PMID: 9529380May 16, 1998Paper

Folding of active beta-lactamase in the yeast cytoplasm before translocation into the endoplasmic reticulum

Molecular Biology of the Cell
E PaunolaM Makarow

Abstract

Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation. We show here that Escherichia coli beta-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells. beta-Lactamase is a globular trypsin-resistant molecule in authentic form. For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150delta, which conferred posttranslational translocation and provided sites for O-glycosylation. We devised conditions to retard translocation of Hsp150delta-beta-lactamase. This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles. Both proteins were trypsin resistant and had similar beta-lactamase activity and Km values for nitrocefin. The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium. Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, ...Continue Reading

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Citations

Mar 22, 2001·EMBO Reports·A MatouschekW Voos
Mar 28, 2013·Critical Reviews in Biochemistry and Molecular Biology·Tslil Ast, Maya Schuldiner
Feb 24, 2001·Critical Reviews in Biochemistry and Molecular Biology·A M Benham, I Braakman
Jul 3, 1998·FEBS Letters·H Holkeri, M Makarow
Nov 9, 2001·Annual Review of Genetics·S W FewellJ L Brodsky
Nov 19, 2010·PLoS Pathogens·Jianing N SunScott G Filler
Dec 17, 2003·Journal of Cell Science·Leena Karhinen, Marja Makarow

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