title: Assaying proteasomal degradation in a cell-free system in plants.
abstract: The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions. The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York; elena.garcia-cano@stonybrook.edu.
authors.forename: Elena
authors.initials: E
authors.lastname: García-Cano
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York.
authors.forename: Adi
authors.initials: A
authors.lastname: Zaltsman
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York.
authors.forename: Vitaly
authors.initials: V
authors.lastname: Citovsky
mesh_terms: D060054:Agrobacterium / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism; D002474:Cell-Free System; D044783:F-Box Proteins / Q000378:metabolism; D010944:Plants / Q000201:enzymology / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D013379:Substrate Specificity; D014026:Nicotiana / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D044783:F-Box Proteins; D025801:Ubiquitin; C432104:VIP1 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease
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