Hierarchy of Regulatory Interactions in the Sarcoplasmic Reticulum Calcium Transport Complex

Abstract

We have used fluorescence microscopy to detect interactions of sarcolipin (SLN), phospholamban (PLB), and the SR Ca-ATPase (SERCA1a isoform). SLN and PLB individually regulate SERCA activity through protein-protein interactions controlled by phosphorylation; SLN is highly expressed in fast-twitch muscle and atria, while PLB is highly expressed in slow-twitch muscle and ventricles. When SERCA, SLN, and PLB are expressed in the same human cell (vastus lateralis muscle, Takotsubo cardiomyopathy), the three proteins form a “super-inhibited” ternary complex (SERCA-SLN-PLB), whereby SERCA activity shows 5-fold decreased calcium affinity and 2-fold decreased maximum velocity (MacLennan JBC 2002; Tupling PLOS 2013). Here we used Forster resonance energy transfer (FRET) to quantify the complex equilibria of homo- and hetero-oligomeric interactions between SERCA, SLN, and PLB. The three proteins were tagged with genetically-encoded fluorescent probes (CFP, YFP), and the fluorescent fusion proteins were expressed in Sf21 cells via baculovirus infection. Five protein-protein interactions were assayed (SLN-SLN, SLN-PLB, PLB-PLB, SERCA-SLN, SERCA-PLB), and three parameters were calculated per interaction (binding affinity, oligomer number, interprobe distance). Results indicate (a) SLN and PLB show high-affinity self-association into homo-oligomers and low-affinity cross-assembly as hetero-dimers, and (b) SERCA forms 1:1 binary complexes with SLN or PLB when co-expressed with either subunit individually, with SERCA having 3-fold higher affinity for SLN over PLB. We conclude that SLN and PLB monomers bind independently to SERCA, and that each subunit shows competing self-association versus regulatory complex formation. Molecular modeling based on FRET parameters was used to examine the SERCA-SLN-PLB complex. We propose that equilibrium allocation of SERCA between binary and ternary complexes depends on the expression level and phosphorylation state of each regulatory subunit in muscle. Acknowledgments: This work was funded by NIH grants to DDT (GM27906, AR0507220, AR007612) and UMN awards to JMA (CBS-HHMI).