Searching for MG53-Interacting Proteins in Skeletal Muscle

Abstract

Mitsugumin 53 (MG53, a muscle-specific tripartite motif family protein) contributes to cell membrane repair in skeletal muscle by facilitating vesicle trafficking. To examine possible involvement of MG53 in skeletal excitation-contraction coupling, GST-fused MG53 proteins purified using GST pull-down assay were incubated with solubilized triad preparation from rabbit skeletal muscle. The protein complexes with GST-fused MG53 obtained from the incubation were separated on SDS-PAGE. Major bands on the gel were subjected to in-gel digestions with trypsin for tandem mass spectrometric characterization and database searches for the identification of proteins on the bands. SERCA (sarco/endoplasmic reticulum Ca2+-ATPase) was found as a MG53-interacting protein, and the interaction between SERCA and MG53 was confirmed by immunoprecipitation assays with anti-MG53 antibody in samples intrinsically expressing SERCA and MG53.