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Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae
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A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP
Jiwon Hwang1, Diedre Ribbens1, Sumana Raychaudhuri1
1Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
The EMBO Journal
|September 23, 2016
Summary
Fungal growth under low oxygen requires sterol regulatory element-binding protein (SREBP) processing. A novel protease, Rbd2, and Cdc48 are essential for SREBP activation, controlling its balance between processing and degradation.
Area of Science:
- Molecular Biology
- Cell Biology
- Mycology
Background:
- Fungal pathogens require sterol regulatory element-binding protein (SREBP) activation for virulence.
- Hypoxic growth necessitates SREBP transcription factors.
- Proteolytic release of SREBP from the membrane under low oxygen involves the Dsc E3 ligase complex.
Purpose of the Study:
- Identify novel factors involved in SREBP processing under hypoxia.
- Elucidate the mechanism of SREBP proteolytic release.
- Investigate the role of Rbd2 and Cdc48 in SREBP regulation.
Main Methods:
- Genetic interaction arrays
- Epistasis analysis
- APEX2 proximity biotinylation
- Proteolytic cleavage assays
Main Results:
- Rbd2, a rhomboid family protease, is identified as essential for SREBP proteolytic processing.
- Rbd2 is a Golgi-localized protease that cleaves the transmembrane segment of substrates.
- Dsc E3 ligase acts on SREBP before Rbd2-mediated cleavage.
- Rbd2 binds the AAA-ATPase Cdc48 via a C-terminal SHP box, and this interaction is crucial for SREBP cleavage.
- Cdc48 likely recruits ubiquitinylated substrates for processing.
- In the absence of Rbd2, SREBP precursor is proteasomally degraded.
Conclusions:
- Rbd2 is a key protease regulating SREBP activation under hypoxia.
- The Rbd2-Cdc48 interaction is critical for SREBP processing, balancing activation and degradation.
- Rbd2 activity determines the fate of SREBP, preventing its proteasomal degradation.
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