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Published 6 August 2001. doi:10.1083/jcb.200103002
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© The Rockefeller University Press, 0021-9525/2001/8/525 $5.00
The Journal of Cell Biology, Volume 154, Number 3, August 6, 2001 525-534

Article

Head-to-tail oligomerization of calsequestrin

: a novel mechanism for heterogeneous distribution of endoplasmic reticulum luminal proteins



Giuliana Gatti1, Sara Trifari2, Nasrin Mesaeli3, J.M. Robert Parker3, Marek Michalak3 and Jacopo Meldolesi2

1 Department of Pharmacology, University of Milan, 20129 Milan, Italy
2 DIBIT, Department of Neuroscience, Vita-Salute University and Scientific Institute San Raffaele, 20132 Milan, Italy
3 Canadian Institutes of Health, Research Group in Molecular Biology of Membrane Proteins, and Department of Biochemistry, University of Alberta, Edmonton, Canada T6G 2H

Address correspondence to Jacopo Meldolesi, DIBIT, Department of Neuroscience, Vita-Salute University and Scientific Institute San Raffaele, Via Olgettina 58, 20132 Milan, Italy. Tel.: 39-02-2643-2770. Fax: 39-02-2643-4813. E-mail: meldolesi.jacopo{at}hsr.it

Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.

Key Words: calsequestrin; condensation; endo/sarcoplasmic reticulum; calsequestrin mutants; L6 and HeLa cells


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