IN-VIVO IODINATION OF A MISFOLDED PROINSULIN REVEALS CO-LOCALIZED SIGNALS FOR BIP BINDING AND FOR DEGRADATION IN THE ER

The signal for degradation of proteins in the endoplasmic reticulum (E R) is thought to be the exposure of internal domains which are buried when the protein has adopted its correct conformation and which are al so exposed in assembly intermediates. This raises the question of why the intermediates are not degraded. We developed a system based on the peroxidase-catalyzed iodination of tyrosine residues which continuous ly monitors the exposure of internal domains of proinsulin. In CHO cel ls this system discriminated between assembly intermediates of wild ty pe (wt) proinsulin and misfolded proinsulin, as shown by the exclusive iodination of a misfolded mutant which was finally degraded in the ER . Iodination in vitro showed that the assembly intermediates of wt pro insulin also exposed internal domains. This iodination was inhibited b y the addition of the molecular chaperone Bip which was co-immunopreci pitated with proinsulin in CHO cells. The results obtained with the mu tant proinsulin support the assumption that exposed internal domains r epresent the signal for degradation in the ER. Observations of wt proi nsulin show that Bip masks internal domains of normal assembly interme diates during the entire assembly process, thereby suppressing their d egradation. We propose that internal domains contain co-localized sign als for Bip binding and for degradation.