Extended Data Fig. 1: Mass spectrometry analysis of Vms1 required for release of Ub–PrANS–tRNA from ribosomes. | Nature

Extended Data Fig. 1: Mass spectrometry analysis of Vms1 required for release of Ub–PrANS–tRNA from ribosomes.

From: Vms1 and ANKZF1 peptidyl-tRNA hydrolases release nascent chains from stalled ribosomes

Extended Data Fig. 1

a, Serial tenfold dilutions of exponential cultures were spotted on YPD plates with or without 100 μg ml−1 hygromycin B and allowed to grow at 30 °C for 3 days. Data shown are representative of three biological replicates. b, Ponceau S stained nitrocellulose filter used as loading control for Fig. 1c. c, Lysate from vms1Δ cells expressing PrANS (input lysate, lane five) was fractionated on a sucrose gradient and 60S fractions were pooled and mock-treated (lanes one and three) or pretreated with the deubiquitylating enzyme Usp2c (1 μM; lane two) or 100 μg ml−1 RNase A (lane four) at 30 °C for 20 min before CTAB precipitation. The pellets were immunoblotted with PAP. d, Ribosomes from the indicated strains were isolated using sucrose cushions and aliquots were immunoblotted to detect PrANS and Rpl3. The remainder was bound to TUBE resin. The adsorbed fractions were immunoblotted with PAP. All lanes in the left and right panels are from the same blots. The dashed lines indicate cropping of lanes not pertinent to the current study. Uncropped gel source images are provided in Supplementary Fig. 1. Data in c and d are representative of two biological replicates. e–h, Mass spectrometry analysis of TAP-tagged Cdc48, Vms1 and Ufd1. e, Relative abundance of each Cdc48 adaptor co-immunoprecipitated with Cdc48 relative to all adaptor proteins identified. f, Schematic illustrating the estimated stoichiometry of Cdc48–adaptor complexes. g, Relative stoichiometry of associated proteins that co-immunoprecipitated with Cdc48, Vms1 and Ufd1. Samples were normalized to the iBAQ value of the bait protein and are presented as percentage of the bait protein. Protein iBAQ values from the untagged control were subtracted from the tagged immunoprecipitation samples. h, Coomassie blue-stained gel of samples used for mass spectrometry analysis. All mass spectrometry data (eh) are representative of two biological replicates.