The finding that Ctk1 is associated with translating ribosomes and that cells deleted or
depleted for Ctk1 show a reduced translational activity in vivo and in vitro, pinpointed to a
novel function of Ctk1 in the translation of mRNA. However, by which means Ctk1 precisely exhibits its function in translation was unclear.
2.10.1 Ctk1 is not involved in ribosome biogenesis
Even though it could be demonstrated that the mRNA of ∆ctk1 cells is exported to the
cytoplasm (Figure 14) and that these mRNAs were fully translatable in in vitro translation
assays (Figure 22), one likely explanation for the deficient translation could be that Ctk1 functions in ribosome biogenesis, especially since it was shown that the RNAP I transcription
pattern and most likely as an effect the structure of the nucleolus is altered in ∆ctk1 cells
(Bouchoux et al. 2004; Grenetier et al. 2006). Defects in ribosome biogenesis can be investigated by several methods. It is known that polysome profiles of dissociated ribosomes can be indicative for a defect in ribosome biogenesis as the ratio of 40S to 60S would change in case of ribosome maturation defects (Dong et al. 2005). When gradients of EDTA-treated
extracts from wt and ∆ctk1 cells were analyzed with regard to the relative ratio between 40S
and 60S, it turned out that this ratio did not change significantly in ∆ctk1 cells (Figure 26a),
suggesting that the deletion of CTK1 does not lead to a severe defect in 40S or 60S ribosome
biogenesis.
Furthermore, defects in early ribosome biogenesis can be observed by accumulation of GFP- tagged ribosomal proteins in the nucleolus or nucleoplasm, under conditions where the export is either directly blocked or maturation defects prevent the proper export of the ribosomal particles in the cytoplasm (for review see Tschochner and Hurt 2003). As the export of ribosomal subunits is dependent on the nuclear export receptor Xpo1, a ts mutant of Xpo1 served as a positive control for the nuclear accumulation of GFP-tagged reporter plasmids for the small, Rps2, and the large, Rpl25, ribosomal subunits (Stade et al. 1997; Moy and Silver
1999; Gadal et al. 2001). Figure 26b shows that in the xpo1 mutant the proteins of the small
and the large ribosomal subunits clearly accumulated inside the nucleus, whereas in ∆ctk1 and
wt cells the GFP signal was evenly distributed all over the cytoplasm, indicating that the loss of Ctk1 function does not lead to a nuclear export defect of the subunits. Although it cannot be completely ruled out that deletion of Ctk1 leads to a minor ribosome biogenesis defect, a major function of Ctk1 in ribosome biogenesis is rather unlikely.
Figure 26: Deletion of Ctk1 does not lead to a defect in ribosome biogenesis.
(a) The ratio of 40S to 60S subunits does not change significantly in ∆ctk1 cells in
comparison to wt cells. 15-30% sucrose density gradients were performed with extracts of wt and ∆ctk1 cells treated with EDTA to disrupt the ribosomes into the subunits. (b) Ribosomal
subunits are exported into the cytoplasm in ∆ctk1 cells. The localization of GFP-tagged Rps2
as a protein of the small ribosomal subunit and GFP-tagged Rpl25 as a protein of the large ribosomal subunit was analyzed in living cells.
2.10.2 Ctk1 influences translation initiation
The in vitro translation system described above mainly measures translation initiation as prior
to addition of L-[35S]-methionine no methionine is present in the extract and consequently, the
translation process is mainly arrested at the stage of initiation when the assay is started. Defects in initiation can be visualized by profiles of polysome density gradients because a
defect in translation initiation results in an increased 80S peak and reduced polysome level (Zhong and Arndt 1993; Nielsen et al. 2004; Dong et al. 2005). When polysome profiles of wt and ∆ctk1 cells were compared, it was obvious that the deletion of Ctk1 lead to an increase of monosomes (80S) and a concomitant reduction in polysomes (Figure 27), suggesting that Ctk1 has a function in initiating translation.
Figure 27: Deletion of Ctk1 leads to an increase in 80S monosomes and a simultaneous reduction in polysomes.
Extracts of WT (a) and ∆ctk1 cells (b) were analyzed by sucrose density centrifugation.
2.10.3 Ctk1 depleted cells are sensitive towards drugs that influence
translation
To uncover an unknown function of a protein, it is a common approach to analyze sensitivity or resistance of a mutant strain towards drugs that affect the process of interest. Thus, the
sensitivity of wt and GAL1::CTK1-TAP cells grown for 18 hours in YPD, i.e. Ctk1 depleted,
were tested for sensitivity towards the translational inhibitors paromomycin, hygromycin B, geneticin, cycloheximide and anisomycin. Cells depleted for Ctk1 are significantly more sensitive to all drugs tested than an isogenic wild type strain (Figure 28). Hypersensitivity towards paromomycin, hygromycin B and geneticin has often been linked to loss of translational accuracy, as these aminoglycoside antibiotics bind to the decoding region of the ribosomal A-site and stimulate the stable association of a near-cognate aminoacyl tRNA (Palmer et al. 1979; Singh et al. 1979; Moazed and Noller 1987). This result suggests that
besides the putative function in translation initiation, Ctk1 might be needed for the correct decoding of the mRNA.
Figure 28: Cells that are depleted for Ctk1 are sensitive towards translation inhibitors.
Wt and GAL1::CTK1-TAP cells were grown for 18 hours in glucose-containing medium and
then spread on YPD plates containing a filter on which paromomycin, hygromycin B, geneticin, cycloheximide, or anisomycin were applied. The size of the halo indicates the sensitivity of the strain towards this drug.