Calsequestrin-1 is Required for Association but not for Targeting of Triadin to the Junctional Sarcoplasmic Reticulum Membranes

Abstract

In striated muscles two membrane systems, the sarcolemma and the sarcoplasmic reticulum (SR) are typically arranged in contiguous junctional membrane complexes (JMCs), called triads. Triads are key components in the excitation-contraction coupling mechanism, as they supply the structural elements necessary for efficient crosstalk between voltage-dependent calcium channels (DHPR) on the T-tubules and the ryanodine receptors calcium channels (RyRs), on the SR. Other proteins including triadin, junctin, calsequestrin and junctophillins are selectively localized to JMCs and assemble into regularly arranged complexes. Mechanisms explaining membrane compartmentalization include selective targeting and “trapping” machineries, which may prevent the spread of molecules throughout the membrane surface. However, no specific mechanisms have been yet associated with either assembly or maintenance of JMCs in striated muscles. In previous studies we showed that the mobile fraction of triadin progressively reduced during differentiation, suggesting that a trapping mechanism may be efficiently active in striated muscle and indicate triadin as a good candidate to study protein localization and association to the JMCs.Expression of GFP-tagged triadin proteins and deletion mutants in rat myotubes showed that three regions (named 1, 2 and 3) were required for protein targeting to the JMCs. On the contrary, analysis of protein mobility revealed that association of triadin to the JMC was mediated by region 3 only. GST-pull down and FRET experiments showed that region 3 was able to interact with calsequestrin-1. Interestingly, experiments performed on myotubes from calsequestrin-1 knockout mice revealed that in the absence of calsequestrin, triadin was properly localized to the JMC, although its mobile fraction was dramatically increased. These results suggest that calsequestrin-1 is directly involved in trapping triadin to the JMCs, whereas selective targeting is likely to be mediated by interactions with other proteins.