Chaperone Function of Calreticulin When Expressed in the Endoplasmic Reticulum as the Membrane-anchored and Soluble Forms

A unique type of chaperone that requires glucose trimming of the target proteins has been shown to be important for their maturation in the endoplasmic reticulum (ER). Calnexin, an ER membrane chaperone, is the first example of such a class. Here, we focus on calreticulin, a major ER luminal protein, which shares with calnexin two sets of characteristic sequence repeat. We evaluated the chaperone function of calreticulin by expressing it on the ER luminal membrane surface. We constructed a membrane-anchored calreticulin chimera by fusing truncated calreticulin to the membrane-anchoring tagged segment of calnexin. When expressed in HepG2 cells, the calreticulin chimera transiently interacted with a set of nascent secretory proteins in a castanospermine-sensitive manner. The spectrum of proteins recognized by the membrane-anchored calreticulin was remarkably similar to that observed with calnexin. Next, we tested if such a chaperone function of calreticulin is expressed at its physiological location. Luminally expressed calreticulin preferentially bound to nascent transferrin and released it upon chase. Association with other calnexin ligands was observed, however, at low efficiencies. Interactions were abrogated by castanospermine treatment. We conclude that calreticulin per se is another chaperone with apparently the same characteristics as calnexin and selectively interacts with nascent transferrin in the lumen, suggesting that calreticulin may cover the diversity of maturations. A unique type of chaperone that requires glucose trimming of the target proteins has been shown to be important for their maturation in the endoplasmic reticulum (ER). Calnexin, an ER membrane chaperone, is the first example of such a class. Here, we focus on calreticulin, a major ER luminal protein, which shares with calnexin two sets of characteristic sequence repeat. We evaluated the chaperone function of calreticulin by expressing it on the ER luminal membrane surface. We constructed a membrane-anchored calreticulin chimera by fusing truncated calreticulin to the membrane-anchoring tagged segment of calnexin. When expressed in HepG2 cells, the calreticulin chimera transiently interacted with a set of nascent secretory proteins in a castanospermine-sensitive manner. The spectrum of proteins recognized by the membrane-anchored calreticulin was remarkably similar to that observed with calnexin. Next, we tested if such a chaperone function of calreticulin is expressed at its physiological location. Luminally expressed calreticulin preferentially bound to nascent transferrin and released it upon chase. Association with other calnexin ligands was observed, however, at low efficiencies. Interactions were abrogated by castanospermine treatment. We conclude that calreticulin per se is another chaperone with apparently the same characteristics as calnexin and selectively interacts with nascent transferrin in the lumen, suggesting that calreticulin may cover the diversity of maturations.