BIP KAR2P SERVES AS A MOLECULAR CHAPERONE DURING CARBOXYPEPTIDASE-Y FOLDING IN YEAST/

Although transiently associated with numerous newly synthesized protei ns, BiP has not been shown to be an essential component directly linke d to the folding and oligomerization of newly synthesized proteins in the endoplasmic reticulum. To determine whether it is needed as a mole cular chaperone, we analyzed the maturation of an endogenous yeast gly coprotein, carboxypeptidase Y (CPY) in several yeast strains with temp erature-sensitive mutations in BiP. These kar2 mutant strains have pre viously been found to be defective in translocation at the nonpermissi ve temperature (Vogel, J. P., L. M. Misra, and M. D. Rose. 1990. J. Ce ll Biol. 110:1885-1895). To circumvent the translocation block, we use d DTT at permissive temperature to delay folding and intracellular tra nsport. We then followed the maturation of the ER-retained CPY after s hifting to the nonpermissive temperature and dilution of the DTT. With out the functional chaperone, CPY aggregated, failed to be oxidized, a nd remained in the ER. In contrast to wild-type cells, in which BiP bi nding was transient with no more than 10-15% of labeled CPY associated at any time, 30-100% of the CPY remained associated with BiP in the m utant strains. In a heterozygous diploid strain, CPY matured and exite d the ER normally. Taken together, the results provide clear evidence that BiP plays a critical role as a molecular chaperone in CPY folding .