Localization of Junctophilin-1 at the Junctional Sarcoplasmic Reticulum Requires a Sequence in the Transmembrane Domain

Membrane contact sites (MCSs) are peculiar structures where two cellular membranes come into close proximity. Such contacts create microdomains that favour the exchange between two organelles. MCSs are established and maintained in durable or transient states by tethering structures without membrane fusion. In skeletal muscle, the most important MCS is the triad, formed by an invagination of the sarcolemma called T-tubule sandwiched between two terminal cisternae (TC) of the sarcoplasmic reticulum (SR). Junctophilins (JPHs) play a direct role in the formation and maintenance of triads acting as molecular bridges between the membranes of the SR and the T-tubule. JPHs are encoded by four genes (JPH1, JPH2, JPH3, and JPH4) that are expressed in a tissue-specific manner and are known to form Endoplasmic Reticulum-Plasma Membrane (ER–PM) junctions in excitable cells. JPHs contain four specific domains: eight N-terminal MORN motifs, which bind the sarcolemma phospholipids; an alpha helix domain followed by a divergent region; and a C-terminal transmembrane domain (TMD) that anchors the protein to the SR. In order to study which of these domains is responsible for targeting JPH1 to the triad, we expressed a number of deletion mutants in frame with GFP in mouse muscle fibers in vivo, and their localization was evaluated by confocal microscopy. We found that the TMD of JPH1 alone can localize at the TC in muscle fibers. To further extend this finding we expressed GFP fusion proteins of the TMD of the other JPH isoforms, JPH2, JPH3 and JPH4 in mouse muscle fibers and observed that the TMDs of JPH1 and JPH2 are able to localize at the triads, while the TMDs of JPH3 and JPH4 are not, suggesting that the TMD sequence of JPH1 and JPH2 may contain specific determinants contributing to protein localization at the triads. We thus compared the primary sequence of the TMD of all JPH isoforms. These studies revealed that the TMD of JPH1 and JPH2 have a high level of sequence identity, while JPH3 and JPH4 show lower levels of sequence identity compared to both JPH1 and JPH2. In particular, two amino acids are completely conserved in JPH1 and JPH2 but are replaced by different residues in JPH3 and JPH4 sequence. Using site-directed mutagenesis, we substituted these two amino acids in the TMD of JPH3 with the corresponding residues of JPH1. The same approach was applied to the TMD of JPH1. Replacement of the two amino acids in the TMD of JPH1 resulted in a complete loss of triadic localization. On the contrary, the replacement of the two amino acids in the TMD of JPH3, resulted in a mutant protein which acquired the ability to localize at the TC, indicating that these two residues have a key role in JPH localization at the triad.