title: The conformation of calreticulin is influenced by the endoplasmic reticulum luminal environment.
abstract: In order to understand the dynamics of the endoplasmic reticulum (ER) luminal environment, we investigated the role of Ca(2+), Zn(2+), and ATP on conformational changes of calreticulin. Purified calreticulin was digested with trypsin in the presence or absence of Ca(2+), Zn(2+), and ATP. At low Ca(2+) concentration (<100 micrometer), calreticulin is rapidly and fully degraded by trypsin, indicating that under these conditions the protein is in a highly trypsin-susceptible conformation. Increasing Ca(2+) concentration up to 500 micrometer or 1 mm resulted in protection of the full-length calreticulin and in generation of the 27-kDa fragment highly resistant to trypsin digestion. The 27-kDa protease-resistant core of the protein represented the NH(2)-terminal half of calreticulin and was identified by its reactivity with specific antibodies and by NH(2)-terminal amino acid sequence analysis. Ca(2+)-dependent changes in calreticulin's sensitivity to proteolysis indicate that agonist-induced fluctuation in the free ER luminal Ca(2+) concentration may affect the protein conformation and function. Trypsin digestion of calreticulin in the presence of Zn(2+) resulted in the formation of a 17-kDa central protease-resistant core in the protein corresponding to the central region of the protein, indicating that under these conditions the N- and C-domains of the protein are in an extended conformation. Here we also show that calreticulin is an ATP-binding protein but that it does not contain detectable ATPase activity. Digestion of the protein with trypsin in the presence of Mg(2+)-ATP protects the full-length protein. These results indicate that calreticulin may undergo frequent, ion-induced conformation changes, which may affect its function and its ability to interact with other proteins in the lumen of the ER.
authors.affiliation: Canadian Institutes of Health Research Group in Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: E F
authors.initials: EF
authors.lastname: Corbett
authors.forename: K M
authors.initials: KM
authors.lastname: Michalak
authors.forename: K
authors.initials: K
authors.lastname: Oikawa
authors.forename: S
authors.initials: S
authors.lastname: Johnson
authors.forename: I D
authors.initials: ID
authors.lastname: Campbell
authors.forename: P
authors.initials: P
authors.lastname: Eggleton
authors.forename: C
authors.initials: C
authors.lastname: Kay
authors.forename: M
authors.initials: M
authors.lastname: Michalak
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D037282:Calreticulin; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D002135:Calcium-Binding Proteins; D037282:Calreticulin; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D000255:Adenosine Triphosphate; D002118:Calcium