Abstract
Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.
Highlights
Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57
PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity
circular dichroism (CD) analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins
Summary
Materials—The FluroTag FITC Conjugation Kit, Mops, Pipes, DTPA, RNase, and EGTA were obtained from Sigma. Fluorescence Labeling—Proteins were labeled with Cascade Blue (CB) acetyl azide by adaptation to a FluroTag FITC Conjugation Kit procedure as recommended by the manufacturer. Six hundred g of purified PDI or ERp57 (2.6 mg/ml) in a 100 mM sodium carbonate/bicarbonate buffer, pH 9.0, was used directly for labeling. Determination of the stoichiometry of the labeling revealed that there were 4 and 5 molecules of CB conjugated per each molecule of ERp57 and PDI, respectively, indicating that over 90% of the protein was labeled with the dye. Fluorescence intensities were measured with constant stirring in 1.5 ml of a binding buffer containing 10 mM Mops, pH 7.0, 100 mM KCl, 2 mM MgCl2, 0.5 mM EGTA. Quantum yields of CB or CB-labeled proteins were calculated from the emission spectra (420 – 460 nm) obtained at the excitation of 385 nm. Free Ca2ϩ concentrations were calculated using Max Chelator, Winmaxc version 1.70
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