title: Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study.
abstract: Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca(2+) release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.
authors: Paulina J
authors: PJ
authors: Stanczyk
authors: F Anthony
authors: FA
authors: Lai
authors: ZissimopoulosS@cardiff.ac.uk.
authors: Spyros
authors: S
authors: Zissimopoulos
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D055503:Protein Multimerization; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry*; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D001616:beta-Galactosidase