The CPI motif of Bsp1 is required for localization of CP to the AMR. (A) Live-cell fluorescence microscopy showing colocalization of Bsp1-GFP and Cap1-mCherry at the bud neck between the mother and daughter cell (SDY1520). Scale bar, 1 µm. (B) Live-cell fluorescence microscopy showing colocalization of Bsp1-GFP (top) or Cap1-GFP (bottom) with the AMR marker Myo1-mCherry, respectively (SDY1647 and SDY1649). Scale bars, 1 µm. (C) Live-cell fluorescence microscopy of cells expressing Bsp1-GFP, Cap1-GFP, Aim21-GFP, and Twf1-GFP along with the AMR marker Myo1-mCherry, respectively (SDY1647, SDY1649, SDY1654, SDY1655). Bsp1-GFP and Cap1-GFP are recruited to the AMR just prior to contraction. In contrast, Aim21-GFP and Twf1-GFP do not colocalize with Myo1-mCherry at the AMR. AMRs used for construction of kymographs to right are indicated by arrowheads. Arrows indicate the arrival and departure of the GFP-tagged protein. Scale bars, 1 µm. (D) Top: Live-cell fluorescence microscopy showing colocalization of Bsp1^CPI*-GFP with the AMR marker Myo1-mCherry (SDY1656). Bottom: Live-cell fluorescence microscopy showing no recruitment of Cap1-GFP to the AMR in cells with the CPI motif of Bsp1 mutated (SDY1657). AMRs used for construction of kymographs to the right are indicated by arrowheads. Arrows indicate the arrival and departure of the GFP-tagged protein. Scale bars, 1 µm. (E) Live-cell fluorescence microscopy of cells expressing Bsp1-GFP (top) or Bsp1^CPI*-GFP (bottom) with Cap1-mCherry, respectively (SDY1647, SDY1649). Cap1-mCherry shows strong colocalization with Bsp1-GFP at the AMR. In contrast, Cap1-mCherry does not colocalize with Bsp1^CPI*-GFP at the AMR. (F) Quantification of the PCC between Bsp1-GFP or Bsp1^CPI*-GFP at the AMR with Cap1-mCherry, respectively. From left to right, mean PCC = 0.544, −0.023, and n = 25, 22. Error bars, mean ± SD.