Expression of muscle calsequestrin in epithelial HeLa cells: distribution and functional role.
Calsequestrin is a Ca2+ binding protein expressed by a few cell types (mostly muscle fibers). In these cells the distribution of the protein is within the endoplasmic/sarcoplasmic reticulum, however, not uniformly throughout but at discrete sites of the lumen. In order to investigate the mechanisms of this unusual intracellular distribution together with the possible functions of the protein, we have studied stable transfected clones of epithelial HeLa cells. Treatment of these cells with butyric acid induced a rapid (24 h) and massive (approx. 10-fold) increase of the transfected protein, whereas the other lumenal and membrane proteins of the endoplasmic reticulum were either modified slightly or unchanged. When butyric acid treatment was interrupted the calsequestrin levels returned rapidly (within 24 h) to the pre-treatment level. Such a rapid turnover was due in part to secretion, sustained by both spontaneous and Ca(2+)-dependent release of calsequestrin to the extracellular medium. From the physiological point of view, the transfected cells exhibited only moderate increases of the Ca2+ release responses triggered by either ATP (a ligand addressed to the P2u receptor and working through IP3 generation) or thapsigargin (a blocker of the endoplasmic reticulum Ca2+ ATPase), with no further increase after butyric acid induction of calsequestrin. This result appears to correlate with the occurrence of only small amounts of calsequestrin within the endoplasmic reticulum lumen of all transfected cells. The bulk of calsequestrin, in contrast, was found sequestered within large vacuoles distributed both near the cell surface and, after butyric acid treatment, also in the deep cytoplasm. These vacuoles (possibly a lysosomal subcompartment) appear to contain no Ca2+ as no difference in 45Ca release from transfected cells was observed without or with butyric acid pretreatment when exposed to ionomycin, alone or combined with monensin. We conclude that HeLa cells possess no adequate mechanisms to keep calsequestrin in its physiologically relevant location, the endoplasmic reticulum. In the transfected cell the protein seems therefore to be diverted (possibly by default) to vacuoles destined to be rapidly eliminated by the cell.
Papazafiri
P
P
Department of Pharmacology, B. Ceccarelli Center, University of Milan, Italy.
Bossi
M
M
Meldolesi
J
J
D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D002118:Calcium / Q000378:metabolism; D002155:Calsequestrin / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D014162:Transfection
D002087:Butyrates; D002155:Calsequestrin; D020148:Butyric Acid; D002118:Calcium