PROTEIN-FOLDING AND ASSEMBLY IN-VITRO PARALLEL INTRACELLULAR FOLDING AND ASSEMBLY - CATALYSIS OF FOLDING AND ASSEMBLY OF THE HUMAN CHORIONIC-GONADOTROPIN ALPHA-BETA DIMER BY PROTEIN DISULFIDE-ISOMERASE

Although purified proteins will refold and assemble in vitro, it is no t known if cellular factors change the mechanisms of these processes. Based on the gel migration of folding intermediates, the kinetic relat ionships between these intermediates, and on the order of formation of six disulfide bonds, we have found that the in vitro folding pathway of the human chorionic gonadotropin beta subunit (hCG-beta) is indisti nguishable from the intracellular folding pathway. The same rate-limit ing event was found in both folding environments; however, the t1/2 fo r this step in a cell is 4 min, whereas in vitro the t1/2 was greater- than-or-equal-to 80 min. Protein disulfide isomerase (PDI) increased t he in vitro rate of this event (t1/2 = 25 min) without changing the or der of disulfide bond formation. PDI also catalyzed the in vitro rate of assembly of hCG subunits. In intact cells, assembly of the alphabet a heterodimer occurs before all of the intramolecular disulfide bonds of beta are formed. In vitro, assembly was increased after reduction o f two of the carboxyl-terminal disulfide bonds of hCG-beta by PDI. The se results strongly suggest that both in intact cells and in vitro, pa rtially unfolded hCG-beta is more assembly-competent than is fully fol ded hCG-beta. The comparison of in vitro and intracellular hCG-beta fo lding and hCG subunit assembly which is shown in this report indicates that the assisted folding and assembly pathway that occurs in cells, where proteins such as PDI play a role, differs only in rate but not i n the order of disulfide bond formation or in the precursor-product re lationships among the folding intermediates.