Figure 3: ARD1 acetylates K77 of Hsp70.
From: ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation
(a) ARD1 acetylates the NBD of Hsp70 in vitro. Top, deletion mutants of GST-Hsp70 were subjected to in vitro acetylation assays with GST-ARD1. Bottom, construction of Hsp70 deletion mutants. NBD; nucleotide-binding domain, SBD; substrate-binding domain, CTD; C-terminal domain. (b) K77 in GST-NBD is acetylated by ARD1 in vitro. The acetylation site in GST-NBD was identified by LC–MS/MS. (c) ARD1 acetylated K77 of Hsp70 in vitro. GST-Hsp70 WT and GST-Hsp70 K77R recombinants were subjected to in vitro acetylation assays with or without GST-ARD1 recombinant. Acetylation levels of GST-Hsp70 recombinants were determined using an anti-Lys-Ac antibody. (d) K77 acetylation of Hsp70 was increased by cellular stress. After treatment of HEK293T cells with 1 mM H2O2, K77 acetylation of GFP-Hsp70 was assessed by western blotting using an anti-Hsp70-K77-Ac antibody. (e) ARD1 and Hsp70 are acetylated by cellular stress. After treatment with 1 mM MPP+ in SH-SY5Y cells, K77 acetylation of GFP-Hsp70 was determined by western blotting using an anti-Hsp70-K77-Ac antibody. Acetylation of FLAG-ARD1 was assessed by immunoprecipitation using an anti-Lys-Ac antibody. (f) ARD1 autoacetylation at K136 is required for stress-induced Hsp70 acetylation. HEK293T cells expressing the indicated ARD1 plasmids were treated with 1 mM H2O2. Acetylation levels of FLAG-ARD1 and GFP-Hsp70 were assessed by immunoprecipitation using an anti-Lys-Ac antibody and western blotting using an anti-Hsp70-K77-Ac antibody, respectively. DN; dominant-negative mutant.
