THE ROLE OF THE CARRIER PROTEIN AND DISULFIDE FORMATION IN THE FOLDING OF BETA-LACTAMASE FUSION PROTEINS IN THE ENDOPLASMIC-RETICULUM OF YEAST

We have studied the relationship between folding and secretion compete nce of hsp150-beta-lactamase fusion proteins in Saccharomyces cerevisi ae. hsp150 is a secretory protein of yeast, and beta-lactamase was cho sen, since its folding can be monitored by assaying its enzymatic acti vity. The hsp150 pre-pro-protein consists of a signal peptide, subunit I, a repetitive region, and a unique C terminus. Fusion of beta-lacta mase to the C terminus of hsp150 produced Cla-bla protein, which was s ecretion-competent but inactive. The Pst-bla protein, where p-lactamas e was fused to subunit I, was also inactive and mostly secreted, but p art of it remained in the pre-Golgi compartment. When beta-lactamase w as fused to the C terminus of the repetitive region, the fusion protei n (Kpn-bla) was translocated to the endoplasmic reticulum, acquired di sulfide bonds, and adopted an enzymatically active conformation. Kpn-b la was secreted to the medium without decrease of specific activity or retention in the cell. Folding of Kpn-bla to an active and transport competent form required co-translational disulfide for formation, sinc e treatment of cells with dithiothreitol resulted in endoplasmic retic ulum-retained inactive Kpn-bla. When dithiothreitol was removed, Kpn-b la resumed transport competence but remained inactive. Reduction of pr efolded Kpn-bla did not inhibit enzymatic activity or transport. The r epetitive hsp150 carrier may have use in heterologous protein producti on by conferring secretion competence to foreign proteins in yeast.