Nucleophagy in Aspergillus oryzae is Mediated by Autophagosome Formation and Vacuole-Mediated Degradation

Abstract

We previously reported autophagy-mediated degradation of nuclei, nucleophagy, in the filamentous fungus Aspergillus oryzae. In this study, we examined whether nuclei are degraded as a whole. We generated A. oryzae mutants deleted for orthologs of Saccharomyces cerevisiae YPT7 and ATG15 which are required, respectively, for autophagosome-vacuole fusion and vacuolar degradation of autophagic bodies. Degradation of histone H2B-EGFP under starvation conditions was greatly decreased in the ΔAoypt7 and ΔAoatg15 mutants. Fluorescence and electron microscopic observations showed that autophagosomes and autophagic bodies surrounding the entire nuclei were accumulated in the cytoplasm of ΔAoypt7 and the vacuole of ΔAoatg15, respectively. These results indicate that nuclei are engulfed in the autophagosomes as a whole and transported/released into the vacuolar lumen where they are degraded.

Fig. 1

EGFP-AoAtg8 processing assay. a Wild type (PA8GAtg8), ΔAoatg1 (DA1EA8), and ΔAoypt7 (DAoypt7AoAtg8) strains were inoculated into DPY liquid medium and incubated at 30 °C for 24 h. Cells were then transferred to CD + CA (1% casamino acid), CD-N, or CD-C medium, and incubated at 30 °C for 6 h. The mycelia were then frozen in liquid nitrogen, disrupted, and proteins were extracted. Western blotting was performed using an anti-EGFP antibody. b Fluorescence intensities of the detected bands were quantified by ImageJ, and the degradation ratio (EGFP/(EGFP-AoAtg8 + EGFP)) was calculated. To quantify the degree of degradation, only the fluorescent intensity of the band for free EGFP (27 kDa) in (a) was measured as an indicator of complete degradation of EGFP-AoAtg8. Error bars, standard deviation (n = 3). Statistical difference by t test was detected between the following pairs: a–b, a–c, p < 0.01; a–d, a–g, b–h, c–f, c–i, p < 0.001; b–e, p < 0.0001

Fig. 2

AnH2B-EGFP processing assay. a Wild-type strain (NSRku70-1-1A-AnH2B-EGFP), ΔAoatg1 strain (DAoatg1AnH2B), ΔAoatg8 strain (PA8GAtg8), ΔAoypt7 strain (DAoypt7AnH2B), and ΔAoatg15 strain (Aoatg15-AnH2B-EGFP) were inoculated into DPY liquid medium and incubated at 30 °C for 24 h. Then, shifted to CD + CA (1% casamino acid) medium, CD-N medium, and CD-C medium, and incubated at 30 °C for 6 h. The mycelia were then frozen in liquid nitrogen, disrupted, and proteins were extracted. Western blotting was performed using the anti-EGFP antibody. b The fluorescence intensity of the detected bands was quantified by ImageJ, and the degradation ratio (EGFP/(AnH2B-EGFP + EGFP)) was calculated. To quantify the degree of degradation, only the fluorescent intensity of the band for free EGFP (27 kDa) in (a) was measured as an indicator of complete degradation of AnH2B-EGFP. Error bars, standard deviation (WT, n = 6; ΔAoatg8, ΔAoypt7, ΔAoatg15, n = 3; ΔAoatg1, n = 5). Statistical difference by t test was detected between the following pairs: b–n, p < 0.05; a–b, c–f, c–l, p < 0.01; a–c, b–k, b–q, c–l, p < 0.001; b–e, p < 0.0001

Fig. 3

Fluorescence microscopy of EGFP-AoAtg8-expressing strains. Wild type (PA8GAtg8; (a)), ΔAoypt7 (DAoypt7; (b)), and ΔAoatg15 (c) strains were grown in DPY liquid medium for 24 h at 30 °C. Cells were then shifted to CD + CA (1% casamino acid), CD-N, or CD-C medium and incubated for 6 h at 30 °C. They were fixed with 4% paraformaldehyde, and the nuclei were stained with DAPI. PC, phase contrast. White open arrows, PAS; white arrowheads, autophagosomes; white arrows, autophagosomes surrounding the entire nucleus; dotted circles, vacuoles; yellow arrowheads, autophagic bodies; yellow arrows, the entire nucleus is surrounded by autophagic bodies. Bars = 20 µm, 5 µm (zoom) (Color figure online)

Fig. 4

Observation using transmission electron microscopy. After inoculation of wild-type strain (NSRku70-1-1A), ΔAoypt7 strain (DAoypt7), and ΔAoatg15 strain (ΔAoatg15) into DPY liquid medium and incubated at 30 °C for 24 h in a glass base dish and incubated. Then, shifted to CD + CA (1% casamino acid) medium, CD-N medium, and CD-C medium, and incubated at 30 °C for 6 h. Samples were prepared for observation and observed by transmission electron microscopy. N Nucleus, V vacuole, AB autophagic body; yellow-dotted circle, autophagic bodies surrounding the entire nucleus. Bars. 2 µm, 1 µm (lower right photo only)