Multi-chaperone complexes regulate the folding of interferon-γ in the endoplasmic reticulum

The quality control mechanisms directing the folding of cytokines in the endoplasmic reticulum (ER) are poorly understood. We have investigated ER chaperone usage by the cytokine interferon-γ (IFN-γ). ATP-depletion or inhibition of N-glycosylation was found to cause IFN-γ to accumulate into detergent-insoluble aggregates in the ER. Six chaperones, GRP94, GRP78, ERp72, PDI, CaBP1/P5 and CRT were found to associate with IFN-γ during its steady state folding. Interaction of the five first chaperones with IFN-γ was regulated co-ordinately by ATP. These chaperones were recently reported to be part of a multi-chaperone complex involved in the folding of complex, multi-subunit proteins. Our data suggest that also proteins with a relatively simple quaternary structure such as cytokines may fold in association with this complex. In addition, we identified calreticulin as the major chaperone interacting with IFN-γ, and the related class II cytokine interleukin-10, during heat-shock in vivo. IFN-γ was maintained in a folding-competent form by calreticulin during heat-shock and released during subsequent recovery at 37 °C. This interaction was observed in both recombinant (CHO-F11) and natural producer cells (Jurkat, NK-92MI) of IFN-γ. Since cytokines such as IFN-γ and IL-10 are frequently produced in the course of inflammatory conditions associated with fever, the thermo-protective effect of calreticulin may constitute a previously unrecognized component of the cellular cytokine production machinery, of likely relevance in sustaining cytokine folding and secretion in pathophysiological conditions.