The C-terminal domain of Spc2 is important for N-length-dependent signal sequence cleavage. (A) Structures of human SPCS2 (PDB: 7P2P) and yeast Spc2 (predicted by AlphaFold2, UniProt ID Q04969) are overlaid. (B) Secondary structures of the predicted Spc2. (C) Cleavage efficiencies of N#CPYt(h) variants in spc2Δ cells with SPC2, spc2-ΔCD(58), and spc2-TM2*. Data of N#CPYt(h) variants in the spc2Δ cells in Fig. 1 (C) are shown for comparison. At least three independent experiments were carried out (n = 3/data point), and the average is shown with the standard deviation. P values between spc2Δ+SPC2 and spc2Δ+ spc2-ΔCD(58) strains 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. (D) Cleavage efficiency of N26CPYt(h) in spc2Δ cells with EV, spc2-ΔCD(58), spc2-ΔCD(23) or SPC2 (under the GPD promoter). The expression levels of Spc2 in the indicated strains were assessed by western blotting using anti-FLAG antibodies recognizing Spc2-FLAG. At least three independent experiments were carried out (n = 3/data point), and the average is shown with the standard deviation. P values were calculated by multiple two-tailed t tests; n.s., P > 0.05; ***, P ≤ 0.001. (E) Whole-cell lysates from the spc2Δ,SPC3HA strain carrying an empty vector (EV), SPC2 (under its own promoter), spc2-ΔCD(58), and spc2-TM2* were analyzed by western blotting. PgK is a loading control. Source data are available for this figure: SourceData F2.