The Transmembrane Domain is Sufficient to Direct Junctophilin-1 Localization at the Junctional SR

Abstract

The sarcoplasmic reticulum (SR) of striated muscle cells is mainly dedicated to Ca2+ homeostasis and regulation of muscle contraction. The SR is organized in longitudinal and junctional SR (j-SR). In skeletal muscle, this latter domain together with the T-tubules form specific junctions called triads, where proteins regulating the excitation-contraction coupling mechanism assemble. Junctophilins (JPs) are directly involved in the formation and maintenance of triads. Basically, they are anchored to the SR via their C-terminal transmembrane domain (TMD), while their N-terminus contains eight MORN motifs, which associate with the phospholipids of the T-tubules. Nevertheless, how JPs are targeted to triads is not known. The roles of the N-terminal and the C-terminal regions of JP1 in this process were investigated. Expression in primary myotubes and/or muscle fibers of JP1 deletion mutants lacking the TMD resulted in protein distribution at both the surface sarcolemma and the T-tubules, confirming that MORN motifs are involved in JP1 interaction with the sarcolemma, but are not sufficient to restrict its localization at the T-tubules.On the other hand, progressive deletion of MORN motifs I-VI, MORN motifs I-VIII or of the entire region 1-635, did no affect JP1 localization at triads, indicating that the presence of the TMD is sufficient for JP1 localization at the j-SR.These results indicate that the localization of JP1 at the triads appears to be mainly directed by mechanisms acting at the level of the SR, rather than at T-tubules. FRAP analysis performed on a GFP-TMD fusion protein expressed in myotubes indicated that this protein has a high mobility, suggesting the absence of strong protein-protein interactions occurring at the j-SR. Further work is needed to better understand the molecular mechanisms driving TMD-mediated JP1 localization at triads.