Effects of polar residues in Spc2 TM on membrane thickness. (A) Localization of the polar (green) and charged (blue for negative, red for positive) residues of Spc2 within the TM window allow for deeper-lying phosphate headgroups within the membrane. (B) LepCC(17L) in spc2Δ cells with an empty vector (−), SPC2, spc2-Y79A, S83A were analyzed by pulse labeling for 10 and 30 min. Bottom: Expression of Spc2 (under its endogenous promoter in the CEN plasmid) and spc2-Y79A,S83A (under the GPD promoter in 2 µm plasmid) is shown. (C) Cleavage (%) was quantified and plotted as in Fig. 6 B. Three independent experiments were carried out, and the average is shown with the standard deviation. (D) Membrane thickness in the TM window for yeast SPC without Spc2 (spc2Δ), with Spc2 (WT) and with a double mutated variant of Spc2 (spc2_Y79A, S83A), embedded in a model of the yeast ER membrane, computed from Martini 3 CGMD simulations. Average values across five 20 μs simulations per system for spc2Δ and WT, and four 4 μs simulations for spc2_Y79A, S83A are shown with the standard error of the mean. Source data are available for this figure: SourceData F6.