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. 2020 Jan 28;9:e54355. doi: 10.7554/eLife.54355

Figure 1. GdnHCl rescues the temperature-sensitive defects of cdc10(D182N) mutants in a manner that does not require Hsp104 or Hsp78.

(A) Dilution series of yeast cells spotted on rich (YPD) agar medium with or without 3 mM GdnHCl and incubated at the indicated temperatures for 3 days before imaging. Strains were: BY4741, CBY07236, CBY06417, CBY08756, and CBY05110. Effects of GdnHCl on septin ring formation and on the growth strains carrying other mutant alleles are provided in Figure 1—figure supplement 1. (B) As in (A), but only at 37°C and with strains of the indicated genotypes. (C) Amino acid alignment of regions S. cerevisiae Hsp104 and Hsp78 with Thermus thermophilus ClpB, showing conservation of residues (in red) that contact Gdm in the ClpB crystal structure (PDB 4HSE). (D) As in (A), but at 37°C and with BY4741 (‘WT’) or CBY06417 (‘cdc10(D182N)') cells and the indicated concentrations of GdnHCl. The cells were streaked with a toothpick, rather than spotted from dilutions. (E) Growth curves for the cdc10(D182N) strain CBY06417 at 37°C in the indicated concentrations of GdnHCl. The optical density at 600 nm (‘OD600’) was measured at 5 min intervals. Each line shows the mean for 12 replicate cultures in the same row of the 96-well plate. Duplicate rows were monitored for each GdnHCl concentration.

Figure 1—source data 1. Optical density readings of culture growth for Figure 1E.
elife-54355-fig1-data1.xlsx (151.8KB, xlsx)

Figure 1.

Figure 1—figure supplement 1. GdnHCl rescues high-temperature septin function and neck filament assembly in cdc10 mutants.

Figure 1—figure supplement 1.

(A) Dilution series of cells of the indicated genotypes (strains BY4741, CBY06417, CBY06420, or CBY06424) spotted on YPD plates with or without 3 mM GdnHCl and incubated at the indicated temperatures for 3–4 days. (B) anti-Cdc11 antibodies were used to stain cdc10∆ cells cultured at 37°C in 3 mM GdnHCl. Arrowhead, septin ring. (C) Cells of the cdc10(D182N)-GFP strain JTY3986 were cultured at 37°C in liquid YPD with 3 mM GdnHCl, cryo-fixed, cryo-substituted, sectioned, and stained with uranyl acetate prior to imaging by EM. Arrowheads indicate neck filaments tightly apposed to the plasma membrane. (D) As in (A), but with cells of strain BY4741 (‘WT’) or MMY0370 (‘cdc10∆ dpl1∆'). An intervening area of the plates was cropped from the images.