Figure 1.
Puf5 has Pop2-independent roles in response to HU.
A) Time course experiments were performed in 0.2 M HU. Shown are averages from three independent cultures and the standard deviation. B) Cells were processed for FACS analysis as described in Materials and Methods. C) Cells of the indicated strains were dropped on plates with or without HU and photographed after 3 days at 30°C. D) Time course experiments were performed as in A). Shown are averages of at least six independent colonies for each strain. The error bar is the standard deviation.
Figure 2.
Puf5 has roles in response to HU that are independent of deadenylation.
Survival in 0.2 M HU of the indicated strains was assayed over a 24 h time course. Shown are averages of six independent colonies and the standard deviation.
Figure 3.
puf5 mutants display a Swe1-independent elongated cell morphology in response to HU.
Cells of the indicated strains were grown in the presence or absence of 0.2 M HU for 16 h, fixed in 70% ethanol, rehydrated in PBS and viewed.
Figure 4.
POP2 and PUF5 act in the same genetic pathway in response to caffeine.
A) Cells of the indicated strains were dropped on plates with or without caffeine and incubated for 2–3 days at 30°C. B) wt or pop2Δ mutants (strain YAT179 in Table 1) were transformed with vector only (pRS426) or a 2 µ plasmid expressing Puf5 (pMPT5, 19). Cells were dropped on YPD plates with or without HU and caffeine and the indicated doses and photographed after five days at 30°C. Similar results were obtained on −Ura selective plates (Figure S2).
Figure 5.
LRG1 is not the relevant mRNA target for the roles of Puf5 in response to HU.
Cells of the indicated strains were dropped on plates with or without HU and photographed after three days of growth at 30°C.
Figure 6.
A functional Puf5 RNA binding domain is required and cytoplasmic localization is sufficient for resistance to HU treatment.
The experiments were performed in the W3031-A background, in which deletion of PUF5 alone leads to a strong HU sensitivity phenotype. A) 1-wild type cells transformed with empty vector; 2–4 puf5Δ mutants transformed with vector only, or vector expressing wild type Puf5 or the RNA binding domain mutant PUM mt. GFP is fused to the N-terminus of the Puf5 proteins (wild type and PUM mt) for monitoring localization. For survival experiments, cells were grown and spotted on YPD plates ±HU. A −Ura control plate shows that the cells did not lose the plasmid during cell division. For microscopy, cells were grown in −Ura media. Live cells were viewed and photographed. B) The experiments were performed as in A), with wild type Puf5 or an NLS-Puf5 fusion. C) Survival experiments were performed as in A). Micrographs show immunofluorescence experiments to detect Tom70-Puf5 with an anti-Tom70 antibody.
Table 1.
Yeast strains.