Assays for studying normal versus suppressive ERAD-associated retrotranslocation pathways in yeast
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Published on: October 25, 2019
Satarupa Bhaduri1, Sonya E Neal1
1Division of Biological Sciences, the Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093, USA.
Abstract:
In S. cerevisiae, we identified rhomboid pseudoprotease Dfm1 as the major mediator for removing or retrotranslocating misfolded membrane substrates from the ER (endoplasmic reticulum). Long-standing challenges with rapid suppression of dfm1-null cells have limited the biochemical study of Dfm1's role in ER protein quality control. Here, we provide a protocol for the generation and handling of dfm1-null cells and procedures for studying normal vs. suppressive alternative retrotranslocation pathways. Our methods can be utilized to study other components involved in retrotranslocation. For complete information on the generation and use of this protocol, please refer to Neal et al. (2017, 2018); Neal et al. (2019); Neal et al. (2020).
Insights
We identified Dfm1 as key for removing misfolded ER proteins in yeast. This protocol enables studying Dfm1
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Misfolded membrane proteins in the endoplasmic reticulum (ER) pose a cellular challenge.
- Rhomboid pseudoprotease Dfm1 is implicated in ER-associated degradation.
- Studying Dfm1's function has been hindered by difficulties with dfm1-null yeast cells.
Purpose of the Study:
- To provide a reliable protocol for generating and handling dfm1-null yeast cells.
- To enable biochemical studies of Dfm1's role in ER protein quality control.
- To investigate alternative retrotranslocation pathways.
Main Methods:
- Development of a protocol for creating and maintaining dfm1-null Saccharomyces cerevisiae strains.
- Establishment of procedures to analyze normal and suppressive retrotranslocation pathways.
- Utilizing these methods for biochemical investigation of Dfm1.
Main Results:
- Successfully generated and characterized dfm1-null yeast cells.
- Established methods for studying ER protein retrotranslocation pathways.
- Demonstrated the utility of the protocol for investigating Dfm1's function.
Conclusions:
- Dfm1 is a major mediator of misfolded membrane substrate removal from the ER in S. cerevisiae.
- The provided protocol facilitates the study of Dfm1 and other retrotranslocation components.
- This work overcomes previous limitations in studying ER protein quality control mechanisms.
