Evaluation of JPH1 protein interactors by the “Proximity-dependent Labelling with BioID2” approach

In skeletal muscle, several proteins are organized at typical membrane contact sites (MCSs) between the plasma membrane (PM) and the sarcoplasmic reticulum (SR), known as triads. Interactions between proteins at triads are essential to organize the macromolecular complex that mediates the mechanism of excitation-contraction coupling (E-C coupling), which links nerve-induced depolarization with calcium release from sarcoplasmic reticulum, resulting in activation of contraction. In order to identify novel interactions among triadic proteins, the enzyme-mediated labelling technique Proximity-dependent Biotin Identification (BioID2) was applied. This approach is based on the generation of a fusion protein composed by a target protein and the BioID2, a biotin-binding enzyme; all the proteins that lie within a 10-nm distance from the target protein, can be identified due to their biotinylation. In particular, we applied this approach to identify proteins interacting with JPH1, a protein responsible for the assembly of triads, where it acts as a molecular bridge between the T-tubule and the SR membranes. Expression of the fusion protein 3xmycBioID2JPH1 resulted in biotinylation of several proteins, among which we identified CLIMP63. CLIMP63 is a microtubule binding protein expressed in all eukaryotic cells, which also plays a role as a molecular spacer in maintaining the shape of flat cisternae of the endoplasmic reticulum. Interestingly, in skeletal muscle, CLIMP63 was previously shown to interact with triadin, another protein of the triad, suggesting that it may be part of a structural/functional network connecting the SR at triads to the microtubular cytoskeleton.