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Updated: Aug 26, 2025
Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae
Published on: April 18, 2016
A two-tiered system for selective receptor and transporter protein degradation
Charlotte Kathleen Golden1, Thomas David Daniel Kazmirchuk1, Erin Kate McNally1
1Department of Biology, Concordia University, Montreal, Quebec, Canada.
Abstract:
Diverse physiology relies on receptor and transporter protein down-regulation and degradation mediated by ESCRTs. Loss-of-function mutations in human ESCRT genes linked to cancers and neurological disorders are thought to block this process. However, when homologous mutations are introduced into model organisms, cells thrive and degradation persists, suggesting other mechanisms compensate. To better understand this secondary process, we studied degradation of transporter (Mup1) or receptor (Ste3) proteins when ESCRT genes (VPS27, VPS36) are deleted in Saccharomyces cerevisiae using live-cell imaging and organelle biochemistry. We find that endocytosis remains intact, but internalized proteins aberrantly accumulate on vacuolar lysosome membranes within cells. Here they are sorted for degradation by the intralumenal fragment (ILF) pathway, constitutively or when triggered by substrates, misfolding or TOR activation in vivo and in vitro. Thus, the ILF pathway functions as fail-safe layer of defense when ESCRTs disregard their clients, representing a two-tiered system that ensures degradation of surface polytopic proteins.
Insights
The ESCRT pathway degrades cell surface proteins. When ESCRT genes are deleted, the intralumenal fragment (ILF) pathway compensates, degrading proteins via a two-tiered system.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Protein degradation is crucial for cellular homeostasis and relies on pathways like ESCRT.
- Loss-of-function mutations in ESCRT genes are linked to diseases, but compensatory mechanisms in model organisms remain unclear.
Purpose of the Study:
- To investigate compensatory protein degradation pathways when ESCRT function is impaired.
- To elucidate the role of the intralumenal fragment (ILF) pathway in degrading surface proteins in Saccharomyces cerevisiae.
Main Methods:
- Utilized live-cell imaging and organelle biochemistry in Saccharomyces cerevisiae.
- Studied the degradation of transporter (Mup1) and receptor (Ste3) proteins.
- Investigated ESCRT gene deletions (VPS27, VPS36).
Main Results:
- Endocytosis remained functional despite ESCRT gene deletion.
- Internalized proteins accumulated on vacuolar/lysosome membranes.
- The ILF pathway mediated protein degradation, both constitutively and upon specific triggers.
Conclusions:
- The ILF pathway acts as a fail-safe mechanism for surface polytopic protein degradation when ESCRTs are absent.
- This study reveals a two-tiered system ensuring protein degradation, crucial for diverse physiology.
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