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-HA24NH2, CS-HA2D, CS-HA3D, and CS-HAHT, 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-HAHT was largely associated to longitudinal SR 
whereas CS-HA was concentrated in TC. In C2C12 myotubes, as judged by 
immunofluorescence, not only CS-HAHT but also CS-HA3D and CS-HA2D 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-HA3D 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.