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Intracellular context affects levels of a chemically dependent destabilizing domain
Mark A Sellmyer1, Ling-chun Chen, Emily L Egeler
1Department of Chemical and Systems Biology, Stanford University, Stanford, California, United States of America.
Plos One
|September 18, 2012
Summary
We developed a destabilizing domain (DD) to control protein levels in cells. Its effectiveness varies by cellular location, suggesting DD can monitor protein quality control machinery.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Protein regulation is vital for understanding cellular functions.
- Existing tools for protein perturbation need enhancement.
- Protein quality control (QC) machinery varies across cellular compartments.
Purpose of the Study:
- To develop and characterize a novel protein destabilizing domain (DD) for controlled protein degradation.
- To investigate the impact of cellular localization on DD-mediated protein regulation.
- To assess the DD's utility as a probe for cellular protein QC.
Main Methods:
- Fusion of the destabilizing domain (DD) to proteins of interest.
- Expression of DD-fusion proteins in mammalian cells, including various cellular compartments (cytoplasm, nucleus, mitochondrial matrix, ER lumen).
- Treatment with Shield-1 (a small molecule stabilizer) and other perturbants affecting protein production, degradation, and ER unfolded protein response.
Main Results:
- DD fusions in the cytoplasm and nucleus were efficiently degraded.
- DD fusions in the mitochondrial matrix or ER lumen accumulated, even without Shield-1.
- ER-targeted DD levels decreased upon induction of the unfolded protein response, showing sensitivity to the degradation environment.
Conclusions:
- The destabilizing domain (DD) is an effective tool for protein perturbation.
- Cellular localization and local protein QC machinery significantly influence DD-mediated protein regulation.
- The DD shows potential as a probe for monitoring cellular protein quality control mechanisms.
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