Ist1 is ubiquitinated in vivo. (A) Simplified representation of known interactions (dotted lines) between Cdc48, Npl4, and ubiquitin (Ub). We hypothesize this Npl4-Cdc48 enzyme module functionally connects with Ist1 via Ist1 ubiquitination. (B) Alphafold structural model of yeast Ist1 with lysine (blue) and arginine (red) residues indicated. (C) Immunoblot of lysates from WT, ist1∆, and Ist1-HA-His₆ strains using ⍺-Ist1, ⍺-HA, and ⍺-GAPDH antibodies. (D) Whole cell lysates were generated from cells expressing Ist1-HA-His₆ and run on the same SDS-PAGE gel as 0.5% of the purified elution from 2 liters culture followed by immunoblotting using ⍺-Ist1 antibodies (left). A separate gel with 10% of purified sample was stained with Coomassie (right) and the bands excised for MS-based identification indicated. (E) Flow diagram depicting sequence of sample preparation for MS analysis of Ist1 targeted at identifying ubiquitinated peptides. (F) Ist1 amino acid sequence annotated with identified peptides (green) and potentially ubiquitinated lysine residue (pink) shown from PEAKS analysis. (G) Simplified schematic of Ni²⁺-NTA purification of the ubiquitome to test is Ist1 is ubiquitinated. (H) Immunoblot using ⍺-Ist1 antibodies of lysates generated from His₆-ubiquitin with (+) or lacking (∆) IST1 (left). 2 liters from each of these cells was purified via Ni²⁺-NTA twice and analyzed by immunoblot and levels indicated by Coomassie staining of SDS-PAGE gels (right). Source data are available for this figure: SourceData F6.