Small Ankyrin 1 Interacts with Phospholamban and Forms a Three-Way Complex with SERCA1.

The activity of the sarco(endo)plasmic reticulum Ca²⁺-ATPase 1 (SERCA1) of muscle is inhibited by small ankyrin1 (sAnk1), a ∼17 kDa transmembrane protein that stabilizes the network compartment of the sarcoplasmic reticulum (SR). sAnk1 binds to sarcolipin (SLN), a 31 amino acid peptide inhibitor of SERCA1, to ablate its inhibitory activity. SERCA1 is also inhibited by phospholamban (PLN), which shares homology with both sAnk1 and SLN. Here we use cotransfection of COS7 cells, coimmunoprecipitation and bimolecular fluorescent complementation (BiFC) to show that sAnk1 associates with PLN and forms a 3-way complex with PLN and SERCA1. Anisotropy-based FRET (AFRET) studies of Cerulean-SERCA1 with Venus-tagged sAnk1 and PLN confirmed the presence of a 3-way complex. ATPase assays showed that, unlike its effects on SLN, sAnk1 does not ablate PLN's inhibition of SERCA1 activity. Our results are consistent with a model in which, in forming a three-way complex, PLN binds to SERCA1 first, followed by binding to sAnk1. This modeling suggests that the interactions of PLN, SLN and sAnk1 with SERCA1, either alone or in pairs, are distinct and have different effects on SERCA1's enzymatic activity.