Microtubule Integrity is Essential to Junctional SR Protein Delivery

Abstract

Cardiac muscle contraction has special reliance upon ER subcompartments to regulate Ca2+ cycling and homeostasis. A unique ER subcompartment, the junctional sarcoplasmic reticulum (jSR) plays a critical role in this process, through the concentration of a unique group of resident proteins that regulate Ca2+ release. Impaired function of jSR has been shown to lead to cardiac pathologies; however, cellular mechanisms and pathways via which proteins traffic to jSR remain undiscovered. To investigate their delivery mechanisms, canine forms of junctin (JCT) or triadin (TRD) were overexpressed in adult rat cardiomyocytes, and their accumulation was visualized by confocal fluorescence microscopy using species-specific antibodies. Both JCT and TRD exhibited patterns of juxtanuclear buildup after 24h. By 48h, higher-intensity punctae were detected across the width of the cell. With microtubule depolymerization, anterograde movement ceased, and newly synthesized proteins did not migrate away from the nucleus. CSQ2-DsRed forms a polymeric complex that localizes early in biosynthesis to juxtanuclear cisternae. To determine whether all jSR proteins follow a common biosynthetic and transport route to jSR, TRD or JCT were co-overexpressed with CSQ2-DsRed. Both TRD and JCT transport from juxtanuclear sites was prohibited. In contrast, triadin in which CSQ2 binding sites were deleted proceeded to the cell periphery. These data suggest that TRD and JCT are biosynthesized at juxtanuclear sites and can interact in situ with CSQ2 at early stages in biosynthesis, and are delivered to jSR sites along a microtubule dependent pathway that lies close to Z-lines, with little or no accumulation at other sarcomeric locations. We conclude that TRD and JCT share the same direct microtubule dependent route, not via free SR compartments.