Interaction of calreticulin with protein disulfide isomerase.
We report here that calreticulin interacts with protein disulfide isomerase (PDI). The PDI-calreticulin complex can be dissociated by Zn(2+)-iminodiacetate-substituted Sepharose-agarose chromatography, suggesting that these interactions may be Zn2+-dependent. Direct interaction between calreticulin and PDI is also documented by calreticulin affinity chromatography. PDI was the only pancreatic microsomal protein retained on the calreticulum affinity column. Calreticulin and PDI were identified by their NH2-terminal amino acid sequence analysis, mobilities in SDS-polyacrylamide gel electrophoresis, binding of 45Ca2+, and their reactivity with specific antibodies. Using glutathione S-transferase-calreticulin fusion proteins, we show that PDI interacts strongly with the P-domain and only weakly with the N-domain of calreticulin. Expression of calreticulin domains and PDI as fusion proteins with GAL4 in the yeast two-hybrid system revealed that calreticulin interacted with PDI also under normal cellular conditions. Interaction with PDI required only the NH2-terminal region of the N-domain (amino acid residues 1-83) and the P-domain (amino acid residues 150-240) of calreticulin. Importantly, interaction between calreticulin and PDI led to the modulation of their activities. In the presence of PDI, calreticulin does not bind Ca2+ with high affinity. Calreticulin or the N-domain of calreticulin inhibited PDI ability to refold scrambled RNase A.
Medical Research Council Group in Molecular Biology of Membrane, University of Alberta, Edmonton, Canada.
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Baksh
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Burns
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Andrin
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Michalak
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D037282:Calreticulin; D002614:Chelating Agents; D002846:Chromatography, Affinity; D017931:DNA Primers; D004285:Dogs; D005982:Glutathione Transferase / Q000378:metabolism; D007535:Isomerases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D002614:Chelating Agents; D017931:DNA Primers; D012261:Ribonucleoproteins; D005982:Glutathione Transferase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D015032:Zinc