Targeting of Calsequestrin to the Sarcoplasmic Reticulum of Skeletal Muscle upon Deletion of Its Glycosylation Site

Abstract

The glycoprotein calsequestrin (CS) is segregated to the junctional sarcoplasmic reticulum (jSR) and is responsible for intraluminal Ca2+ binding. A chimeric CS–hemoagglutinin 1 (HA1), obtained by adding the nine amino acid viral epitope hemoagglutinin to the carboxy terminal of CS and shown to be correctly segregated to skeletal muscle jSR [A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe (1997). Chimeric calsequestrin and its targeting to the junctional sarcoplasmic reticulum of skeletal muscle. Am. J. Physiol. 272, C1420–C1428] lends itself as a molecular tool to investigate the targeting domains of CS. A putative targeting mechanism of CS to jSR implies glycosylation-dependent steps in the endoplasmic reticulum (ER) and Golgi complex. To test this hypothesis, CS-HA1ΔGly, a mutant in which the unique N-glycosylation site Asn316 was changed to Ile, was engineered by site-directed mutagenesis. The mutant cDNA was transiently transfected in either HeLa cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of CS-HA1ΔGly was studied by double-labeling epifluorescence by means of antibodies against either CS, HA1, or the ryanodine receptor calcium release channel. CS-HA1ΔGly was expressed and retained to ER and ER/sarcoplasmic reticulum of HeLa cells and myotubes, respectively, and expressed, sorted, and correctly segregated to jSR of regenerating soleus muscle fibers. Thus, the targeting mechanism of CS in vivo appears not to be affected by glycosylation—that is, the sorting, docking, and segregation of CS are independent of cotranslational and posttranslational glycosylation or glycosylations.