Targeting of calsequestrin to sarcoplasmic reticulum after deletions of its acidic carboxy terminus

Calsequestrin (CS) is theCa²⁺ binding protein of thejunctional sarcoplasmic reticulum (jSR) lumen. Recently, a chimericCS-HA1, obtained by adding the nine-amino-acid viral epitopehemagglutinin (HA1) to the COOH terminus of CS, was shown to becorrectly segregated to the sarcoplasmic reticulum [A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe.Am. J. Physiol. 272 (Cell Physiol. 41): C1420-C1428,1997]. A putative targeting mechanism of CS to jSR implieselectrostatic interactions between negative charges on CS and positivecharges on intraluminal domains of jSR integral proteins, such astriadin and junctin. To test this hypothesis, 2 deletion mutants ofchimeric CS were engineered: CS-HA1Glu-Asp, in which the 14 acidicresidues[-Glu-(Asp)₅-Glu-(Asp)₇-] of the COOH-terminal tail were removed, andCS-HA149_COOH, in which thelast, mostly acidic, 49 residues of the COOH terminus were removed.Both mutant cDNAs were transiently transfected in HeLa cells, myoblastsof rat skeletal muscle primary cultures, or regenerating soleus musclefibers of adult rats. The expression and intracellular localization ofCS-HA1 mutants were studied by epifluorescence microscopy with use ofantibodies against CS or HA1. CS-HA1 mutants were shown to beexpressed, sorted, and correctly segregated to jSR. Thus short or longdeletions of the COOH-terminal acidic tail do not influence thetargeting mechanism of CS.