Recognition of signal sequence cleavage sites in the spc2Δ strain. (A) Signal sequences of CPYt and N#CPYt(h) cleavage site (CS) variants. (B) N16CPYt(h) CS1/2, CS1 and CS2 in the spc3-4, WT, and spc2Δ strains were pulse-labeled and subjected to Endo H treatment prior to SDS-PAGE. (C) N16CPYt(h) CS1/2 and CS0 in the spc3-4 and WT strains were assessed by pulse labeling. (D) Cleavage efficiencies of N#CPYt(h) CS2 variants in the spc2Δ strain (green). (E) CS2 variants in the spc2Δ strain (green). Cleavage profiles of CS2 or CS1 variants in the spc1Δ strain (red) (Yim et al., 2021) are shown for comparison. Three independent experiments were carried out (n = 3/data point), and the average is shown with the standard deviation. P values between CS1 variants in the WT and spc2Δ, and between CS2 variants in the WT and spc2Δ were calculated by multiple two-tailed t tests; n.s., P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (F and G) Signal sequences and the downstream residues of Rrt6 (F) and its CS variants (G). Mutated residues are colored in red, and potential cleavage sites are indicated with an arrow (↓). The indicated Rrt6 CS variants in the spc3-4, WT, and spc2Δ strains were analyzed by pulse labeling. A representative of at least three experiments is shown. De-glycosylated full-length and cleaved products are indicated in unfilled black and red arrows, respectively. Source data are available for this figure: SourceData F4.