Ags1 localization depends on the polarity establishment proteins, polarized actin patches, and polarized exocytosis. (A) Ags1 localization depends on the polarity proteins but not on the microtubule cytoskeleton. Left: ags1⁺-GFP mutant cells were shifted to 36°C for 6 h. Right and bottom: ags1⁺-RFP GFP-atb2⁺ cells were transferred to medium containing 25 µg/ml MBC for 30 min and imaged. Arrow: Ags1 displacement to the middle of the cell. Elapsed time is shown in minutes. (B) Ags1 localization depends on the correct actin patch polarization, but not on the polymerized actin, actin cables, and type V myosins. Left: cps8-188 ags1⁺-GFP cells were shifted to 32°C for 4 h. Middle: ags1⁺-RFP crn1⁺-GFP cells were transferred to medium containing 100 µM Lat A for 30 min. Right and bottom: ags1⁺-RFP crn1⁺-GFP mutant cells were grown at 28°C (for3Δ and myo52Δ) or at 25°C and shifted to 37°C for 2 h (cdc3-6). (C) Ags1 localization depends on the exocytosis. sec8-1 ags1⁺-GFP cells were shifted to 37°C for 2 h. Bars, 5 µm.