Different endoplasmic reticulum trafficking and processing pathways for calsequestrin (CSQ) and epitope-tagged CSQ.
Cardiac calsequestrin (CSQ) is a protein that traffics to and concentrates inside sarcoplasmic reticulum (SR) terminal cisternae, a protein secretory compartment of uncertain origin. To investigate trafficking of CSQ within standard ER compartments, we expressed CSQ in nonmuscle cell lines and examined its localization by immunofluorescence and its molecular structure from the mass spectrum of total cellular CSQ. In all cells examined, CSQ was a highly phosphorylated protein with a glycan structure predictive of ER-retained proteins: Man9,8GlcNAc2 lacking terminal GlcNAc. Immunostaining was restricted to polymeric ER cisternae. Secretory pathway disruption by brefeldin A and thapsigargin led to altered CSQ glycosylation and phosphorylation consistent with post-ER trafficking. When epitope-tagged forms of CSQ were expressed in the same cells, mannose trimming of CSQ glycans was far more extensive, and C-terminal phosphorylation sites were nearly devoid of phosphate, in complete contrast to the highly phosphorylated wild-type protein that concentrates in all cells tested. Epitope-tagged CSQ also showed a reduced ER staining compared to wild-type protein, with significant staining in juxta-Golgi compartments. Loss of ER retention due to epitope tags or thapsigargin and resultant changes in protein structure or levels of bound Ca(2+) point to CSQ polymerization as an ER/SR retention mechanism.
Houle
Timothy D
TD
Wayne State University, Elliman Building, Room 1107, 421 East Canfield Avenue, Detroit, MI 48201, USA.
Ram
Michal L
ML
McMurray
Walter J
WJ
Cala
Steven E
SE
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D019556:COS Cells; D002155:Calsequestrin / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008954:Models, Biological; D010766:Phosphorylation; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D002155:Calsequestrin; D000939:Epitopes; D020126:Brefeldin A; D019284:Thapsigargin