Phospholamban oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase binding measured by fluorescence resonance energy transfer in living cells.
Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB and yellow fluorescent protein-PLB in AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 +/- 1.3%. The observed FRET corresponds to a probe separation distance of 58.7 +/- 0.5A(,) according to a computational model of intrapentameric FRET. This is consistent with models of the PLB pentamer in which cytoplasmic domains fan out from the central bundle of transmembrane helices. An I40A mutation of PLB did not alter pentamer conformation but increased the concentration of half-maximal FRET (K(D)) by >4-fold. This is consistent with the previous observation that this putatively monomeric mutant still oligomerizes in intact membranes but forms more dynamic pentamers than wild type PLB. PLB association with SERCA, measured by FRET between cyan fluorescent protein-SERCA and yellow fluorescent protein-PLB, was increased by the I40A mutation without any detectable change in probe separation distance. The data indicate that the regulatory complex conformation is not altered by the I40A mutation. A naturally occurring human mutation (L39Stop) greatly reduced PLB oligomerization and SERCA binding and caused mislocalization of PLB to the cytoplasm and nucleus. Overall, the data suggest that the PLB pentamer adopts a "pinwheel" shape in cell membranes, as opposed to a more compact "bellflower" conformation. I40A mutation decreases oligomerization and increases PLB binding to SERCA. Truncation of the transmembrane domain by L39Stop mutation prevents anchoring of the protein in the membrane, greatly reducing PLB binding to itself or its regulatory target, SERCA.
Kelly
Eileen M
EM
Department of Physiology, Loyola University Chicago, Maywood, Illinois 60153,USA.
Hou
Zhanjia
Z
Bossuyt
Julie
J
Bers
Donald M
DM
Robia
Seth L
SL
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002460:Cell Line; D002463:Cell Membrane Permeability; D002470:Cell Survival; D004058:Diffusion; D004285:Dogs; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D000099211:Phospholamban
D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C054562:yellow fluorescent protein, Bacteria