Abstract
The role of the ubiquitin–proteasome system (UPS) in maintaining protein homeostasis has generated a demand for assays that quantify UPS function in the presence of chemical and protein UPS inhibitors. Here, we describe protocols that measure changes in UPS reporter levels in response to changes in the expression level, localization, or aggregation state of a second protein. We utilize cell lines stably expressing fluorescent UPS substrates that are transfected with a second protein tagged with a compatible fluorophore. We describe protocols to correlate levels of UPS substrates with changes in the levels or properties of the transfected protein.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schwartz AL, Ciechanover A (1999) The ubiquitin-proteasome pathway and pathogenesis of human diseases. Annu Rev Med 50:57–74.
Bence NF, Sampat RM, Kopito RR (2001) Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292:1552–1555.
Dantuma NP, Lindsten K, Glas R et al (2000) Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nat Biotechnol 18:538–543.
Li X, Zhao X, Fang Y et al (1998) Generation of destabilized green fluorescent protein as a transcription reporter. J Biol Chem 273:34970–34975.
Fiebiger E, Story C, Ploegh HL et al (2002) Visualization of the ER-to-cytosol dislocation reaction of a type I membrane protein. EMBO J21:1041–1053.
DeLaBarre B, Christianson JC, Kopito RR et al (2006) Central pore residues mediate the p97/VCP activity required for ERAD. Mol Cell 22:451–462.
Bennett EJ, Bence NF, Jayakumar R et al (2005) Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation. Mol Cell 17:351–365.
Wang J, Wang CE, Orr A et al (2008) Impaired ubiquitin-proteasome system activity in the synapses of Huntington’s disease mice. J Cell Biol 180:1177–1189.
Fiebiger E, Hirsch C, Vyas JM et al (2004) Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin. Mol Biol Cell 15:1635–1646.
Keyomarsi K, Efuet ET, Bui TN (2010) Semi-high throughput method of measuring proteasome inhibition in vitro and in cultured cells. Cell Biol Toxicol.
Salomons FA, Menendez-Benito V, Bottcher C et al (2009) Selective accumulation of aggregation-prone proteasome substrates in response to proteotoxic stress. Mol Cell Biol 29:1774–1785.
Riley BE, Kaiser SE, Shaler TA et al (2010) Ubiquitin accumulation in autophagy-deficient mice is dependent on the Nrf2-mediated stress response pathway: a potential role for protein aggregation in autophagic substrate selection. J Cell Biol 191:537–552.
Shaner NC, Campbell RE, Steinbach PA et al (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572.
Kessler BM, Tortorella D, Altun M et al (2001) Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits. Chem Biol 8:913–929.
Lindsten K, de Vrij FM, Verhoef LG et al (2002) Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation. J Cell Biol 157:417–427.
Mitra S, Tsvetkov AS, Finkbeiner S (2009) Single neuron ubiquitin-proteasome dynamics accompanying inclusion body formation in huntington disease. J Biol Chem 284:4398–4403.
Bence NF, Bennett EJ, Kopito RR (2005) Application and analysis of the GFPu family of ubiquitin-proteasome system reporters. Methods Enzymol 399:481–490.
Davis JM, (Ed.) (2002) Basic Cell Culture, Second ed., Oxford University Press.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hipp, M.S., Bersuker, K., Kopito, R.R. (2012). Live-Cell Imaging of Ubiquitin–Proteasome System Function. In: Dohmen, R., Scheffner, M. (eds) Ubiquitin Family Modifiers and the Proteasome. Methods in Molecular Biology, vol 832. Humana Press. https://doi.org/10.1007/978-1-61779-474-2_33
Download citation
DOI: https://doi.org/10.1007/978-1-61779-474-2_33
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-473-5
Online ISBN: 978-1-61779-474-2
eBook Packages: Springer Protocols