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 native-like, catalytically active, and trypsin-resistant conformation, and is thereafter translocated into the ER and secreted to the medium. Previously, it was thought that pre-folded proteins cannot be translocated. Here we have studied in living yeast cells whether beta-lactamase, a tight globule in authentic form, must be unfolded for ER translocation. A beta-lactamase mutant (E166A) binds irreversibly benzylpenicillin via Ser(70) in the active site. We fused E166A to the C terminus of a yeast-derived polypeptide having a post-translational signal peptide. In the presence of benzylpenicillin, the E166A fusion protein was not translocated into the endoplasmic reticulum, whereas translocation of the unmutated variant was not affected. The benzylpenicillin-bound protein adhered to the endoplasmic reticulum membrane, where it prevented translocation of BiP, carboxypeptidase Y, and secretory proteins. Although the 321-amino acid-long N-terminal fusion partner adopts no regular secondary structure and should have no constraints for pore penetration, the benzylpenicillin-bound protein remained fully exposed to the cytosol, maintaining its signal peptide. Our data suggest that the beta-lactamase portion must unfold for translocation, that the unfolding machinery is cytosolic, and that unfolding of the remote C-terminal beta-lactamase is required for initiation of pore penetration.
Paunola
E
E
Program in Cellular Biotechnology, Institute of Biotechnology, P.O. Box 56, University of Helsinki, 00014 Helsinki, Finland.
Qiao
M
M
Shmelev
A
A
Makarow
M
M
D001692:Biological Transport / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D010400:Penicillin G / Q000494:pharmacology*; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D001618:beta-Lactamases / Q000378:metabolism*
D001618:beta-Lactamases; D010400:Penicillin G