Ubiquitin Family Modifiers and the Proteasome

Volume 832 of the series Methods in Molecular Biology pp 505-518


Assays to Measure ER-Associated Degradation in Yeast

  • Joseph R. TranAffiliated withDepartment of Biological Sciences, University of PittsburghGraduate Program in Biochemistry and Molecular Genetics, University of Pittsburgh School of Medicine
  • , Jeffrey L. BrodskyAffiliated withDepartment of Biological Sciences, University of Pittsburgh Email author 

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Endoplasmic reticulum-associated degradation (ERAD) is a process that clears the early secretory pathway of misfolded proteins. Though ERAD is of basic biological importance, the clinical importance of this pathway is emphasized by the fact that mutations that render a protein subject to the ERAD quality control pathway underlie the cause of several diseases. The yeast, Saccharomyces cerevisiae, is a valuable and frequently used model system to study biological processes, such as ERAD, as it is a relatively simple model system for which numerous biochemical and genetic tools are available. In addition, the ERAD system is highly conserved between yeast and man. In this chapter, we describe two methods for the analysis of model substrates that undergo catabolism via the ERAD pathway using S. cerevisiae. In particular, we will describe non-radioactive degradation assays and the analysis of substrate ubiquitylation in vivo with or without the use of ubiquitin overexpression systems. We also describe technical hurdles, which we have encountered in our research, and highlight remedies to overcome them.

Key words

Yeast ER-associated degradation Ubiquitin Proteasome Cycloheximide chase