title: Oligomeric interactions of sarcolipin and the Ca-ATPase.
abstract: We have detected directly the interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring fluorescence resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) and yellow fluorescent protein (acceptor). SLN is a membrane protein that helps control contractility by regulating SERCA activity in fast-twitch and atrial muscle. Here we used FRET microscopy and spectroscopy with baculovirus expression in insect cells to provide direct evidence for: 1) oligomerization of SLN and 2) regulatory complex formation between SLN and the fast-twitch muscle Ca-ATPase (SERCA1a isoform). FRET experiments demonstrated that SLN monomers self-associate into dimers and higher order oligomers in the absence of SERCA, and that SLN monomers also bind to SERCA monomers in a 1:1 binary complex when the two proteins are coexpressed. FRET experiments further demonstrated that the binding affinity of SLN for itself is similar to that for SERCA. Mutating SLN residue isoleucine-17 to alanine (I17A) decreased the binding affinity of SLN self-association and converted higher order oligomers into monomers and dimers. The I17A mutation also decreased SLN binding affinity for SERCA but maintained 1:1 stoichiometry in the regulatory complex. Thus, isoleucine-17 plays dual roles in determining the distribution of SLN homo-oligomers and stabilizing the formation of SERCA-SLN heterodimers. FRET results for SLN self-association were supported by the effects of SLN expression in bacterial cells. We propose that SLN exists as multiple molecular species in muscle, including SERCA-free (monomer, dimer, oligomer) and SERCA-bound (heterodimer), with transmembrane zipper residues of SLN serving to stabilize oligomeric interactions.
authors: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors: Joseph M
authors: JM
authors: Autry
authors: John E
authors: JE
authors: Rubin
authors: Sean D
authors: SD
authors: Pietrini
authors: Deborah L
authors: DL
authors: Winters
authors: Seth L
authors: SL
authors: Robia
authors: David D
authors: DD
authors: Thomas
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D016367:Baculoviridae / Q000235:genetics; D003001:Cloning, Molecular; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006325:Heart Atria / Q000166:cytology; D007313:Insecta / Q000166:cytology; D008164:Luminescent Proteins; D018656:Muscle Fibers, Fast-Twitch; D009124:Muscle Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D055503:Protein Multimerization; D011510:Proteolipids / Q000378:metabolism*; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D009124:Muscle Proteins; D011510:Proteolipids; C054562:yellow fluorescent protein, Bacteria; C077258:sarcolipin; D049452:Green Fluorescent Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases