Journal: Molecular Biology of the Cell
Article Title: Substrate binding by the yeast Hsp110 nucleotide exchange factor and molecular chaperone Sse1 is not obligate for its biological activities
Figure Lengend Snippet: A novel SBD mutant exhibits impaired chaperone holdase activity but retains Hsp70 nucleotide exchange capacity. (A) Crystal structure of the Sse1 β-domain, with amino acids selected for mutations highlighted in red ( Xu et al. , 2012 ). (B) Fluorescence anisotropy was performed with increasing concentrations of chaperone (Sse1 or Sse1 sbd ) binding fluorescently labeled ATP-FAM. (C) Nucleotide exchange activity assays using HSPA8 (Hsp70) prebound to α- 32 P-ATP in the presence or absence of Sse1. (D) Holdase experiments were conducted using chemically denatured FFL (200 nM) diluted into refolding buffer without chaperone (no chap), with Sse1 (400 nM), or with Sse1 sbd (400 nM). FFL diluted into denaturing buffer instead of folding buffer was used as an aggregation control (denat). (E) Differential centrifugation analysis of FFL aggregation in the absence of chaperone or with Sse1 or Sse1 sbd after a 30-min holdase assay. Samples were visualized by SDS–PAGE, followed by Coomassie stain, and scanning densitometry quantitation was performed to determine FFL aggregation under each condition. (F) Endpoint analysis of holdase experiments performed as in D, using denatured FFL with varying ratios of chaperone to substrate, quantified as fraction of total aggregation.
Article Snippet: HSPA8 (70 µg) was loaded with 100 µCi of α-32 P-ATP in a total volume of 120 µl of complex buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES]–KOH, pH 7.5, 100 mM KCl, 11 mM MgOAc, and 25 µM ATP) for 30 min at 4°C, and HSPA8–32 P-ATP complex was obtained by centrifugation through a Microspin G-25 column (GE Healthcare, Chicago, IL).
Techniques: Mutagenesis, Activity Assay, Fluorescence, Binding Assay, Labeling, Centrifugation, SDS Page, Staining, Quantitation Assay