Identification of an N-domain histidine essential for chaperone function in calreticulin

Calreticulin is an endoplasmic reticulum ( ER) luminal Ca2+- binding chaperone involved in folding of newly synthesized glycoproteins via the calreticulin- calnexin cycle. We reconstituted ER of calreticulin- deficient cells with N- terminal histidine ( His(25), His(82), His(128), and His(153)) calreticulin mutants and carried out a functional analysis. In crt (-/-) cells bradykinin- dependent Ca2+ release is altered, and the reestablishment of bradykinin-dependent Ca2+ release was used as a marker for calreticulin function. Bradykinin- dependent Ca2+ release from the ER was rescued by wild type calreticulin and by the His(25), His(82), or His(128) mutant but not by the His(153) mutant. Wild type calreticulin and the His(25), His(82), and His(128) mutants all prevented in vitro thermal aggregation of malate dehydrogenase and IgY, whereas the His(153) mutant did not, indicating that His(153) chaperone function was impaired. Biophysical analysis of His(153) mutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders.