;met media were prepared as described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. CD medium containing 1% casamino acids (CD\u0026thinsp;+\u0026thinsp;A) and CD lacking NaNO\u003csub\u003e3\u003c/sub\u003e (CD-N) or glucose (CD-C) were used for induction of autophagy.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProcessing assay\u003c/h2\u003e \u003cp\u003eMycelia were inoculated in 50 mL of DPY medium and cultured at 30℃ for 24 h. Then medium was replaced with 20 mL of CD\u0026thinsp;+\u0026thinsp;A, CD-N, or CD-C medium, and the mycelia were further cultured at 30℃ for 6 h. After cultivation, the mycelia were frozen in liquid nitrogen and disrupted using ShakeMan6 (Bio Medical Science). The mycelial powder was suspended in 400 \u0026micro;L of extraction buffer [50 mM Tris-HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, Protease Inhibitor Cocktail (Promega)], and the suspension was centrifuged at 4℃ at 13,000 rpm for 5 min. The supernatant was recovered, mixed with 5\u0026times; Laemmli sample buffer, and boiled at 100℃ for 3 min. SDS-PAGE, immunoblotting, and band quantification were conducted as described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFluorescence microscopy observation\u003c/h3\u003e\n\u003cp\u003eConidia or hyphae were cultured in a glass-bottom dish in 200 \u0026micro;L DPY medium at 30℃ for 24 h. Then the medium was replaced with CD\u0026thinsp;+\u0026thinsp;A or CD-N medium, and the mycelia were further cultivated at 30℃ for 6 h. When vacuoles were stained, 7-amino-4-chloromethylcoumain (CMAC) was added 30 min before observation to achieve a final concentration of 10 \u0026micro;M. When nuclei were stained, cells were fixed in 4% paraformaldehyde, stained by 4\u0026rsquo;,6-diamidino-2-phenylimdole (DAPI) at 2.5 \u0026micro;g/mL, and observed using a fluorescence microscope BX53-33-PH (Olympus, Tokyo, Japan) equipped with a 100\u0026times; objective lens. Images were captured by DP73 CCD camera and analyzed by cellSens software (Olympus, Tokyo, Japan).\u003c/p\u003e"},{"header":"Results and Dscussion","content":"\u003cp\u003e \u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003epepE\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eAoprb1\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eA. oryzae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify Pep4 ortholog in \u003cem\u003eA. oryzae\u003c/em\u003e, a BLAST search was performed using Pep4 amino acid sequence as the query on FungiDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://fungidb.org/fungidb/app\u003c/span\u003e\u003cspan address=\"https://fungidb.org/fungidb/app\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Two proteins, PepE (AO090003000693) and Pep2 (AO090038000229), were identified as orthologs in \u003cem\u003eA. oryzae\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B). In Pep4 of \u003cem\u003eS. cerevisiae\u003c/em\u003e, D109 and D294 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B, squares in red) are known as the main catalytic residues, and W116 and Y152 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B, squares in blue) are known as residues involved in the catalytic site hydrogen bonding [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The amino acid sequence identity between Pep4 and PepE is 61%, and D109, D294, W116, and Y152 in Pep4 are all conserved in PepE. On the other hand, the amino acid sequence identity between Pep4 and Pep2 is 48% and D294 is not conserved in Pep2. In addition, the expression level of \u003cem\u003epep2\u003c/em\u003e is less than 1% of that of \u003cem\u003epepE\u003c/em\u003e when grown in the dextrin peptone medium according to the data in the Comprehensive Aspergillus oryzae genome database across the clades (CAoGDX) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://nribf21.nrib.go.jp/CAoGDX/\u003c/span\u003e\u003cspan address=\"https://nribf21.nrib.go.jp/CAoGDX/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, we focused only on PepE in this study and \u003cem\u003epepE\u003c/em\u003e gene was disrupted by homologous recombination using the \u003cem\u003eadeA\u003c/em\u003e selection marker (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eSimilarly, to identify Prb1 ortholog in \u003cem\u003eA. oryzae\u003c/em\u003e, a BLAST search was performed using Prb1 amino acid sequence as the query on FungiDB, and AO090020000517, named AoPrb1, was identified as an ortholog in \u003cem\u003eA. oryzae\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). In Prb1 of \u003cem\u003eS. cerevisiae\u003c/em\u003e, D325, H357, and S519 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC, squares in red) are thought to be the main active residues. The alignment score between Prb1 and AoPrb1 was 49%, and the main active residues in Prb1 were all conserved in AoPrb1. To analyze the function of AoPrb1 in autophagy, \u003cem\u003eAoprb1\u003c/em\u003e gene was disrupted by homologous recombination using the \u003cem\u003esC\u003c/em\u003e selection marker (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Furthermore, \u003cem\u003eAoprb1\u003c/em\u003e gene was disrupted using Δ\u003cem\u003epepE\u003c/em\u003e as a host, generating Δ\u003cem\u003epepE\u003c/em\u003eΔ\u003cem\u003eAoprb1\u003c/em\u003e double deletion strain.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhenotypic analysis of Δ\u003c/b\u003e \u003cb\u003epepE\u003c/b\u003e, \u003cb\u003eΔ\u003c/b\u003e\u003cb\u003eAoprb1\u003c/b\u003e, \u003cb\u003eand Δ\u003c/b\u003e\u003cb\u003epepE\u003c/b\u003e\u003cb\u003eΔ\u003c/b\u003e\u003cb\u003eAoprb1\u003c/b\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA decrease in the number of conidia is a phenotype commonly observed in \u003cem\u003eA. oryzae\u003c/em\u003e autophagy-deficient strains [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, the numbers of conidia in the control, Δ\u003cem\u003eAoatg8\u003c/em\u003e, Δ\u003cem\u003epepE\u003c/em\u003e, Δ\u003cem\u003eAoprb1\u003c/em\u003e, and Δ\u003cem\u003epepE\u003c/em\u003eΔ\u003cem\u003eAoprb1\u003c/em\u003e strains were measured and compared. When conidia were inoculated at the center of PD medium and cultured at 30℃ for 4 days, radial growth was almost similar among the strains tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast, the conidiation in Δ\u003cem\u003eAoatg8\u003c/em\u003e was severely impaired, again indicating that autophagy is required for conidia formation in \u003cem\u003eA. oryzae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Surprisingly, Δ\u003cem\u003epepE\u003c/em\u003e did not show any change in conidiation compared to the control strain. Conidia formation over time up to 4 days in culture was measured, but there was no difference between the control and Δ\u003cem\u003epepE\u003c/em\u003e strains (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). In contrast, the number of conidia in Δ\u003cem\u003eAoprb1\u003c/em\u003e significantly decreased, which further decreased in Δ\u003cem\u003epepE\u003c/em\u003eΔ\u003cem\u003eAoprb1\u003c/em\u003e. These results suggest that AoPrb1, but not Pep4, plays a role in autophagy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDeletion of\u003c/b\u003e \u003cb\u003eAoprb1\u003c/b\u003e, \u003cb\u003ebut not\u003c/b\u003e \u003cb\u003epepE\u003c/b\u003e, \u003cb\u003ecauses accumulation of autophagic bodies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eS. cerevisiae\u003c/em\u003e, it is known that when \u003cem\u003ePEP4\u003c/em\u003e or \u003cem\u003ePRB1\u003c/em\u003e is disrupted, autophagic bodies accumulate in the vacuole [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This is because Pep4 proteolytically activates Prb1, and Prb1 directly activates Atg15, the sole phospholipase in the vacuole that is responsible for the degradation of membranes of autophagic bodies [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. To investigate whether Δ\u003cem\u003epepE\u003c/em\u003e, Δ\u003cem\u003eAoprb1\u003c/em\u003e, and Δ\u003cem\u003epepE\u003c/em\u003eΔ\u003cem\u003eAoprb1\u003c/em\u003e also accumulate autophagic bodies in the vacuoles, we observed EGFP-AoAtg8-expressing strains by fluorescence microscopy. Atg8 localizes to autophagosomes and fluorescent protein-labeled Atg8 has been used as a marker for autophagic membranes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Cells pre-grown in the nutrient-rich medium for 24 h were shifted to and grown for 6 h in the CD\u0026thinsp;+\u0026thinsp;A (synthetic CD medium containing casamino acids) or CD-N (CD without nitrogen source) medium. When grown in CD\u0026thinsp;+\u0026thinsp;A medium, the control strain displayed EGFP fluorescence in the cytoplasm, but not in the vacuoles labeled by CMAC, a dye commonly used for vacuole staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Similar cytoplasmic EGFP fluorescence was observed in the Δ\u003cem\u003eAoatg1\u003c/em\u003e and Δ\u003cem\u003epepE\u003c/em\u003e strains. In some cells EGFP puncta which were thought to be aggregates of AoAtg8 oligomers [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] were also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, white arrowheads). In contrast, irregularly-shaped fluorescent clusters, some of which colocalized with vacuoles, were present in the Δ\u003cem\u003eAoprb1\u003c/em\u003e and Δ\u003cem\u003epepE\u003c/em\u003eΔ\u003cem\u003eAoprb1\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, yellow arrowheads). Although the identity of these structures is unclear, they were likely to be autophagic bodies, as autophagic bodies involved in the homeostatic recycling of cellular components might accumulate in the vacuole in the absence of vacuolar protease activity.\u003c/p\u003e \u003cp\u003eIn the nitrogen starvation condition, EGFP fluorescence was uniformly detected in the vacuoles in the control strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, arrows), indicating that the autophagosomes containing EGFP-AoAtg8 formed upon nitrogen starvation were delivered to vacuoles where they were degraded, releasing free EGFP in the vacuolar lumen. In Δ\u003cem\u003eAoatg1\u003c/em\u003e, the fluorescence of EGFP-AoAtg8 remained in the cytoplasm, indicating that the autophagic delivery of EGFP-AoAtg8 to the vacuole did not occur. In Δ\u003cem\u003epepE\u003c/em\u003e, EGFP fluorescence was detected in the vacuoles as observed in the control strain. In marked contrast, in Δ\u003cem\u003eAoprb1\u003c/em\u003e and Δ\u003cem\u003epepE\u003c/em\u003eΔ\u003cem\u003eAoprb1\u003c/em\u003e, autophagic body-like clustered structures accumulated in the vacuoles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, yellow arrowheads). It should be noted, however, that some EGFP fluorescence appeared diffuse within the vacuoles, suggesting that EGFP-AoAtg8 was at least partially degraded. Collectively, these results indicate that AoPrb1, but not PepE, is necessary for the degradation of autophagic bodies in \u003cem\u003eA. oryzae\u003c/em\u003e. Another ortholog of Pep4, Pep2, might substitute the function of PepE, but Pep2 lacks one of the putative catalytic residues corresponding to D294 in Pep4 and the expression level of \u003cem\u003epep2\u003c/em\u003e is far lower than that of \u003cem\u003epepE\u003c/em\u003e. We thus speculate that Pep2 is not involved in the activation AoPrb1 and the activation mechanism of AoPrb1 differs from that of Prb1. It is of note, however, that the orthologs of PepE and Pep2 are conserved in other filamentous fungi such as \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, \u003cem\u003eAspergillus nidulans\u003c/em\u003e, and \u003cem\u003eNeurospora crassa\u003c/em\u003e, and the four residues related to the catalytic function of Pep4 are all conserved in these orthologs. Since it seems that the expression level of PepE orthologs is higher than that of Pep2 orthologs, as observed in \u003cem\u003eA. oryzae\u003c/em\u003e, Pep2 orthologs might have overlapping and compensatory functions with PepE orthologs in these species.\u003c/p\u003e\n\u003ch3\u003eDegradation of marker proteins in the processing assay\u003c/h3\u003e\n\u003cp\u003eTo investigate the effect of disrupting \u003cem\u003epepE\u003c/em\u003e and/or \u003cem\u003eAoprb1\u003c/em\u003e on the overall autophagy activity, the processing assay using EGFP-AoAtg8 as a marker was performed. When the target protein tagged with EGFP is transported to vacuole and degraded via autophagy, only the EGFP moiety, which is resistant to degradation by vacuolar proteases, accumulates in the vacuole. Since Atg8 is transported to vacuole along with substrates via autophagy, GFP-Atg8 has been used in the processing assay to quantify the overall activity of autophagy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe degradation ratio of EGFP-AoAtg8 in the control strain increased under nitrogen and carbon starvation conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In Δ\u003cem\u003eAoatg1\u003c/em\u003e, the degradation ratio significantly decreased compared to the control strain under all nutrient conditions, indicating that AoAtg8 is degraded through autophagy. In Δ\u003cem\u003epepE\u003c/em\u003e, although the degradation ratio under nutrient-rich condition partially decreased, in the nitrogen and carbon starvation conditions no decrease in the degradation was observed; rather, the degradation ratio was slightly increased. These results again indicate that PepE is not essential for autophagy. In Δ\u003cem\u003eAoprb1\u003c/em\u003e, however, unexpected from its characteristic phenotypes of autophagy deficiency such as severely impaired conidiation and accumulation of autophagic bodies, only a marginal decrease in the degradation ratio was observed compared to the control strain in the carbon starvation condition, and in the nitrogen starvation condition no significant difference was detected. These results are apparently inconsistent with poor conidiation phenotype and accumulation of autophagic bodies observed in Δ\u003cem\u003eAoprb1\u003c/em\u003e. The reason for this discrepancy is not clear, but previous studies have shown that the deletion of \u003cem\u003ePRB1\u003c/em\u003e or its ortholog alone leads to a leaky phenotype in terms of degradation of substrate proteins. First, in contrast to the complete blockade of degradation of Pex11-GFP in \u003cem\u003epep4\u003c/em\u003eΔ strain of \u003cem\u003eS. cerevisiae\u003c/em\u003e, some degree of degradation was observed in \u003cem\u003eprb1\u003c/em\u003eΔ [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Second, while the degradation of proteasomal subunits was abolished in \u003cem\u003epep4\u003c/em\u003eΔ \u003cem\u003eprb1\u003c/em\u003eΔ, degradation was evident in \u003cem\u003eprb1\u003c/em\u003eΔ [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Third, in \u003cem\u003eFusarium graminearum\u003c/em\u003e, even though autophagic bodies accumulated in the vacuoles of Δ\u003cem\u003eFgprb1\u003c/em\u003e strain, degradation of GFP-FgAtg8 was still observed, albeit at a reduced level compared to the wild type [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study, some EGFP fluorescence appeared diffuse within the vacuoles, suggesting that EGFP-AoAtg8 was at least partially degraded in Δ\u003cem\u003eAoprb1\u003c/em\u003e. Thus, it is likely that loss of \u003cem\u003ePRB1\u003c/em\u003e or its orthologs reduces, but does not completely abolish, autophagy activity, thereby affecting conidiation. In contrast, in Δ\u003cem\u003epepE\u003c/em\u003eΔ\u003cem\u003eAoprb1\u003c/em\u003e, the degradation ratio partially but significantly decreased under all nutrient conditions.\u003c/p\u003e\n\u003ch3\u003eNucleophagy in the stains deficient for vacuolar proteases\u003c/h3\u003e\n\u003cp\u003eFinally we investigated the roles of \u003cem\u003epepE\u003c/em\u003e and \u003cem\u003eAoprb1\u003c/em\u003e on nucleophagy using the cells expressing \u003cem\u003eAspergillus nidulans\u003c/em\u003e histone H2B (AnH2B)-EGFP. When AnH2B-EGFP is expressed in \u003cem\u003eA. oryzae\u003c/em\u003e, it localizes to nuclei and therefore the degradation of AnH2B-EGFP has been used as a hallmark to quantify nucleophagy activity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. As reported in the previous study [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the degradation ratio increased under nitrogen and carbon starvation conditions in the control strain, while in Δ\u003cem\u003eAoatg8\u003c/em\u003e the degradation ratio significantly decreased under all nutrient conditions, indicating that nuclei were degraded via autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In Δ\u003cem\u003epepE\u003c/em\u003e and Δ\u003cem\u003eAoprb1\u003c/em\u003e, no significant difference from the control strain was observed, except for a slight decrease when Δ\u003cem\u003epepE\u003c/em\u003e was starved for nitrogen source. In contrast, the degradation ratio significantly decreased in Δ\u003cem\u003epepE\u003c/em\u003eΔ\u003cem\u003eAoprb1\u003c/em\u003e, although the decrease was not as evident as that observed for EGFP-AoAtg8. These results suggest that vacuolar protease(s) other than PepE and AoPrb1 were involved in the degradation of nuclei.\u003c/p\u003e \u003cp\u003eIt is enigmatic that almost no decrease was detected in the degradation of EGFP-AoAtg8 and AnH2B-EGFP in the Δ\u003cem\u003eAoprb1\u003c/em\u003e strain compared to the wild type strain. One possible explanation for this is that if the membranes of autophagic bodies accumulated in the vacuoles of Δ\u003cem\u003eAoprb1\u003c/em\u003e were fragile and quickly disrupted during sample preparation for processing assay, proteins contained in the autophagic bodies were readily exposed to and degraded by surrounding vacuolar proteases; it has been shown that processing by Pep4p alone could result in the generation of active vacuolar proteases [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In contrast, in Δ\u003cem\u003epepE\u003c/em\u003eΔ\u003cem\u003eAoprb1\u003c/em\u003e, the degradation of both EGFP-AoAtg8 and AnH2B-EGFP was significantly decreased. In this case, even if autophagic body membranes were similarly disrupted, proteins were not degraded due to greatly reduced vacuolar protease activity in the cells lacking both PepE and AoPrb1. It should be noted that the degradation of AnH2B-EGFP was not as severely affected as EGFP-AoAtg8, suggesting that protease(s) besides PepE and AoPrb1 were involved in the degradation of nuclei.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHY conducted the research, analyzed the data, and wrote the manuscript. MA supervised the research and corrected the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful for the support by the grant (20K05783) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to MA.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOhsumi Y (1999) Molecular mechanism of autophagy in yeast, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Philos Trans R Soc Lond B Biol Sci 354:1577\u0026ndash;1580. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1098/rstb.1999.0501\u003c/span\u003e\u003cspan address=\"10.1098/rstb.1999.0501\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGatica D, Lahiri V, Klionsky DJ (2018) Cargo recognition and degradation by selective autophagy. 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EMBO J 6:2157\u0026ndash;2163. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/j.1460-2075.1987.tb02483.x\u003c/span\u003e\u003cspan address=\"10.1002/j.1460-2075.1987.tb02483.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aspergillus oryzae, autophagy, autophagic body, nucleophagy, Pep4, Prb1, vacuolar protease","lastPublishedDoi":"10.21203/rs.3.rs-8802980/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8802980/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe previously reported that in the filamentous fungus \u003cem\u003eAspergillus oryzae\u003c/em\u003e 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 \u003cem\u003eA. oryzae\u003c/em\u003e. We constructed \u003cem\u003eA. oryzae\u003c/em\u003e mutants disrupted for \u003cem\u003epepE\u003c/em\u003e and \u003cem\u003eAoprb1\u003c/em\u003e encoding the orthologs of major vacuolar proteases Pep4 and Prb1, respectively, in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Surprisingly, disruption of \u003cem\u003epepE\u003c/em\u003e did not result in any autophagy-related phenotypic alterations such as reduced conidiation and decrease in starvation-induced substrate degradation. In contrast, \u003cem\u003eAoprb1\u003c/em\u003e 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 \u003cem\u003epepE\u003c/em\u003e and \u003cem\u003eAoprb1\u003c/em\u003e 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.\u003c/p\u003e","manuscriptTitle":"Roles of vacuolar proteases in the autophagic degradation of nuclei and other substrates in Aspergillus oryzae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-16 17:17:11","doi":"10.21203/rs.3.rs-8802980/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ecbf1731-5556-42df-9e45-1b40c758e210","owner":[],"postedDate":"March 16th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-04T18:04:10+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T18:09:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-16 17:17:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8802980","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8802980","identity":"rs-8802980","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}