A Method to Monitor Protein Turnover by Flow Cytometry and to Screen for Factors that Control Degradation by Fluorescence-Activated Cell Sorting
The protein quality control network consists of multiple proteins or protein complexes that monitor proteome integrity by mediating protein folding and the removal of proteins that cannot be folded. An integral component of this network is the ubiquitin-proteasome system, which controls the degradation of thousands of cellular proteins. A number of questions remain unanswered regarding the degradation of misfolded proteins. For example, how are substrates recognized and triaged? What are the identities of the components involved? And finally, what substrates are targeted by any given component of the quality control network? Finding answers to these questions is what inspires our work in protein quality control. Further characterization of protein quality control mechanisms requires methods that can reliably quantify turnover rates of model substrates. One such method is based on flow cytometry. Here, we present protocols detailing how to assess protein stability with flow cytometry and how fluorescence-activated cell sorting (FACS) can be used to screen for factors important for protein quality control and protein turnover.
Key wordsGreen fluorescent protein (GFP) Flow cytometry Fluorescence-activated cell sorting (FACS) High-throughput screen Protein quality control Protein misfolding Ubiquitin-proteasome system
We appreciate the insightful discussions and comments by all members, former and current, of the Mayor lab. We thank Justin Wong and Andy Johnson of the UBC Flow Cytometry Facility for their assistance with cell sorting, analysis, and training. Finally, we are grateful to the Hieter lab for access to their FACSCalibur flow cytometer. This research was funded by a Canadian Institutes of Health Research (CIHR) grant, and TM is a MSFHR new investigator.
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