Direct Detection of Phospholamban and Sarcoplasmic Reticulum Ca-ATPase Interaction in Membranes Using Fluorescence Resonance Energy Transfer

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• 作者：Benjamin Mueller ; Christine B. Karim ; Igor V. Negrashov ; Howard Kutchai ; and David D. Thomas
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：July 13, 2004
• 年：2004
• 卷：43
• 期：27
• 页码：8754 - 8765
• 全文大小：158K
• 年卷期：v.43,no.27(July 13, 2004)
• ISSN：1520-4995

文摘

We used fluorescence resonance energy transfer (FRET) to detect and quantitate the interactionof the sarcoplasmic reticulum Ca-ATPase (SERCA) with phospholamban (PLB) in membranes. PLB inhibitsSERCA only at submicromolar Ca. It has been proposed that relief of inhibition at micromolar Ca is dueto dissociation of the inhibitory complex. To test this hypothesis, we co-reconstituted donor-labeled SERCAand acceptor-labeled I40A-PLB (superinhibitory, monomeric PLB mutant) in membranes of defined lipidand protein composition, with full retention of Ca-dependent ATPase activity and inhibitory regulationby PLB. FRET from SERCA to PLB was measured as a function of membrane concentrations of PLBand SERCA, and functional activity was measured on the same samples. The data revealed clearly thatthe stoichiometry of binding is one PLB per SERCA, and that binding is a strict function of the ratio oftotal PLB to SERCA in the membrane. We conclude that the dissociation constant of PLB binding toSERCA is far less than physiological PLB membrane concentrations. Binding at low Ca (pCa 6.5), whereI40A-PLB inhibits SERCA, was virtually identical to that at high Ca (pCa 5.0), where no inhibition wasobserved. However, the limiting energy transfer at saturating PLB was less at high Ca, indicating a greaterdonor-acceptor distance. We conclude that (a) the affinity of PLB for SERCA is so great that PLB isessentially a SERCA subunit under physiological conditions and (b) relief of inhibition at micromolar Cais due to a structural rearrangement within the SERCA-PLB complex, rather than dissociation.