Rough endoplasmic reticulum to junctional sarcoplasmic reticulum trafficking of calsequestrin in adult cardiomyocytes.
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.
McFarland
Timothy P
TP
Department of Physiology, Wayne State University, Detroit, MI 48201, USA.
Milstein
Michelle L
ML
Cala
Steven E
SE
D000818:Animals; D001145:Arrhythmias, Cardiac / Q000378:metabolism*; D002155:Calsequestrin / Q000378:metabolism*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism*; D015151:Immunoblotting; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000378:metabolism; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D002155:Calsequestrin