The transmembrane helix of Ssh4 is important for Ypq1 degradation. (A) Conceptual model of competition assay. Full-length Ssh4 can interact with and ubiquitinate Ypq1, whereas Ssh4^NT can only interact but not ubiquitinate. Ub, ubiquitin. (B) Left: Ypq1-GFP degradation kinetics after Ssh4^NT overexpression. Right: Quantification (±SD, n = 3). Vec, empty vector. (C) Ypq1-GFP degradation conferred by Ssh4 TM Ala (A) or Trp (W) mutants based on Western blot. Red bar set at 50% as cutoff for strong blocking mutants. WT, L51A, and V69W are nonblockers (±SD, n = 3). Also see Fig. S4, D and E, for blots. (D) Degradation phenotype of Ypq1-GFP when Ssh4 TM residues were mutated to Ala (blue) or Trp (red), based on quantitative Western blots (n = 3). ─, normal degradation; +, partial block; ++, strong block; n/a, not mutated. (E) Heat map showing Ypq1-GFP degradation in the presence of Ssh4 mutants. Degradation-blocking mutants are noted as "Hits." Ssh4 mutants that had low expression levels or were lumenal are also noted. (F) Helical wheel of Ssh4 TM showing residues conferring a strong block when mutated to Ala or Trp. (G) Predicted structure of Ssh4 TM showing critical residues (red).