We found that the exocyst component Sec8 has similar dynamics and localises equally at the site of division in the presence or absence of Hof1, which implies that Hof1 has no role in the docking of the exocyst at the site of division, although it is involved in Spa2 localisation

We found that the exocyst component Sec8 has similar dynamics and localises equally at the site of division in the presence or absence of Hof1, which implies that Hof1 has no role in the docking of the exocyst at the site of division, although it is involved in Spa2 localisation. Given that motor type V myosin, Myo2, can be pulled down by Spa2 in the absence of Hof1, we analysed the contribution of the secretory vesicle system to the localisation of Spa2 at the site of division. (i) YMF837, (ii) YMF824 and (iii) YMF1261 shows the synthetic lethality of cells.(EPS) pgen.1007299.s001.eps (3.9M) GUID:?98ACB087-BEFB-4F69-A032-38A317C81032 S2 Fig: (YMF167) and (YMF183) strains were arrested in G1 phase at 24C in YPRaff and then shifted to YPGal at 37C to deplete Iqg1-td. Subsequently, cells were released to allow progression through the cell cycle and DNA content was measured by flow cytometry. Examples of cells are shown for specific time-points. Scale bar: 10 m.(EPS) pgen.1007299.s002.eps (6.1M) GUID:?C993ACA7-27A8-46F0-AD8E-8304A8577F51 S3 Fig: (A) (YMF167) and (YMF185) strains were arrested in G1 phase at 24C in YPRaff and then shifted to YPGal at 37C to deplete Myo1-td. 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 single Spa2 rings at the cleavage site (ii). (B) (YMF167) and (YMF164) strains were grown as in (A). 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 single Spa2 rings at the cleavage site (ii).(EPS) pgen.1007299.s003.eps (1.9M) GUID:?826150DC-CFA2-4608-A6E3-5F910325C06A S4 Fig: (A) strain (YMF1104) was grown in YPRaff at 24C in YPRaff and then shifted to YPGal at 37C to determine Cyk3 protein stability. Samples were taken as indicated and protein extracts prepared before immunoblotting with anti-Cyk3 antibodies. (B) (YMF716) strain was E3 ligase Ligand 9 grown in YPRaff at 24C in YPRaff and then shifted to YPGal at 37C to study Myo2 protein levels at restrictive conditions. Samples were taken as indicated and protein extracts prepared before immunoblotting with anti-DHFR antibodies.(EPS) pgen.1007299.s004.eps (973K) GUID:?37826570-E584-45B6-B4C9-0E311F6DDE05 S5 Fig: (A) (YMF872) and (YMF1432) strains were arrested in G1 phase at 24C in YPRaff and then shifted to YPGal at 37C to deplete Iqg1-td. 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 single Sec8 rings at the cleavage site (ii). (B) (YMF872) and (YMF909) strains were grown in YPRaff as in (A) and cells were then 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 Sec8 rings at the cleavage site (ii).(EPS) pgen.1007299.s005.eps (2.0M) GUID:?CB2A459A-76A1-496A-85B8-E05B7A5E0D84 S6 Fig: (YMF330) and (YMF869) strains were arrested in G1 phase at 24C in YPRaff and then synchronously shifted to YPRaff medium containing 0.2 M hydroxyurea. Therefore cells were arrested at the early S phase, but bud growth continued. Cells were then transferred to YPGal containing 0.2 M hydroxyurea at 37C in order to deplete Myo2-td. 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 Chs2 rings at the cleavage site (ii).(EPS) pgen.1007299.s006.eps (1.0M) GUID:?7D0D5CCC-6084-4A7A-8489-B80B0811F52A S7 Fig: (YMF167) and (YMF1418) strains were arrested in G1 phase at 24C in YPRaff and then synchronously shifted to YPRaff medium containing 0.2 M hydroxyurea and arrested in early S phase. Before E3 ligase Ligand 9 Rabbit Polyclonal to FUK cells were transferred to YPGal containing 0.2 M hydroxyurea at 37C in order to deplete Hof1-td and Myo2-td, they were allowed to grow their buds. Subsequently, cells were released to allow progression E3 ligase Ligand 9 through the cell cycle and DNA content was measured by flow cytometry. Examples of cells are shown for specific time-point. Scale bar: 10 m.(EPS) pgen.1007299.s007.eps (5.8M) GUID:?3D8622C3-B035-4CA1-B054-4154AFC721D0 S8 Fig: (A) Examples of cells depicted in Fig 5D are shown with Spa2-GFP single ring at the bud-neck at 90 minutes for Spa2-GFP and 105 E3 ligase Ligand 9 minutes for Spa2-1-552-GFP and Spa2-553-1466-GFP. Scale bar: 2 m. (B) (YMF117), (YMF967) and (YMF1023) strains were grown asynchronously at 24C in YPD. Samples to monitor the level of corresponding proteins expressed under the control of promoter were collected. Protein extracts were prepared before immunoblotting with anti-GFP antibodies. As protein levels varied, two different exposure times are presented.(EPS) pgen.1007299.s008.eps (2.8M) GUID:?8D5F3274-5AED-4605-AD77-F3E80BF62119 S9 Fig: (A) (YMF167) and (YMF1088) strains were arrested in G1 phase at 24C in YPRaff and then shifted to YPGal at 37C to deplete Hof1-td and Cyk3-td simultaneously. Subsequently, cells were released to allow progression through the cell cycle and samples were taken and protein extracts prepared before immunoblotting with anti-GFP antibodies to detect Spa2 protein level. (B) (YMF330) and (YMF1076) strains were grown in YPRaff as in (A). Samples were taken at the indicated times to determine the level of Chs2 protein using anti-Chs2 antibodies. (C) (YAD380) and (YMF1076) strains were arrested in G1 phase at 24C in YPRaff and then.