Extended Data Fig. 3: Role of Arx1 in the recruitment of Drg1 to the pre-60S particle.
From: Visualizing maturation factor extraction from the nascent ribosome by the AAA-ATPase Drg1
(a) Domain organization of Drg1. (b) GST-Arx1 pulldown using Drg1 wildtype and the D1 Walker B mutant Drg1EQ1 (E346Q) as prey in the presence of 1 mM ATP, ATPγS or no nucleotide (-). GST served as control for unspecific binding, GST-Rlp24C as binding reference. (c) In vitro ATPase activity assay of Drg1 in the presence of purified Arx1. Relative activity normalized to Drg1 wildtype basal activity. Rlp24C = HIS6-tagged C-terminal 53 amino acids of Rlp24. Mean ± standard deviation of three biological replicates (n = 3). (d) Composition of Bud20-TAP particles from the Leptomycin B (LmB) sensitive crm1T539C mutant strain with either ARX1 wildtype or Δarx1 background (Western blot). (e) Spot assay. For plasmid shuffling, a Δdrg1 Δarx1 double shuffle strain carrying wildtype DRG1 on a URA3 plasmid (pRS316) as well as either pRS313-ARX1 or the empty vector control (pRS313) was transformed with the indicated plasmids carrying mutant drg1 alleles and spotted on selective media and 5-FOA agar plates.
