HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 37, 34553-34559, September 14, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/37/34553    most recent M102056200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Paunola, E. Articles by Makarow, M. Search for Related Content PubMed PubMed Citation Articles by Paunola, E. Articles by Makarow, M. Social Bookmarking                      What's this?

Inhibition of Translocation of -Lactamase into the Yeast Endoplasmic Reticulum by Covalently Bound Benzylpenicillin^*

Eija Paunola, Mingqiang Qiao, Anton Shmelev, and Marja Makarow

From the Program in Cellular Biotechnology, Institute of Biotechnology, P.O. Box 56, University of Helsinki, 00014 Helsinki, Finland

We found recently that -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 -lactamase, a tight globule in authentic form, must be unfolded for ER translocation. A -lactamase mutant (E166A) binds irreversibly benzylpenicillin via Ser⁷⁰ 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 -lactamase portion must unfold for translocation, that the unfolding machinery is cytosolic, and that unfolding of the remote C-terminal -lactamase is required for initiation of pore penetration.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				L. Karhinen and M. Makarow Activity of recycling Golgi mannosyltransferases in the yeast endoplasmic reticulum J. Cell Sci., January 15, 2004; 117(2): 351 - 358. [Abstract] [Full Text] [PDF]

				M. Yabal, S. Brambillasca, P. Soffientini, E. Pedrazzini, N. Borgese, and M. Makarow Translocation of the C Terminus of a Tail-anchored Protein across the Endoplasmic Reticulum Membrane in Yeast Mutants Defective in Signal Peptide-driven Translocation J. Biol. Chem., January 24, 2003; 278(5): 3489 - 3496. [Abstract] [Full Text] [PDF]