Skip to main content
. 2009 Jan 6;7(1):e1000008. doi: 10.1371/journal.pbio.1000008

Figure 3. The Catalytically Inactive swt1-D135N Mutant Resembles the swt1 Null Mutation In Vivo.

Figure 3

(A) The swt1-D135N mutant does not complement the synthetically lethal/enhanced phenotypes of the indicated swt1Δ double-mutant strains. Strains were transformed with wild-type SWT1 or mutant swt1-D135N, and transformants were spotted in 10-fold serial dilutions on the indicated plates. The strains were incubated for 5 d at 30 °C (tho2Δ swt1Δ) or for 3 d at 36 °C (thp2Δ swt1Δ and nup60Δ swt1Δ).

(B) The expression levels of wild-type and mutant Swt1 proteins are similar. Whole-cell lysates prepared from a swt1Δ strain or a swt1Δ strain expressing wild-type Swt1 or Swt1-D135N were analyzed by SDS-PAGE and western blotting using polyclonal anti-Swt1 antibodies. The detection of Arc1 protein by an anti-Arc1 polyclonal antibody was used as a loading control.

(C) The Swt1-D135N mutant cannot rescue the poly(A)+ export defect of the mlp1Δ swt1Δ strain. mlp1Δ swt1Δ cells expressing wild-type Swt1 or Swt1-D135N were analyzed for poly(A)+ RNA localization as in Figure 2A or stained with DAPI to detect DNA.

(D) Swt1-D135N-expressing cells show a nuclear pre-mRNA leakage phenotype. The swt1Δ and mlp1Δ swt1Δ strains were transformed with the indicated plasmids and assayed for the leakage of the lacZ pre-mRNA reporter (mutBP) as described in Figure 2B. The results of four independent experiments are shown. Error bars indicate standard deviations.

(E) Swt1-D135N cells accumulate elevated levels of mutated lacZ pre-mRNA. Total RNA was isolated from the swt1-D135N and isogenic wild-type strain expressing the intron-containing lacZ reporter transcript (norm., for normal) or the intron-containing lacZ transcript mutated in the branch-point splicing site (mut., for mutated). The lacZ levels were quantified by real-time RT-PCR using the GAL1 mRNA as a reference transcript. The results of four independent experiments are shown. Error bars indicate standard errors of the mean.

(F) Overexpression of wild-type Swt1, but not overexpression of the catalytically inactive Swt1-D135N mutant, is toxic to the cells. Swt1Δ cells containing GAL1::SWT1, GAL1::swt1-D135N, or empty plasmid were spotted in 10-fold serial dilutions on plates containing either galactose or glucose and incubated for 3 d at 30 °C. Only the last two dilutions of glucose plate are shown.

(G) Overexpression of wild-type Swt1, but not overexpression of the catalytically inactive Swt1-D135N mutant, induces strong nuclear accumulation of poly(A)+ RNA. The same strains as used in (F) were grown in raffinose-containing medium to early-log phase and then induced by 2% galactose for 5 h. The localization of poly(A)+ RNA was analyzed by in situ hybridization using a Cy3-oligo(dT) probe. DNA was stained with DAPI. The percentage of the cells showing an accumulation of nuclear poly(A)+ signal is shown (n > 150 cells).