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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.

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Related in: MedlinePlus

The Elp1 C-terminal domain can drive nuclear import of GFP dependent on the conserved basic region. Representative images of cells expressing Nic96-4mCherry (red) to indicate the nuclear periphery (YRDS84) containing either pUG34 (GFP control) expressing GFP or its derivative plasmids expressing GFP fused at its C-terminus to residues 1181–1349 from wild-type (Elp1–CTD) or the mutant (Elp1–KR4A-CTD, Elp1–KR5A–CTD, Elp1–KR9A–CTD) versions of Elp1 shown in Fig. 1 (green). Cells were grown to log phase in DOA-Met-His medium to induce GFP expression and then fixed for imaging. Both GFP (27 kDa) and the fusion proteins (47 kDa) are expected to be present in both the nucleus and cytoplasm in the absence of active nuclear localization signals as they are small enough to enter the nucleus by diffusion. CTD, C-terminal domain; scale bar: 5 μm.
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fig02: The Elp1 C-terminal domain can drive nuclear import of GFP dependent on the conserved basic region. Representative images of cells expressing Nic96-4mCherry (red) to indicate the nuclear periphery (YRDS84) containing either pUG34 (GFP control) expressing GFP or its derivative plasmids expressing GFP fused at its C-terminus to residues 1181–1349 from wild-type (Elp1–CTD) or the mutant (Elp1–KR4A-CTD, Elp1–KR5A–CTD, Elp1–KR9A–CTD) versions of Elp1 shown in Fig. 1 (green). Cells were grown to log phase in DOA-Met-His medium to induce GFP expression and then fixed for imaging. Both GFP (27 kDa) and the fusion proteins (47 kDa) are expected to be present in both the nucleus and cytoplasm in the absence of active nuclear localization signals as they are small enough to enter the nucleus by diffusion. CTD, C-terminal domain; scale bar: 5 μm.

Mentions: In agreement with earlier work (Fichtner et al., 2003), we found that the Elp1 C-terminal domain (amino acids 1181–1349) was capable of driving nuclear import of GFP and that this was dependent specifically on the basic region. Thus GFP tagged with the wild-type Elp1 C-terminus was concentrated exclusively in the cell nucleus (Fig. 2), while the elp1–KR9A, elp1–KR5A and elp1–KR4A mutants all showed diffuse localization of GFP throughout the cell that was comparable to that of GFP alone, suggesting that when the basic region is mutated the C-terminus can no longer influence GFP localization. The basic region in the Elp1 carboxy-terminal domain therefore has the potential to drive nuclear import.


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 Elp1 C-terminal domain can drive nuclear import of GFP dependent on the conserved basic region. Representative images of cells expressing Nic96-4mCherry (red) to indicate the nuclear periphery (YRDS84) containing either pUG34 (GFP control) expressing GFP or its derivative plasmids expressing GFP fused at its C-terminus to residues 1181–1349 from wild-type (Elp1–CTD) or the mutant (Elp1–KR4A-CTD, Elp1–KR5A–CTD, Elp1–KR9A–CTD) versions of Elp1 shown in Fig. 1 (green). Cells were grown to log phase in DOA-Met-His medium to induce GFP expression and then fixed for imaging. Both GFP (27 kDa) and the fusion proteins (47 kDa) are expected to be present in both the nucleus and cytoplasm in the absence of active nuclear localization signals as they are small enough to enter the nucleus by diffusion. CTD, C-terminal domain; scale bar: 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: The Elp1 C-terminal domain can drive nuclear import of GFP dependent on the conserved basic region. Representative images of cells expressing Nic96-4mCherry (red) to indicate the nuclear periphery (YRDS84) containing either pUG34 (GFP control) expressing GFP or its derivative plasmids expressing GFP fused at its C-terminus to residues 1181–1349 from wild-type (Elp1–CTD) or the mutant (Elp1–KR4A-CTD, Elp1–KR5A–CTD, Elp1–KR9A–CTD) versions of Elp1 shown in Fig. 1 (green). Cells were grown to log phase in DOA-Met-His medium to induce GFP expression and then fixed for imaging. Both GFP (27 kDa) and the fusion proteins (47 kDa) are expected to be present in both the nucleus and cytoplasm in the absence of active nuclear localization signals as they are small enough to enter the nucleus by diffusion. CTD, C-terminal domain; scale bar: 5 μm.
Mentions: In agreement with earlier work (Fichtner et al., 2003), we found that the Elp1 C-terminal domain (amino acids 1181–1349) was capable of driving nuclear import of GFP and that this was dependent specifically on the basic region. Thus GFP tagged with the wild-type Elp1 C-terminus was concentrated exclusively in the cell nucleus (Fig. 2), while the elp1–KR9A, elp1–KR5A and elp1–KR4A mutants all showed diffuse localization of GFP throughout the cell that was comparable to that of GFP alone, suggesting that when the basic region is mutated the C-terminus can no longer influence GFP localization. The basic region in the Elp1 carboxy-terminal domain therefore has the potential to drive nuclear import.

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