Direct detection of phospholamban and sarcoplasmic reticulum Ca-ATPase interaction in membranes using fluorescence resonance energy transfer.
We used fluorescence resonance energy transfer (FRET) to detect and quantitate the interaction of the sarcoplasmic reticulum Ca-ATPase (SERCA) with phospholamban (PLB) in membranes. PLB inhibits SERCA only at submicromolar Ca. It has been proposed that relief of inhibition at micromolar Ca is due to dissociation of the inhibitory complex. To test this hypothesis, we co-reconstituted donor-labeled SERCA and acceptor-labeled I40A-PLB (superinhibitory, monomeric PLB mutant) in membranes of defined lipid and protein composition, with full retention of Ca-dependent ATPase activity and inhibitory regulation by PLB. FRET from SERCA to PLB was measured as a function of membrane concentrations of PLB and SERCA, and functional activity was measured on the same samples. The data revealed clearly that the stoichiometry of binding is one PLB per SERCA, and that binding is a strict function of the ratio of total PLB to SERCA in the membrane. We conclude that the dissociation constant of PLB binding to SERCA is far less than physiological PLB membrane concentrations. Binding at low Ca (pCa 6.5), where I40A-PLB inhibits SERCA, was virtually identical to that at high Ca (pCa 5.0), where no inhibition was observed. However, the limiting energy transfer at saturating PLB was less at high Ca, indicating a greater donor-acceptor distance. We conclude that (a) the affinity of PLB for SERCA is so great that PLB is essentially a SERCA subunit under physiological conditions and (b) relief of inhibition at micromolar Ca is due to a structural rearrangement within the SERCA-PLB complex, rather than dissociation.
Mueller
Benjamin
B
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Karim
Christine B
CB
Negrashov
Igor V
IV
Kutchai
Howard
H
Thomas
David D
DD
D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D007532:Isoleucine; D000099211:Phospholamban