N-Ethylmaleimide inactivates a nucleotide-free Hsp70 molecular chaperone.

The Journal of Biological Chemistry
Q LiuW J Chirico

Abstract

Hsp70 molecular chaperones are ATPases that bind to hydrophobic regions of proteins and guide their folding, assembly, and translocation across membranes. The ability of purified Hsp70s to uncoat clathrin-coated vesicles or to stimulate the post-translational translocation of precursor proteins into the endoplasmic reticulum, mitochondria, and the nucleus was previously shown not to be sensitive to the sulfhydryl-modifying reagent N-ethylmaleimide (NEM). During purification of factors required for protein folding in the cytosol, we found that the ATP-agarose binding activity of the yeast Hsp70 Ssa1p in postribosomal supernatants was inhibited by NEM. We also found that completely removing nucleotides from purified Ssa1p rendered its ATP-agarose binding activity, ATPase activity, and post-translational translocation-stimulating activity sensitive to NEM. We modified nucleotide-free Ssa1p with [14C]NEM and then digested it with proteases. Purification and sequencing of the radiolabeled proteolytic fragments revealed that each of Ssa1p's three cysteine residues (Cys-15, Cys-264, and Cys-303) was modified with [14C]NEM. ADP protected each of the cysteine residues from modification and protected Ssa1p from inactivation. The cysteine...Continue Reading

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Citations

Jan 9, 2001·Archives of Biochemistry and Biophysics·G NardaiP Csermely
Aug 16, 2011·Chemical Research in Toxicology·Lixin MiFung-Lung Chung
Jul 20, 2012·Molecular Biology of the Cell·Yanyu WangKevin A Morano
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Jun 24, 2016·Proceedings of the National Academy of Sciences of the United States of America·Gregory S OrfRobert E Blankenship
Mar 16, 2018·Chembiochem : a European Journal of Chemical Biology·Fuzhong OuyangJian Liu
Jul 4, 2019·Current Pharmaceutical Design·Dominik KołodziejskiAgnieszka Bartoszek
Aug 27, 1999·Archives of Biochemistry and Biophysics·A Hermawan, W J Chirico
Jan 5, 2018·Biochemistry·John P O'DonnellCarolyn S Sevier

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