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Am J Physiol Cell Physiol 291: C245-C253, 2006. First published March 29, 2006; doi:10.1152/ajpcell.00370.2005
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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Calsequestrin targeting to sarcoplasmic reticulum of skeletal muscle fibers

Alessandra Nori,* Giorgia Valle,* Elena Bortoloso, Federica Turcato, and Pompeo Volpe

Dipartimento di Scienze Biomediche Sperimentali dell'Università di Padova, Istituto Interuniversitario di Miologia, Padua, Italy

Submitted 21 July 2005 ; accepted in final form 18 March 2006

Calsequestrin (CS) is the low-affinity, high-capacity calcium binding protein segregated to the lumen of terminal cisternae (TC) of the sarcoplasmic reticulum (SR). The physiological role of CS in controlling calcium release from the SR depends on both its intrinsic properties and its localization. The mechanisms of CS targeting were investigated in skeletal muscle fibers and C2C12 myotubes, a model of SR differentiation, with four deletion mutants of epitope (hemagglutinin, HA)-tagged CS: CS-HA{Delta}24NH2, CS-HA{Delta}2D, CS-HA{Delta}3D, and CS-HA{Delta}HT, a double mutant of the NH2 terminus and domain III. As judged by immunofluorescence of transfected skeletal muscle fibers, only the double CS-HA mutant showed a homogeneous distribution at the sarcomeric I band, i.e., it did not segregate to TC. As shown by subfractionation of microsomes derived from transfected skeletal muscles, CS-HA{Delta}HT was largely associated to longitudinal SR whereas CS-HA was concentrated in TC. In C2C12 myotubes, as judged by immunofluorescence, not only CS-HA{Delta}HT but also CS-HA{Delta}3D and CS-HA{Delta}2D were not sorted to developing SR. Condensation competence, a property referable to CS oligomerization, was monitored for the several CS-HA mutants in C2C12 myoblasts, and only CS-HA{Delta}3D was found able to condense. Together, the results indicate that 1) there are at least two targeting sequences at the NH2 terminus and domain III of CS, 2) SR-specific target and structural information is contained in these sequences, 3) heterologous interactions with junctional SR proteins are relevant for segregation, 4) homologous CS-CS interactions are involved in the overall targeting process, and 5) different targeting mechanisms prevail depending on the stage of SR differentiation.

protein-protein interactions; oligomerization; intracellular sorting


Address for reprint requests and other correspondence: P. Volpe, Dipartimento di Scienze Biomediche Sperimentali, Università degli Studi di Padova, viale G. Colombo 3, 35121 Padua, Italy (e-mail address: pompeo.volpe{at}unipd.it)






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