Protein Components of the Ryanodine Receptor Complex Traffic Directly from Rough ER to Concentrate in Junctional SR

Abstract

Interactions of the junctional SR (jSR) proteins calsequestrin (CSQ2), triadin (TRD), and junctin (JCT) with the ryanodine receptor have been demonstrated in vitro as essential for proper SR Ca2+ release. However, the intracellular pathway(s) by which these critical proteins traffic to jSR sites remains unknown. To investigate pathways of trafficking for jSR proteins in adult cardiomyocytes, wild-type canine CSQ2, TRD, and JCT were overexpressed in cultured adult rat cardiomyocytes using recombinant adenoviruses. The overexpressed proteins were detected using either species-specific antibodies, or by fusion with fluorescent proteins. After 24h and 48h, cardiomyocytes were examined using confocal fluorescence microscopy. By the time overexpressed CSQ2 was first detected by immunofluorescence (24h), it was already distributed across jSR sites. In contrast, TRD and JCT exhibited patterns of perinuclear accumulation after 24h. By 48h, they became increasingly concentrated along a transverse pathway that extended from perinuclear puncta towards the cell surface. This novel transverse jSR trafficking pathway co-localized with desmin, a prominent protein component of cardiac intermediate filaments. It was previously found that CSQ2-DsRed forms a polymeric complex that prematurely polymerizes at perinuclear sites of rough ER. We reasoned that this proximal CSQ2 polymer should then bind to newly synthesized TRD and JCT in situ, leading to their enhanced concentration around the nucleus. Indeed, when canine TRD and JCT were co-overexpressed with CSQ2-DsRed, both of these small transmembrane proteins bound to perinuclear CSQ2-DsRed, and failed to exhibit anterograde trafficking to peripheral jSR puncta. In contrast, TRD in which CSQ2 binding sites were deleted readily trafficked towards the cell periphery. Rates of anterograde trafficking of CSQ2 and other jSR proteins may depend upon the assembly of CSQ2 into polymers, which may be regulated by luminal Ca2+ concentrations.