Targeting of calsequestrin to sarcoplasmic reticulum after deletions of its acidic carboxy terminus.
Calsequestrin (CS) is the Ca(2+) binding protein of the junctional sarcoplasmic reticulum (jSR) lumen. Recently, a chimeric CS-HA1, obtained by adding the nine-amino-acid viral epitope hemagglutinin (HA1) to the COOH terminus of CS, was shown to be correctly 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 implies electrostatic interactions between negative charges on CS and positive charges on intraluminal domains of jSR integral proteins, such as triadin and junctin. To test this hypothesis, 2 deletion mutants of chimeric CS were engineered: CS-HA1DeltaGlu-Asp, in which the 14 acidic residues [-Glu-(Asp)(5)-Glu-(Asp)(7)-] of the COOH-terminal tail were removed, and CS-HA1Delta49(COOH), in which the last, mostly acidic, 49 residues of the COOH terminus were removed. Both mutant cDNAs were transiently transfected in HeLa cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of CS-HA1 mutants were studied by epifluorescence microscopy with use of antibodies against CS or HA1. CS-HA1 mutants were shown to be expressed, sorted, and correctly segregated to jSR. Thus short or long deletions of the COOH-terminal acidic tail do not influence the targeting mechanism of CS.
Nori
A
A
Centro di Studio per la Biologia e la Fisiopatologia Muscolare del Consiglio Nazionale delle Ricerche, Dipartimento di Scienze Biomediche Sperimentali dell'Università di Padova, 35121 Padua, Italy.
Gola
E
E
Tosato
S
S
Cantini
M
M
Volpe
P
P
D000143:Acids; D000367:Age Factors; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002155:Calsequestrin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003461:Crystallography; D018076:DNA, Complementary; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D015870:Gene Expression / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008297:Male; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology; D016296:Mutagenesis / Q000502:physiology; D021382:Protein Sorting Signals / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012038:Regeneration; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D000143:Acids; D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D018076:DNA, Complementary; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins