ERcalcistorin/Protein-disulfide Isomerase Acts as a Calcium Storage Protein in the Endoplasmic Reticulum of a Living Cell

Abstract

Intracellular Ca2ϩ plays an important role in signal transduction mechanisms, motility, secretory processes, and enzyme activity. The cytoplasmic Ca 2ϩ concentration is finely regulated by calcium pumps and channels in the plasma membrane and the endoplasmic reticulum (ER), 1 the lumen of which stores Ca 2ϩ (1, 2). Muscle sarcoplasmic reticulum, a specialized form of the ER, is known to regulate cytoplasmic Ca 2ϩ (3), and the proteins involved were first well characterized in this organelle (4, 5). One of these proteins, calsequestrin, a high capacity low affinity calcium-binding protein (6), is concentrated in the cisternae of the sarcoplasmic reticulum (7) and is linked by triadin to the cisternal membrane (8, 9), which contains the Ca 2ϩ release channel, the ryanodine receptor (10). Ikemoto et al. (11) postulated that Ca 2ϩ -induced conformational changes in calsequestrin affect the junctional face membrane proteins, which in turn regulate the opening and closing of the Ca 2ϩ channel. Calsequestrin's location in the sarcoplasmic reticulum and its Ca 2ϩ binding properties in vitro strongly suggest that it functions as a Ca 2ϩ storage protein, but as yet, no direct evidence has been reported on such a role in a living cell. The ER of smooth muscle and many other cells contains calreticulin, a high capacity low affinity Ca 2ϩ -binding protein postulated to be a calcium storage protein (12, 13).We identified a 58-kDa protein, characterized as calsequestrin-like, in a microsomal fraction of sea urchin eggs (14), in which Ca 2ϩ is released following fertilization (15, 16) and subsequently during phases of the cell cycle (17). By immunocytology, we showed its presence in the ER of sea urchin eggs and also showed, for the first time, the ER undergoing dynamic changes in structure and location, following fertilization, with aggregation occurring around the mitotic apparatus (18). Such movement of the ER was corroborated later by Terasaki and Jaffe (19). Subsequently, we determined the Ca 2ϩ binding properties of this egg protein (23 mol of Ca 2ϩ /mol of protein at low affinity) and identified a unique N-terminal sequence and other features distinct from calsequestrin and calreticulin (20). The two latter proteins have been identified immunocytologically in the egg (21). The amino acid sequence of the 58-kDa protein deduced from cDNA surprisingly shows a 55% identity to mammalian protein-disulfide isomerase (PDI) (22), an enzyme of importance in disulfide bond formation of nascent proteins within the ER (23-25). PDI may also act as a chaperone (26 -28). The sea urchin protein also has PDI activity, and we designated it ERcalcistorin/PDI (ECaSt/PDI), alluding to its putative bifunctional role as a Ca 2ϩ storage protein and an isomerase in the ER (22 Constructs for Expression-Plasmids expressing the precursors of 1) ECaSt/PDI, 2) C-terminally truncated ECaSt/PDI, 3) calreticulin, 4) calsequestrin, and 5) lyso-KDEL as a control were constructed according to standard procedures (33).1) For ECaSt/PDI, the full-length cDNA...

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