Cell polarity protein Spa2 coordinates Chs2 incorporation at the division site in budding yeast
Fig 5
IPC component Hof1 and secretory vesicle transport protein Myo2 both contribute to the localisation of Spa2 at the site of division.
(A) SPA2-GFP (YMF167) and SPA2-GFP hof1-td myo2-td (YMF1418) strains were arrested in G1 phase at 24°C in YPRaff and then synchronously shifted to YPRaff medium containing 0.2 M hydroxyurea and arrested in early S phase. Before cells were transferred to YPGal containing 0.2 M hydroxyurea at 37°C in order to deplete Hof1-td and Myo2-td, they were allowed to grow their buds. Subsequently, cells were released to allow progression through the cell cycle. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with Spa2 rings at the cleavage site (ii). An example of a cell with Spa2-GFP ring at the bud-neck at 60 minutes is shown (iii). Scale bar: 2μm. (B) Indicated diploid strains TAP-SPA2/SPA2-5FLAG (YMF1448) and SPA2/SPA-5FLAG (YMF1449) were grown asynchronously at 24°C in YPD medium and cell extracts were prepared before immunoprecipitation of TAP-Spa2 and detection of the indicated proteins by immunoblotting. (C) Summary of yeast two-hybrid interactions between the different fragments of Spa2, one containing SHD-I region (Spa2-1-145) and another one containing SHD-II (Spa2-421-552). (D) SPA2-GFP (YMF117), ΔC-SPA2-GFP (YMF967, Spa2-1-552-GFP) and ΔN-SPA2-GFP (YMF1023, Spa2-553-1466-GFP) strains were arrested in G1 phase at 24°C in YPD and then released from G1 arrest to allow progression through the cell cycle. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with rings of Spa2 or its truncations at the cleavage site (ii). (E) Summary of yeast two-hybrid interactions between the C-terminal fragments of Spa2 and Hof1.