Wilson, George S.Schoeneich, ChristianHewawasam, Geetha S.2008-09-082008-09-082008-07-292008http://dissertations.umi.com/ku:2490https://hdl.handle.net/1808/4135Bcl-2 regulates apoptosis by controlling luminal Ca2+ concentration of endoplasmic reticulum (ER). Dremina et al. reported that Bcl-2 interacts with SERCA, the Ca2+ pump in SR/ER membrane, causing inactivation and translocation. This work reports the characteristics of the SERCA/Bcl-2 interactions using wild type and three mutants, G145E, S24C/C158S and S205C/C158S, of the truncated protein, Bcl-2Δ21. Protein cross-linking, Ca2+-ATPase activity assay, Sucrose Density Gradient fractionation, immunoprecipitation and the fusion protein binding assay are the approaches used. Results reveal that the two proteins can interact with both 1:1 and 2:1 (Bcl-2Δ21: SERCA) molar ratios. The hydrophobic groove of Bcl-2Δ21 is involved in the interactions. The BH1 domain of Bcl-2Δ21 interacts with the ATP binding domain of SERCA. The G145E mutant is a loss-of-function whereas the two Cys-mutants are gain-of-function on SERCA inactivation and translocation. Therefore the conserved residue G145 is a critical hot spot for the Bcl-2Δ21-mediated inactivation and translocation of SERCA.169 pagesENThis item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.Analytical chemistryBcl-2SercaApoptosisErPhoto cross-linkingProtein interactionsDissertationopenAccess