Processing of CFTR bearing the P574H mutation differs from wild-type and Delta F508-CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) containing the D elta F508 mutation is retained in the endoplasmic reticulum (ER). This defe ct can be partially overcome by a reduction in temperature which allows som e of the Delta F508 protein to exit the ER and move to the cell surface. Ea rlier studies showed that the CF-associated mutants, P574H and A455E, were also misprocessed, In this study, we found that processing of P574H and A45 5E was also temperature-sensitive; at 26 degrees C, some of the protein mat ured. In contrast to other CFTR mutants, P574H accumulated in punctate cyto plasmic bodies that colocalized with endoplasmic reticulum (ER) markers. At 26 degrees C, these bodies were no longer present. P574H showed a prolonge d association with Hsp70 and also colocalized with Hsp70. We used brefeldin A (BFA) to determine which processing step(s) was altered by reduced tempe rature. Unlike wild-type CFTR, which was converted into an intermediate tha t was stable in the presence of BFA at 37 degrees C, Delta F508 and P574H p roduced the intermediate only when the temperature was reduced to 26 degree s C, Furthermore the wild-type intermediate was not associated with Hsp70. These data suggest that formation of the stable intermediate is a key tempe rature-sensitive step and appears to be coincident with release of the wild -type protein from Hsp70.