Limits...
A conserved and essential basic region mediates tRNA binding to the Elp1 subunit of the Saccharomyces cerevisiae Elongator complex.

Di Santo R, Bandau S, Stark MJ - Mol. Microbiol. (2014)

Bottom Line: Since these modifications are required for the tRNAs to function efficiently, a translation defect caused by hypomodified tRNAs may therefore underlie the variety of phenotypes associated with Elongator dysfunction.The Elp1 carboxy-terminal domain contains a highly conserved arginine/lysine-rich region that resembles a nuclear localization sequence (NLS).Thus the conserved basic region in Elp1 may be essential for tRNA wobble uridine modification by acting as tRNA binding motif.

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation & Expression, College of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, Scotland, UK.

Show MeSH

Related in: MedlinePlus

The nucleo-cytoplasmic distribution of Elp1 is unaffected by basic region mutations.A. Representative images of strains expressing Nic96-4mCherry (red) together with GFP-tagged wild-type Elp1, Elp1–KR9A, Elp1–NLS or Elp1–NLS4A (green). Elp1–NLS contains the NLS from Cbp80 and Elp1–NLS4A contains a mutant version containing 4 alanine substitutions, inserted in each case between the Elp1 and GFP sections of the fusion protein. Cells were grown to log phase in YPAD medium and then fixed for imaging. Scale bar: 5 μm.B. Zymocin sensitivity assays of the indicated strains as described in the legend to Fig. 2. Wild-type (WT, BY4741) and elp1Δ (YRDS250) served as controls for zymocin sensitivity and resistance respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150532&req=5

fig03: The nucleo-cytoplasmic distribution of Elp1 is unaffected by basic region mutations.A. Representative images of strains expressing Nic96-4mCherry (red) together with GFP-tagged wild-type Elp1, Elp1–KR9A, Elp1–NLS or Elp1–NLS4A (green). Elp1–NLS contains the NLS from Cbp80 and Elp1–NLS4A contains a mutant version containing 4 alanine substitutions, inserted in each case between the Elp1 and GFP sections of the fusion protein. Cells were grown to log phase in YPAD medium and then fixed for imaging. Scale bar: 5 μm.B. Zymocin sensitivity assays of the indicated strains as described in the legend to Fig. 2. Wild-type (WT, BY4741) and elp1Δ (YRDS250) served as controls for zymocin sensitivity and resistance respectively.

Mentions: However, when the distribution of wild-type full length Elp1 between the nucleus and cytoplasm was examined using strains with GFP-tagged Elp1 and mCherry-tagged Nic96 (to define the nuclear boundary), localization of wild-type Elp1–GFP was largely cytoplasmic (Fig. 3A) in agreement with previous reports (Pokholok et al., 2002; Huh et al., 2003; Tkach et al., 2012), but with low levels of Elp1–GFP fluorescence in the nucleus. Furthermore, when the localization of the Elp1–KR9A–GFP mutant was also assayed it showed no difference in the nucleo-cytoplasmic distribution of Elp1 in comparison with wild-type Elp1–GFP (Fig. 3A). Thus while the basic region of Elp1 can drive nuclear import in isolation, in the context of the whole polypeptide this does not appear to be functionally relevant and the apparent nucleo-cytoplasmic distribution of Elp1 was unaffected by the basic region mutation. The elp1–KR5A and elp1–KR4A mutants also showed no differences in Elp1 localization (data not shown). The fact that the elp1–KR4A mutant was almost normal in terms of Elongator function in comparison with the defective elp1–KR9A allele (Fig. 1) and yet the C-terminal domain from either mutant protein was equally compromised in its ability to drive nuclear import of GFP (Fig. 2) is consistent with the role of this region being unrelated to potential NLS function. Finally, we confirmed that when the NLS from Cbp80 was inserted into our Elp1–GFP construct that Elp1–NLS–GFP localized exclusively to the nucleus (Fig. 3A) and that this was associated with complete loss of Elongator-dependent tRNA modification as assayed by zymocin resistance (Fig. 3B). In contrast, when four basic amino acids in the Cbp80 NLS sequence were mutated, Elp1 localization reverted to a mainly cytoplasmic distribution and full sensitivity to zymocin was restored (Fig. 3). This extends the previous findings (Rahl et al., 2005) by showing that Elongator's tRNA wobble uridine modification function, as assayed by zymocin sensitivity, is abolished when Elp1 is trapped in the nucleus. This supports a tRNA modification role for Elongator that requires cytoplasmic rather than nuclear localization. Taken together, these data are therefore consistent with the functional importance of the Elp1 basic region being not through acting as a determinant of nucleo-cytoplasmic localization but through playing some other role.


A conserved and essential basic region mediates tRNA binding to the Elp1 subunit of the Saccharomyces cerevisiae Elongator complex.

Di Santo R, Bandau S, Stark MJ - Mol. Microbiol. (2014)

The nucleo-cytoplasmic distribution of Elp1 is unaffected by basic region mutations.A. Representative images of strains expressing Nic96-4mCherry (red) together with GFP-tagged wild-type Elp1, Elp1–KR9A, Elp1–NLS or Elp1–NLS4A (green). Elp1–NLS contains the NLS from Cbp80 and Elp1–NLS4A contains a mutant version containing 4 alanine substitutions, inserted in each case between the Elp1 and GFP sections of the fusion protein. Cells were grown to log phase in YPAD medium and then fixed for imaging. Scale bar: 5 μm.B. Zymocin sensitivity assays of the indicated strains as described in the legend to Fig. 2. Wild-type (WT, BY4741) and elp1Δ (YRDS250) served as controls for zymocin sensitivity and resistance respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150532&req=5

fig03: The nucleo-cytoplasmic distribution of Elp1 is unaffected by basic region mutations.A. Representative images of strains expressing Nic96-4mCherry (red) together with GFP-tagged wild-type Elp1, Elp1–KR9A, Elp1–NLS or Elp1–NLS4A (green). Elp1–NLS contains the NLS from Cbp80 and Elp1–NLS4A contains a mutant version containing 4 alanine substitutions, inserted in each case between the Elp1 and GFP sections of the fusion protein. Cells were grown to log phase in YPAD medium and then fixed for imaging. Scale bar: 5 μm.B. Zymocin sensitivity assays of the indicated strains as described in the legend to Fig. 2. Wild-type (WT, BY4741) and elp1Δ (YRDS250) served as controls for zymocin sensitivity and resistance respectively.
Mentions: However, when the distribution of wild-type full length Elp1 between the nucleus and cytoplasm was examined using strains with GFP-tagged Elp1 and mCherry-tagged Nic96 (to define the nuclear boundary), localization of wild-type Elp1–GFP was largely cytoplasmic (Fig. 3A) in agreement with previous reports (Pokholok et al., 2002; Huh et al., 2003; Tkach et al., 2012), but with low levels of Elp1–GFP fluorescence in the nucleus. Furthermore, when the localization of the Elp1–KR9A–GFP mutant was also assayed it showed no difference in the nucleo-cytoplasmic distribution of Elp1 in comparison with wild-type Elp1–GFP (Fig. 3A). Thus while the basic region of Elp1 can drive nuclear import in isolation, in the context of the whole polypeptide this does not appear to be functionally relevant and the apparent nucleo-cytoplasmic distribution of Elp1 was unaffected by the basic region mutation. The elp1–KR5A and elp1–KR4A mutants also showed no differences in Elp1 localization (data not shown). The fact that the elp1–KR4A mutant was almost normal in terms of Elongator function in comparison with the defective elp1–KR9A allele (Fig. 1) and yet the C-terminal domain from either mutant protein was equally compromised in its ability to drive nuclear import of GFP (Fig. 2) is consistent with the role of this region being unrelated to potential NLS function. Finally, we confirmed that when the NLS from Cbp80 was inserted into our Elp1–GFP construct that Elp1–NLS–GFP localized exclusively to the nucleus (Fig. 3A) and that this was associated with complete loss of Elongator-dependent tRNA modification as assayed by zymocin resistance (Fig. 3B). In contrast, when four basic amino acids in the Cbp80 NLS sequence were mutated, Elp1 localization reverted to a mainly cytoplasmic distribution and full sensitivity to zymocin was restored (Fig. 3). This extends the previous findings (Rahl et al., 2005) by showing that Elongator's tRNA wobble uridine modification function, as assayed by zymocin sensitivity, is abolished when Elp1 is trapped in the nucleus. This supports a tRNA modification role for Elongator that requires cytoplasmic rather than nuclear localization. Taken together, these data are therefore consistent with the functional importance of the Elp1 basic region being not through acting as a determinant of nucleo-cytoplasmic localization but through playing some other role.

Bottom Line: Since these modifications are required for the tRNAs to function efficiently, a translation defect caused by hypomodified tRNAs may therefore underlie the variety of phenotypes associated with Elongator dysfunction.The Elp1 carboxy-terminal domain contains a highly conserved arginine/lysine-rich region that resembles a nuclear localization sequence (NLS).Thus the conserved basic region in Elp1 may be essential for tRNA wobble uridine modification by acting as tRNA binding motif.

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation & Expression, College of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, Scotland, UK.

Show MeSH
Related in: MedlinePlus