Inhibition of translocation of beta-lactamase into the yeast endoplasmic reticulum by covalently bound benzylpenicillin

We found recently that beta -lactamase folds in the yeast cytosol to a nati ve-like, catalytically active, and trypsin-resistant conformation, and is t hereafter translocated into the ER and secreted to the medium. Previously, it was thought that pre-folded proteins cannot be translocated. Here we hav e studied in living yeast cells whether beta -lactamase, a tight globule in authentic form, must be unfolded for ER translocation. A beta -lactamase m utant (E166A) binds irreversibly benzylpenicillin via Ser(70) in the active site. We fused E166A to the C terminus of a yeast-derived polypeptide havi ng a post-translational signal peptide. In the presence of benzylpenicillin , the E166A fusion protein was not translocated into the endoplasmic reticu lum, whereas translocation of the unmutated variant was not affected. The b enzylpenicillin-bound protein adhered to the endoplasmic reticulum. membran e, where it prevented translocation of BiP, carboxypeptidase Y, and secreto ry proteins. Although the 321-amino acid-long N-terminal fusion partner ado pts no regular secondary structure and should have no constraints for pore penetration, the benzylpenicillin-bound protein remained fully exposed to t he cytosol, maintaining its signal peptide. Our data suggest that the beta -lactamase portion must unfold for translocation, that the unfolding machin ery is cytosolic, and that unfolding of the remote C-terminal. P-lactamase is required for initiation of pore penetration.