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Identification and functional analysis of NOL7 nuclear and nucleolar localization signals.

Zhou G, Doçi CL, Lingen MW - BMC Cell Biol. (2010)

Bottom Line: In support, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7.These results identify the minimal sequences required for the active targeting of NOL7 to the nucleus and nucleolus.Taken together, these results identify the requisite protein domains for NOL7 localization, the kinetics that drive this targeting, and suggest NOL7 may function in both the nucleus and nucleolus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, The University of Chicago, Chicago, IL, USA.

ABSTRACT

Background: NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.

Results: An in vitro import assay demonstrated that NOL7 requires cytosolic machinery for active nuclear transport. Using sequence homology and prediction algorithms, four putative nuclear localization signals (NLSs) were identified. NOL7 deletion constructs and cytoplasmic pyruvate kinase (PK) fusion proteins confirmed the functionality of three of these NLSs. Site-directed mutagenesis of PK fusions and full-length NOL7 defined the minimal functional regions within each NLS. Further characterization revealed that NLS2 and NLS3 were critical for both the rate and efficiency of nuclear targeting. In addition, four basic clusters within NLS2 and NLS3 were independently capable of nucleolar targeting. The nucleolar occupancy of NOL7 revealed a complex balance of rapid nucleoplasmic shuttling but low nucleolar mobility, suggesting NOL7 may play functional roles in both compartments. In support, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7.

Conclusions: These results identify the minimal sequences required for the active targeting of NOL7 to the nucleus and nucleolus. Further, this work characterizes the relative contribution of each sequence to NOL7 nuclear and nucleolar dynamics, the subnuclear constituents that participate in this targeting, and suggests a functional role for NOL7 in both compartments. Taken together, these results identify the requisite protein domains for NOL7 localization, the kinetics that drive this targeting, and suggest NOL7 may function in both the nucleus and nucleolus.

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NOL7 requires cytosolic factors for efficient nuclear localization. HeLa cells permeabilized with digitonin were incubated at 4°C or 37°C as indicated with (+) or without (-) full length NOL7 expressing a C-terminal GFP tag, cytosol, heat-inactivated cytosol, ATP, or WGA. Localization of NOL7 was confirmed by visualization of GFP.
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Figure 1: NOL7 requires cytosolic factors for efficient nuclear localization. HeLa cells permeabilized with digitonin were incubated at 4°C or 37°C as indicated with (+) or without (-) full length NOL7 expressing a C-terminal GFP tag, cytosol, heat-inactivated cytosol, ATP, or WGA. Localization of NOL7 was confirmed by visualization of GFP.

Mentions: NOL7 is predicted to have a molecular weight of approximately 29 kDa. Some proteins of this size have been reported to enter the nucleus through passive diffusion, while others require active NLS-mediated transport. To distinguish between an active transport and a passive diffusion mechanism of nuclear localization for NOL7, we subjected GFP-tagged NOL7 to an in vitro transport assay using permeabilized HeLa cells [54]. In this assay, the cytoplasmic membranes are permeabilized with digitonin, which depletes the cells of their soluble endogenous cytosolic factors while leaving the nuclear membrane intact. In this fashion, the nuclear import of GFP-tagged proteins can be studied under various conditions. In these experiments, digitonin-permeabilized HeLa cells were incubated with GFP-tagged NOL7 under various conditions to assay the mechanism of its localization. NOL7-GFP alone was not sufficient for nuclear import (Figure 1, first panel). Addition of cytosolic extract or ATP alone was also insufficient for transport (Figure 1, second and third panels), suggesting that NOL7 localization is energy and complex dependent. At 4°C or in the presence of heat-inactivated cytosol, NOL7-GFP was restricted to the cytoplasm (Figure 1, fourth and fifth panels), demonstrating that NOL7 requires cytosolic proteins and is localized via an active transport mechanism. At physiologic temperature, combination of cytosol and ATP was sufficient for NOL7 nuclear localization (Figure 1, sixth panel). However, this was blocked by pretreatment with wheat germ agglutinin (WGA) (Figure 1, seventh panel). Together, these controls demonstrate that NOL7 is actively targeted to the nucleus and nucleolus via an energy-dependent, nucleoporin-mediated mechanism.


Identification and functional analysis of NOL7 nuclear and nucleolar localization signals.

Zhou G, Doçi CL, Lingen MW - BMC Cell Biol. (2010)

NOL7 requires cytosolic factors for efficient nuclear localization. HeLa cells permeabilized with digitonin were incubated at 4°C or 37°C as indicated with (+) or without (-) full length NOL7 expressing a C-terminal GFP tag, cytosol, heat-inactivated cytosol, ATP, or WGA. Localization of NOL7 was confirmed by visualization of GFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2957388&req=5

Figure 1: NOL7 requires cytosolic factors for efficient nuclear localization. HeLa cells permeabilized with digitonin were incubated at 4°C or 37°C as indicated with (+) or without (-) full length NOL7 expressing a C-terminal GFP tag, cytosol, heat-inactivated cytosol, ATP, or WGA. Localization of NOL7 was confirmed by visualization of GFP.
Mentions: NOL7 is predicted to have a molecular weight of approximately 29 kDa. Some proteins of this size have been reported to enter the nucleus through passive diffusion, while others require active NLS-mediated transport. To distinguish between an active transport and a passive diffusion mechanism of nuclear localization for NOL7, we subjected GFP-tagged NOL7 to an in vitro transport assay using permeabilized HeLa cells [54]. In this assay, the cytoplasmic membranes are permeabilized with digitonin, which depletes the cells of their soluble endogenous cytosolic factors while leaving the nuclear membrane intact. In this fashion, the nuclear import of GFP-tagged proteins can be studied under various conditions. In these experiments, digitonin-permeabilized HeLa cells were incubated with GFP-tagged NOL7 under various conditions to assay the mechanism of its localization. NOL7-GFP alone was not sufficient for nuclear import (Figure 1, first panel). Addition of cytosolic extract or ATP alone was also insufficient for transport (Figure 1, second and third panels), suggesting that NOL7 localization is energy and complex dependent. At 4°C or in the presence of heat-inactivated cytosol, NOL7-GFP was restricted to the cytoplasm (Figure 1, fourth and fifth panels), demonstrating that NOL7 requires cytosolic proteins and is localized via an active transport mechanism. At physiologic temperature, combination of cytosol and ATP was sufficient for NOL7 nuclear localization (Figure 1, sixth panel). However, this was blocked by pretreatment with wheat germ agglutinin (WGA) (Figure 1, seventh panel). Together, these controls demonstrate that NOL7 is actively targeted to the nucleus and nucleolus via an energy-dependent, nucleoporin-mediated mechanism.

Bottom Line: In support, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7.These results identify the minimal sequences required for the active targeting of NOL7 to the nucleus and nucleolus.Taken together, these results identify the requisite protein domains for NOL7 localization, the kinetics that drive this targeting, and suggest NOL7 may function in both the nucleus and nucleolus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, The University of Chicago, Chicago, IL, USA.

ABSTRACT

Background: NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.

Results: An in vitro import assay demonstrated that NOL7 requires cytosolic machinery for active nuclear transport. Using sequence homology and prediction algorithms, four putative nuclear localization signals (NLSs) were identified. NOL7 deletion constructs and cytoplasmic pyruvate kinase (PK) fusion proteins confirmed the functionality of three of these NLSs. Site-directed mutagenesis of PK fusions and full-length NOL7 defined the minimal functional regions within each NLS. Further characterization revealed that NLS2 and NLS3 were critical for both the rate and efficiency of nuclear targeting. In addition, four basic clusters within NLS2 and NLS3 were independently capable of nucleolar targeting. The nucleolar occupancy of NOL7 revealed a complex balance of rapid nucleoplasmic shuttling but low nucleolar mobility, suggesting NOL7 may play functional roles in both compartments. In support, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7.

Conclusions: These results identify the minimal sequences required for the active targeting of NOL7 to the nucleus and nucleolus. Further, this work characterizes the relative contribution of each sequence to NOL7 nuclear and nucleolar dynamics, the subnuclear constituents that participate in this targeting, and suggests a functional role for NOL7 in both compartments. Taken together, these results identify the requisite protein domains for NOL7 localization, the kinetics that drive this targeting, and suggest NOL7 may function in both the nucleus and nucleolus.

Show MeSH
Related in: MedlinePlus