Absence of Ags1 alters the cell wall structure and composition and causes a lack of cell wall in the pole lateral region, which leads to lysis and cytoplasm release. (A) Ags1 depletion promotes general cell wall ultrastructure alterations, thicker and uniformly transparent in the absence (top-right panels) or multilayered in the presence of sorbitol (bottom-right panels). Cells were grown in EMM+T for 3 (−S, top panels) or 9 h (+S, bottom panels) and analyzed by transmission electron microscopy (TEM). ICW: internal cell wall layer. Bar, 1 µm. (B) Absence of Ags1 generates a reduction in the outer layer of mannoproteins. hht1⁺-RFP (WT, RFP nuclei) and 81X-ags1⁺ (no RFP nuclei) cells were grown in EMM+T for 3 h, mixed, and visualized for FITC-concanavalin A (mannoproteins) and RFP fluorescence (nuclei, WT cells). FITC fluorescence (mannoproteins amount) was quantified by using arbitrary units (see Materials and methods). Bar, 5 µm. (C) Ags1 depletion causes irregular walls with cavities (open arrowhead) and/or attached sister wall fragments (closed arrowhead) at the pole side region. Cells were analyzed by TEM as in A. Bars, 1 µm. (D) Ags1 depletion promotes lysis and cytoplasm release from the pole side region. Cells were grown as in A. Arrowhead: lateral region of cell lysis. Elapsed time is shown in seconds. The percentage of lateral lysis was quantified. Bars, 5 µm.