Abstract
Cardiac calsequestrin (CSQ) is synthesized on rough endoplasmic reticulum (ER), but concentrates within the junctional sarcoplasmic reticulum (SR) lumen where it becomes part of the Ca(2+)-release protein complex. To investigate CSQ trafficking through biosynthetic/secretory compartments of adult cardiomyocytes, CSQ-DsRed was overexpressed in cultured cells and examined using confocal fluorescence microscopy. By 48h of adenovirus treatment, CSQ-DsRed fluorescence had specifically accumulated in perinuclear cisternae, where it co-localized with markers of rough ER. From rough ER, CSQ-DsRed appeared to traffic directly to junctional SR along a transverse (Z-line) pathway along which sec 23-positive (ER-exit) sites were enriched. In contrast to DsRed direct fluorescence that presumably reflected DsRed tetramer formation, both anti-DsRed and anti-CSQ immunofluorescence did not detect the perinuclear CSQ-DsRed protein, but labeled only junctional SR puncta. These putative CSQ-DsRed monomers, but not the fluorescent tetramers, were observed to traffic anterogradely over the course of a 48h overexpression from rough ER towards the cell periphery. We propose a new model of CSQ and junctional SR protein traffic in the adult cardiomyocyte, wherein CSQ traffics from perinuclear cisternae, along contiguous ER/SR lumens in cardiomyocytes as a mobile monomer, but is retained in junctional SR as a polymer.