Oligomeric Interactions of Sarcolipin and SERCA Detected by FRET Microscopy

Abstract

We have monitored molecular interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca- ATPase (SERCA) by measuring Förster resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) and yellow fluorescent protein (acceptor). SLN is a key membrane protein that controls muscle contractility by regulating the calcium transport activity of SERCA, and perhaps by also acting as an ATP-activated anion channel (Becucci et al., Biophys. J. 2007, 2009). Here we used baculovirus expression of fluorescent fusion proteins in insect cells and FRET microscopy to provide novel evidence for (a) independent oligomerization of SLN and (b) regulatory complex formation between SERCA and SLN. FRET assays demonstrated that SLN monomers self-assemble into dimers and higher-order oligomers in the absence of SERCA, but that SLN monomers also bind to SERCA in a 1:1 binary complex when the two proteins are co-expressed. FRET assays further demonstrated that the binding affinity of SLN:SLN homo-oligomers is greater than the binding affinity of SERCA:SLN hetero-dimers, indicating that SLN monomers favor self-association over SERCA binding. Mutating SLN residue isoleucine-17 to alanine (I17A) decreased FRET for SLN self-association and eliminated multimeric assembly of SLN, converting higher-order oligomers into monomers and dimers. The I17A mutation also decreased FRET for SERCA:SLN binding but maintained 1:1 stoichiometry of hetero-dimer formation. Thus, isoleucine-17 is a residue that plays dual roles in (a) determining the distribution of SLN homo-oligomers and (b) stabilizing the formation of SERCA:SLN hetero-dimers. When expressed in bacterial cells, wild-type SLN prevented colony formation but the I17A-SLN mutant did not, indicating that higher-order oligomers of SLN exhibit antibacterial activity, possibly through channel formation. We propose that SLN exists as multiple molecular species in muscle membranes, including SERCA-free (monomer, dimer, oligomer) and SERCA-bound (hetero-dimer).