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Analysis of a predicted nuclear localization signal: implications for the intracellular localization and function of the Saccharomyces cerevisiae RNA-binding protein Scp160.

Brykailo MA, McLane LM, Fridovich-Keil J, Corbett AH - Nucleic Acids Res. (2007)

Bottom Line: We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus.These localization studies were complemented by a mutational analysis of the predicted NLS.Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics and Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Gene expression is controlled by RNA-binding proteins that modulate the synthesis, processing, transport and stability of various classes of RNA. Some RNA-binding proteins shuttle between the nucleus and cytoplasm and are thought to bind to RNA transcripts in the nucleus and remain bound during translocation to the cytoplasm. One RNA-binding protein that has been hypothesized to function in this manner is the Saccharomyces cerevisiae Scp160 protein. Although the steady-state localization of Scp160 is cytoplasmic, previous studies have identified putative nuclear localization (NLS) and nuclear export (NES) signals. The goal of this study was to test the hypothesis that Scp160 is a nucleocytoplasmic shuttling protein. We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus. These localization studies were complemented by a mutational analysis of the predicted NLS. Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function. This finding has important implications for understanding the function of Scp160, which is likely limited to the cytoplasm. Additionally, our results provide strong evidence that the presence of a predicted nuclear localization signal within the sequence of a protein should not lead to the assumption that the protein enters the nucleus in the absence of additional experimental evidence.

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Scp160 is localized to the cytoplasm at steady state. (A) Schematic representation of Scp160 domain structure and location of putative nuclear targeting motifs. The predicted NLS is indicated by the filled diamond and the predicted NES is indicated by the open diamond. The sequences for the predicted NLS and NES motifs as well as their positions within the open reading frame are indicated. Numbered boxes represent the 14 KH domains. (B) The Scp160-GFP protein was expressed in wild-type cells that express a red-fluorescent protein-tagged nuclear rim protein, Mlp1 (56) to mark the position of the nucleus. Fluorescent protein localization was examined by direct fluorescence microscopy. Cells were also stained with Hoechst dye to mark the position of chromatin within the nucleus. A merged fluorescence image is shown as well as the corresponding DIC image. (C) Immunoblot analysis of GFP in protein lysate from wild-type cells transformed with plasmids encoding FLAG-Scp160-GFP (right lane) or a control NLS-NES-GFP (left lane). Migration of protein standards is indicated to the left of the image. The position of the bands corresponding to FLAG-Scp160-GFP and NLS-NES-GFP is indicated.
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Figure 1: Scp160 is localized to the cytoplasm at steady state. (A) Schematic representation of Scp160 domain structure and location of putative nuclear targeting motifs. The predicted NLS is indicated by the filled diamond and the predicted NES is indicated by the open diamond. The sequences for the predicted NLS and NES motifs as well as their positions within the open reading frame are indicated. Numbered boxes represent the 14 KH domains. (B) The Scp160-GFP protein was expressed in wild-type cells that express a red-fluorescent protein-tagged nuclear rim protein, Mlp1 (56) to mark the position of the nucleus. Fluorescent protein localization was examined by direct fluorescence microscopy. Cells were also stained with Hoechst dye to mark the position of chromatin within the nucleus. A merged fluorescence image is shown as well as the corresponding DIC image. (C) Immunoblot analysis of GFP in protein lysate from wild-type cells transformed with plasmids encoding FLAG-Scp160-GFP (right lane) or a control NLS-NES-GFP (left lane). Migration of protein standards is indicated to the left of the image. The position of the bands corresponding to FLAG-Scp160-GFP and NLS-NES-GFP is indicated.

Mentions: Figure 1A shows a schematic representation of the domain structure and postulated sequence motifs of Scp160. The predicted NES sequence is contained within the N-terminal portion of the protein, while the predicted NLS is located between KH domains three and four (KH3 and KH4), spanning a few residues into both KH3 and KH4. The amino acid sequence for each predicted transport signal is shown.Figure 1.


Analysis of a predicted nuclear localization signal: implications for the intracellular localization and function of the Saccharomyces cerevisiae RNA-binding protein Scp160.

Brykailo MA, McLane LM, Fridovich-Keil J, Corbett AH - Nucleic Acids Res. (2007)

Scp160 is localized to the cytoplasm at steady state. (A) Schematic representation of Scp160 domain structure and location of putative nuclear targeting motifs. The predicted NLS is indicated by the filled diamond and the predicted NES is indicated by the open diamond. The sequences for the predicted NLS and NES motifs as well as their positions within the open reading frame are indicated. Numbered boxes represent the 14 KH domains. (B) The Scp160-GFP protein was expressed in wild-type cells that express a red-fluorescent protein-tagged nuclear rim protein, Mlp1 (56) to mark the position of the nucleus. Fluorescent protein localization was examined by direct fluorescence microscopy. Cells were also stained with Hoechst dye to mark the position of chromatin within the nucleus. A merged fluorescence image is shown as well as the corresponding DIC image. (C) Immunoblot analysis of GFP in protein lysate from wild-type cells transformed with plasmids encoding FLAG-Scp160-GFP (right lane) or a control NLS-NES-GFP (left lane). Migration of protein standards is indicated to the left of the image. The position of the bands corresponding to FLAG-Scp160-GFP and NLS-NES-GFP is indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175298&req=5

Figure 1: Scp160 is localized to the cytoplasm at steady state. (A) Schematic representation of Scp160 domain structure and location of putative nuclear targeting motifs. The predicted NLS is indicated by the filled diamond and the predicted NES is indicated by the open diamond. The sequences for the predicted NLS and NES motifs as well as their positions within the open reading frame are indicated. Numbered boxes represent the 14 KH domains. (B) The Scp160-GFP protein was expressed in wild-type cells that express a red-fluorescent protein-tagged nuclear rim protein, Mlp1 (56) to mark the position of the nucleus. Fluorescent protein localization was examined by direct fluorescence microscopy. Cells were also stained with Hoechst dye to mark the position of chromatin within the nucleus. A merged fluorescence image is shown as well as the corresponding DIC image. (C) Immunoblot analysis of GFP in protein lysate from wild-type cells transformed with plasmids encoding FLAG-Scp160-GFP (right lane) or a control NLS-NES-GFP (left lane). Migration of protein standards is indicated to the left of the image. The position of the bands corresponding to FLAG-Scp160-GFP and NLS-NES-GFP is indicated.
Mentions: Figure 1A shows a schematic representation of the domain structure and postulated sequence motifs of Scp160. The predicted NES sequence is contained within the N-terminal portion of the protein, while the predicted NLS is located between KH domains three and four (KH3 and KH4), spanning a few residues into both KH3 and KH4. The amino acid sequence for each predicted transport signal is shown.Figure 1.

Bottom Line: We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus.These localization studies were complemented by a mutational analysis of the predicted NLS.Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics and Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Gene expression is controlled by RNA-binding proteins that modulate the synthesis, processing, transport and stability of various classes of RNA. Some RNA-binding proteins shuttle between the nucleus and cytoplasm and are thought to bind to RNA transcripts in the nucleus and remain bound during translocation to the cytoplasm. One RNA-binding protein that has been hypothesized to function in this manner is the Saccharomyces cerevisiae Scp160 protein. Although the steady-state localization of Scp160 is cytoplasmic, previous studies have identified putative nuclear localization (NLS) and nuclear export (NES) signals. The goal of this study was to test the hypothesis that Scp160 is a nucleocytoplasmic shuttling protein. We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus. These localization studies were complemented by a mutational analysis of the predicted NLS. Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function. This finding has important implications for understanding the function of Scp160, which is likely limited to the cytoplasm. Additionally, our results provide strong evidence that the presence of a predicted nuclear localization signal within the sequence of a protein should not lead to the assumption that the protein enters the nucleus in the absence of additional experimental evidence.

Show MeSH
Related in: MedlinePlus