Effects of Spc2 deletion on the cleavage of TM segments. (A) Schematics and sequences of LepCC variants. The Leu TM segment is in bold, and the cleavage site is indicated as ↓. LepCC(14L(P1)) carries an A to P mutation in the +1 position of the cleavage site, and the cleavage is inhibited. (B) LepCC(14L) in the spc3-4 and WT strains and LepCC(14L[P1]) in the WT strain were analyzed by 5 min pulse labeling, and Endo H (EH) was added to the samples prior to SDS‒PAGE. (C) The indicated LepCC variants in the spc3-4, WT, and spc2Δ strains were analyzed by pulse labeling. Protein samples were treated with or without Endo H (EH) prior to SDS‒PAGE. FL, full-length; C, cleaved species. (D) The relative amounts of cleaved products over total products (cleavage [%]) were plotted (n = 3). (E) Schematic of Pho8 and its SA sequence (in bold) plus 5 downstream residues. The mutated Pro54 residue is indicated in italics. A representative gel is shown. Average cleavage efficiencies from three independent experiments (n = 3/datapoint) and standard deviation are shown. Unfilled black and red arrows indicate de-glycosylated full-length and cleaved products, respectively. ^# indicates a nonspecific band. P values were calculated by two-tailed unpaired t test with Welch’s correction; P > 0.05; *. Source data are available for this figure: SourceData F3.