![The N-length-dependent signal sequence cleavage profile in the spc2Δ strain. (A) Schematics of N#CPYt(h) constructs. The extended N-terminal sequences are from Dap2, a yeast SA protein (green). N# indicates the number of N-terminally extended residues, t indicates the C-terminal truncation after residue 323 of CPY, (h) denotes hydrophobic version of the CPY signal sequence (Table S1). N-glycan sites are indicated as Y. (B) N16CPYt(h) in the spc3-4 strain was analyzed by pulse labeling at the indicated temperatures, subjected to endoglycosidase H treatment (EH) prior to SDS‒PAGE, and analyzed by autoradiography. (C) N#CPYt(h) constructs in the spc2Δ, spc2Δ + SPC2, and spc2Δ + SPC1 strains were analyzed by pulse labeling. The relative amounts of cleaved products over total products (cleavage [%]) were plotted against the number of n-region residues (N-length). At least three independent experiments were carried out (n = 3/data point), and the average is shown with the standard deviation. P values between WT and spc2Δ and between spc2Δ and spc2Δ+SPC2 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. The cleavage profiles in the WT and spc1Δ strains (Yim et al., 2021) are shown in comparison. (D) The volcano plot of the WT and spc2Δ proteomes as quantified from mass spectrometry (Data S1). The relative abundances of Sec11, Spc3, Spc2, and Sbh2 are indicated in green circles and Pdi1 and Kar2 in red squares. (E and F) SPSuc2-Lep (Hessa et al., 2009) and ppαF, (F) Ecm38, Kar2 in the spc3-4, WT and spc2Δ strains were analyzed by pulse-labeling. Representative gels from at least three independent experiments are shown in F. Average cleavage efficiencies with standard deviation are indicated. Filled black and red arrows indicate glycosylated full-length and cleaved products, respectively, and unfilled black and red arrows indicate deglycosylated full-length and cleaved products, respectively. Source data are available for this figure: SourceData F1.](https://cdn.rupress.org/rup/content_public/journal/jcb/223/12/10.1083_jcb.202211035/1/jcb_202211035_fig1.png?Expires=1768055937&Signature=KAHL-rfhANv7R1X53S3sPN9p-QrI-6TWkPjewhks2Dq9hy1ZJXISyK1zoP7ydKmyh7-aWqwfo9HTOa~Z41K5XNZL7l4lBTb-5JPutdQQVd5dOH-xbaLQRO4mmqGs6uTzRX7Hu8m1g~~Ny5NM6th6DyAZLe~H~VWPniIAYOsSUIaJrNRFAIxvLqyTUaGtLdUjyN34LRL5t5SsEo~NiU~14PQ7LHVJSVnQBuhEkrJq4v05WimuxyHdk0lsADMrKq6HkSD21oZlr6TQDnv15MLX2fAVhA2taYEwBCwlI7af0qDd6zsPF5AHWqBdX99rgNGpCSkGe5OAAd3QA5bw9~Kv3g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The N-length-dependent signal sequence cleavage profile in the spc2Δ strain. (A) Schematics of N#CPYt(h) constructs. The extended N-terminal sequences are from Dap2, a yeast SA protein (green). N# indicates the number of N-terminally extended residues, t indicates the C-terminal truncation after residue 323 of CPY, (h) denotes hydrophobic version of the CPY signal sequence (Table S1). N-glycan sites are indicated as Y. (B) N16CPYt(h) in the spc3-4 strain was analyzed by pulse labeling at the indicated temperatures, subjected to endoglycosidase H treatment (EH) prior to SDS‒PAGE, and analyzed by autoradiography. (C) N#CPYt(h) constructs in the spc2Δ, spc2Δ + SPC2, and spc2Δ + SPC1 strains were analyzed by pulse labeling. The relative amounts of cleaved products over total products (cleavage [%]) were plotted against the number of n-region residues (N-length). At least three independent experiments were carried out (n = 3/data point), and the average is shown with the standard deviation. P values between WT and spc2Δ and between spc2Δ and spc2Δ+SPC2 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. The cleavage profiles in the WT and spc1Δ strains (Yim et al., 2021) are shown in comparison. (D) The volcano plot of the WT and spc2Δ proteomes as quantified from mass spectrometry (Data S1). The relative abundances of Sec11, Spc3, Spc2, and Sbh2 are indicated in green circles and Pdi1 and Kar2 in red squares. (E and F) SPSuc2-Lep (Hessa et al., 2009) and ppαF, (F) Ecm38, Kar2 in the spc3-4, WT and spc2Δ strains were analyzed by pulse-labeling. Representative gels from at least three independent experiments are shown in F. Average cleavage efficiencies with standard deviation are indicated. Filled black and red arrows indicate glycosylated full-length and cleaved products, respectively, and unfilled black and red arrows indicate deglycosylated full-length and cleaved products, respectively. Source data are available for this figure: SourceData F1.