Chimaeric calsequestrin and its targeting to the junctional
sarcoplasmic reticulum of skeletal muscle.
Nori, Alessandra, Katyuscia A. Nadalini, Adelina Martini, Rosario
Rizzuto, Antonello Villa, and Pompeo Volpe.
Dipartimento di Scienze Biomediche Sperimentali
dell'Universit[grave]a di Padova, via Trieste 75, 35121 Padova, and
DIBIT, Istituto Scientifico San Raffaele, Universit[grave]a di
Milano, via Olgettina 58, 20132, Milano
APStracts 3:0367C, 1996.
Calsequestrin (CS) is the junctional sarcoplasmic reticulum (jSR)
Ca2& binding protein responsible for intraluminal Ca2&
storage. The targeting mechanisms of CS to the jSR are yet to be
unraveled. The 9 aminoacid epitope of the influenza virus
haemoagglutinin (referred to as HA1) was added at the COOH-terminal
of CS by PCR cloning. The HA1-tagged CS cDNA was transiently
transfected in either HeLa cells, myogenic cell lines, such as C2 and
L8 cells, myoblasts of rat skeletal muscle primary cultures, or
regenerating soleus muscle fibers of adult rats. The expression and
intracellular localization of chimaeric CS-HA1 were monitored by
epifluorescence and confocal microscopy using either anti-CS
antibodies or anti-HA1 antibodies. About 30% of transfected HeLa
cells and 20-40% of myogenic cells expressed CS-HA1 into
intracellular compartments, such as the perinuclear cisternae of
endoplasmic reticulum (ER). Myoblasts of newborn rat skeletal muscles
were first transfected and subsequently stimulated to differentiate
into myotubes. CS-HA1 was detected in about 20% of transfected
myotubes and did not affect CS distribution in myotubes. In the
soleus muscle of adult rat, intramuscular injection of bupivacaine
induced necrosis followed by regeneration. In vivo transfection of
HA1-tagged CS cDNA in regenerating skeletal muscles determined
expression in a few skeletal muscle fibers; CS-HA1 was localized only
in jSR, as judged by confocal microscopy of longitudinal sections.
The present results show that chimaeric CS-HA1 is correctly sorted to
ER/SR compartments, and that the free COOH-terminal is not requested
for sorting, retention and segregation of CS to the SR.
Received 10 October 1995; accepted in final form 1 November 1996.
APS Manuscript Number C614-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 31 December 1996