Novel conidiation regulatory mechanism by N-end rule pathway
in Aspergillus oryzae
Mizuki TANAKA
Department of Applied Biological Science,
Tokyo University of Agriculture and Technology
Research objective
The stability of intracellular proteins depends on their N-terminal amino acid residues. Ineukaryotes, the ubiquitin ligase Ubr1 generally recognizes intracellular proteins, whose Nterminal amino acids are arginine, lysine, or histidine, leading to their degradation by theproteasome. This protein degradation mechanism, called the N-end rule, is involved in various
cellular processes, such as the regulation of peptide transporter gene expression. We found that
disruption of the Ubr1 ortholog gene (ubrA) in Aspergillus oryzae markedly enhances
conidiospore formation. There have been no reports on the N-end rule in filamentous fungi,
and its involvement in conidiospore formation is unknown. In this study, we analyzed the Nend rule in A. oryzae and its involvement in the degradation of transcription factors that control
conidiospore formation.
Methods
To examine whether the N-end rule is applicable in A. oryzae, we expressed ubiquitin-fused
green fluorescent proteins (GFPs)(1), which can express GFP with different N-terminal aminoacids, in wild-type and ubrA disruption strains. GFP protein levels were compared in the cells
by western blot analysis using an anti-GFP antibody. In addition, to investigate whether the Nend rule is involved in the degradation of transcription factors that control conidiosporeformation, the transcription factors fused 3×FLAG tag to the C-terminus were expressed inwild-type and ubrA disruption strains. The amounts of these transcription factors in the cells
were compared by western blot analysis using an anti-FLAG antibody.
Results
Analysis of N-end rule in A. oryzae
GFPs with N-terminal amino acids methionine or arginine were expressed as a fusion proteinwith ubiquitin in A. oryzae. Since the ubiquitin of this fusion protein is cleaved bydeubiquitinating enzymes, this allowed us to examine the expression level of GFP withdifferent N-terminal amino acids. In the wild-type strain, only GFP with N-terminalmethionine (M-GFP) was detected, whereas both M-GFP and GFP with N-terminal arginine(R-GFP) were detected in the ubrA disruption strain. These results indicate that R-GFP wasdegraded in a UbrA-dependent manner, indicating that the N-end rule is applicable to A. oryzae.
Involvement of the N-end rule in degradation of transcription factors that regulate theconidiospore formation
Conidiospore formation in filamentous fungi is mainly regulated by the master regulator
BrlA. Two transcription start sites of the brlA gene provide two types of translation products:
BrlAα and BrlAβ. The N-terminus of BrlAβ is extended 23 amino acids from that of Brlα.
Each BrlA with a C-terminal 3×FLAG tag was expressed in the wild-type and ubrA disruption
strains. Western blot analysis revealed no significant difference in the abundance of a proteinof approximately 50 kDa, which is presumed to be the full-length translational product of brlA.
In contrast, fragments of approximately 30 kDa of both BrlA proteins were also detected.
These fragments accumulated more in the ubrA disruption strain than in the wild strain. Theseresults suggest that both BrlAa and BrlAb are cleaved, and that their C-terminal cleavage
fragments are degraded in a UbrA-dependent manner.
Conclusion
This study provides the first evidence that the N-end rule is applicable to filamentous fungi.
We also showed that BrlA is cleaved near the middle, and that the resulting C-terminal
fragment may be degraded in a UbrA-dependent manner. Since the DNA-binding domain ofBrlA is located near the C-terminus, we speculate that the accumulated C-terminal fragmentof BrlA promotes conidiospore formation in the ubrA disruption strain. Future studies will be
performed to verify whether the C-terminal cleavage fragment of BrlA maintains
transcriptional ability and to identify the cleavage site of BrlA. The findings will elucidate theregulatory mechanism of conidiospore formation via the N-end rule.
References
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