Abstract
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.
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References
Balch WE, Morimoto RI, Dillin A, Kelly JW (2008) Adapting proteostasis for disease intervention. Science 319(5865):916–919
Kleiger G, Mayor T (2014) Perilous journey: a tour of the ubiquitin-proteasome system. Trends Cell Biol 24(6):352–359
Geiler-Samerotte KA, Dion MF, Budnik BA, Wang SM, Hartl DL, Drummond DA (2011) Misfolded proteins impose a dosage-dependent fitness cost and trigger a cytosolic unfolded protein response in yeast. Proc Natl Acad Sci U S A 108(2):680–685
Finley D, Ulrich HD, Sommer T, Kaiser P (2012) The ubiquitin-proteasome system of Saccharomyces cerevisiae. Genetics 192(2):319–360
Vembar SS, Brodsky JL (2008) One step at a time: endoplasmic reticulum-associated degradation. Nat Rev Mol Cell Biol 9(12):944–957
Gardner RG, Nelson ZW, Gottschling DE (2005) Degradation-mediated protein quality control in the nucleus. Cell 120(6):803–815
Heck JW, Cheusng SK, Hampton RY (2010) Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1. Proc Natl Acad Sci U S A 107(3):1106–1111
Comyn SA, Young BP, Loewen CJ, Mayor T (2016) Prefoldin promotes proteasomal degradation of cytosolic proteins with missense mutations by maintaining substrate solubility. PLoS Genet 12(7):e1006184
Fang NN, Chan GT, Zhu M, Comyn SA, Persaud A, Deshaies RJ, Rotin D, Gsponer J, Mayor T (2014) Rsp5/Nedd4 is the main ubiquitin ligase that targets cytosolic misfolded proteins following heat stress. Nat Cell Biol 16(12):1227–1237
Fang NN, Ng AH, Measday V, Mayor T (2011) Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins. Nat Cell Biol 13(11):1344–1352
Khosrow-Khavar F, Fang NN, Ng AH, Winget JM, Comyn SA, Mayor T (2012) The yeast ubr1 ubiquitin ligase participates in a prominent pathway that targets cytosolic thermosensitive mutants for degradation. G3 (Bethesda) 2(5):619–628
Yewdell JW, Lacsina JR, Rechsteiner MC, Nicchitta CV (2011) Out with the old, in with the new? Comparing methods for measuring protein degradation. Cell Biol Int 35(5):457–462
Gardner RG, Hampton RY (1999) A highly conserved signal controls degradation of 3-hydroxy-3-methylglutaryl-coenzyme a (HMG-CoA) reductase in eukaryotes. J Biol Chem 274(44):31671–31678
Cronin SR, Hampton RY (1999) Measuring protein degradation with green fluorescent protein. Methods Enzymol 302:58–73
Yen HC, Elledge SJ (2008) Identification of SCF ubiquitin ligase substrates by global protein stability profiling. Science 322(5903):923–929
Yen HC, Xu Q, Chou DM, Zhao Z, Elledge SJ (2008) Global protein stability profiling in mammalian cells. Science 322(5903):918–923
Ben-Aroya S, Coombes C, Kwok T, O’Donnell KA, Boeke JD, Hieter P (2008) Toward a comprehensive temperature-sensitive mutant repository of the essential genes of Saccharomyces cerevisiae. Mol Cell 30(2):248–258
Ben-Aroya S, Pan X, Boeke JD, Hieter P (2010) Making temperature-sensitive mutants. Methods Enzymol 470:181–204
Comyn SA, Flibotte S, Mayor T (2017) Recurrent background mutations in WHI2 impair proteostasis and degradation of misfolded cytosolic proteins in Saccharomyces cerevisiae. Sci Rep 7(1):4183
Lee do H, Sherman MY, Goldberg AL (2016) The requirements of yeast Hsp70 of SSA family for the ubiquitin-dependent degradation of short-lived and abnormal proteins. Biochem Biophys Res Commun 475(1):100–106
Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM, Connelly C, Davis K, Dietrich F, Dow SW, El Bakkoury M, Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, Jones T, Laub M, Liao H, Liebundguth N, Lockhart DJ, Lucau-Danila A, Lussier M, M’Rabet N, Menard P, Mittmann M, Pai C, Rebischung C, Revuelta JL, Riles L, Roberts CJ, Ross-MacDonald P, Scherens B, Snyder M, Sookhai-Mahadeo S, Storms RK, Veronneau S, Voet M, Volckaert G, Ward TR, Wysocki R, Yen GS, Yu K, Zimmermann K, Philippsen P, Johnston M, Davis RW (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285(5429):901–906
Gietz RD, Schiestl RH (2007) Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1):35–37
Liu C, Apodaca J, Davis LE, Rao H (2007) Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells. BioTechniques 42(2):158, 160, 162
Acknowledgments
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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Comyn, S.A., Mayor, T. (2018). A Method to Monitor Protein Turnover by Flow Cytometry and to Screen for Factors that Control Degradation by Fluorescence-Activated Cell Sorting. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_10
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DOI: https://doi.org/10.1007/978-1-4939-8706-1_10
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