The bZIP Transcription Factor HapX Is Post-Translationally Regulated to Control Iron Homeostasis in Aspergillus fumigatus
Abstract
:1. Introduction
2. Results
2.1. HapX Is Rapidly Degraded during Adaptation from Iron-Depleted to Iron-Replete Conditions in a Proteasome Dependent Manner
2.2. HapX Turnover Is Very Fast during Iron Limitation
2.3. HapX Affinity Purification Studies Identified Protein Interactors and Post-Translational Modifications during the Adaptation from Iron-Deficiency to Iron-Replete Conditions
2.3.1. HapX Specifically Interacts with Fbx22 and SumO during sFe Conditions
Inactivation of Fbx22 and SumO Affects Iron Resistance, HapX Levels and Degradation and Heme Biosynthesis
2.3.2. HapX Is Ubiquitinated, Sumoylated and Phosphorylated during sFe Conditions
Point Mutations in HapX K161, K242 and T319 Affect Iron Resistance, HapX Levels and Degradation and Heme Biosynthesis
2.4. Overexpression of hapX Negatively Affects Growth and Production of Iron-Related Metabolites
3. Discussion
3.1. HapX Is Regulated at the Protein Level
3.2. HapX Interacts with Fbx22 and SumO and Is Prost-Translationally Modified Specifically under sFe Conditions
4. Materials and Methods
4.1. Fungal Isolates and Culture Conditions
4.2. Generation of Mutant Strains
4.3. Nucleic Acid Manipulations, Quantitative Real-Time RT-PCR and PpIX Analysis
4.4. Western Blotting
4.5. Myc-Trap Immunoprecipitation of MHapX Fusion Protein
4.6. In-Solution Digest of Myc-Trap Eluates
4.7. nLC-MS/MS Analysis
4.8. Protein Database Search
4.9. Western Blot Detection of Proteins after Myc-Trap
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
RIA | Reductive iron assimilation |
CBC | CCAAT-binding complex |
CRR | Cysteine rich region |
−Fe | Iron-deplete |
+Fe | Iron-replete |
sFe | Shift from iron-depleted to iron-replete |
FsC | Fusarinine C |
TAFC | Triacetylfusarinine C |
chx | Cycloheximide |
LFQ | Label-free quantification abundances |
SCF | Skp, Cullin, F-box |
hFe | high iron |
PpIX | Protoporphyrin IX |
NDSM | Negatively charged amino acid-dependent SUMOylation motif |
AMM | Aspergillus minimal medium |
TCA | Trichloroacetic acid |
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López-Berges, M.S.; Scheven, M.T.; Hortschansky, P.; Misslinger, M.; Baldin, C.; Gsaller, F.; Werner, E.R.; Krüger, T.; Kniemeyer, O.; Weber, J.; et al. The bZIP Transcription Factor HapX Is Post-Translationally Regulated to Control Iron Homeostasis in Aspergillus fumigatus. Int. J. Mol. Sci. 2021, 22, 7739. https://doi.org/10.3390/ijms22147739
López-Berges MS, Scheven MT, Hortschansky P, Misslinger M, Baldin C, Gsaller F, Werner ER, Krüger T, Kniemeyer O, Weber J, et al. The bZIP Transcription Factor HapX Is Post-Translationally Regulated to Control Iron Homeostasis in Aspergillus fumigatus. International Journal of Molecular Sciences. 2021; 22(14):7739. https://doi.org/10.3390/ijms22147739
Chicago/Turabian StyleLópez-Berges, Manuel Sánchez, Mareike Thea Scheven, Peter Hortschansky, Matthias Misslinger, Clara Baldin, Fabio Gsaller, Ernst R. Werner, Thomas Krüger, Olaf Kniemeyer, Jakob Weber, and et al. 2021. "The bZIP Transcription Factor HapX Is Post-Translationally Regulated to Control Iron Homeostasis in Aspergillus fumigatus" International Journal of Molecular Sciences 22, no. 14: 7739. https://doi.org/10.3390/ijms22147739