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Mutant Rab24 GTPase is targeted to nuclear inclusions.

Maltese WA, Soule G, Gunning W, Calomeni E, Alexander B - BMC Cell Biol. (2002)

Bottom Line: Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form.If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope.However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, OH 43614, USA. wmaltese@mco.edu

ABSTRACT

Background: Members of the Rab GTPase family regulate intracellular protein trafficking, but the specific function of Rab24 remains unknown. Several attributes distinguish this protein from other members of the Rab family, including a low intrinsic GTPase activity.

Results: The functions of other Rab proteins have been defined through the use of dominant-negative mutants with amino acid substitutions in the conserved N(T)KxD nucleotide binding motif. Surprisingly, when such Rab24 constructs were expressed in cultured cells, they accumulated in nuclear inclusions which disrupted the integrity of the nuclear envelope. The inclusions reacted positively with antibodies against ubiquitin and Hsp70, similar to protein aggregates observed in polyglutamine disorders. They also appeared to sequester importin-beta and GFP-coupled glucocorticoid receptor. Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form. Studies with Rab24/Rab1B chimeras indicated that targeting of the mutant protein to inclusions requires the unique C-terminal domain of Rab24.

Conclusion: These studies demonstrate that mutations in Rab24 can trigger a cytopathic cellular response involving accumulation of nuclear inclusions. If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope. However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.

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Subcellular distribution of wild-type and mutant Rab24 expressed in 293 cells. (A) Cells were transfected with vectors encoding mycRab24wt or mycRab24(D123I) and 24 h later the expressed proteins were localized by immunofluorescence microscopy, using primary antibodies against the myc epitope or the Rab24 C-terminal hypervariable domain, as indicated above each panel. At the exposure setting used for the photograph, the Rab24 antibody did not give a detectable signal in the non-transfected cells (indicated by asterisk). The bar equals10 microns. (B) Cells were transfected with a vector encoding Rab24(D123I) without the myc epitope, and the expressed protein was localized with the Rab24 antibody. (C) Transfected cells expressing mycRab24wt or mycRab24(D123I) were lysed in buffer without detergent and fractionated by centrifugation as described in the Methods. The cytosol and particulate fractions were subjected to SDS-PAGE and immunoblot analysis, using the anti-myc monoclonal antibody to detect the expressed proteins. (D) Transfected cells expressing mycRab24wt or mycRab24(D123I) were lysed in high-detergent buffer (see Methods) and the proteins recovered in the detergent-soluble and insoluble fractions were assayed by SDS-PAGE and immunoblot analysis, using the anti-myc monoclonal antibody.
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Figure 1: Subcellular distribution of wild-type and mutant Rab24 expressed in 293 cells. (A) Cells were transfected with vectors encoding mycRab24wt or mycRab24(D123I) and 24 h later the expressed proteins were localized by immunofluorescence microscopy, using primary antibodies against the myc epitope or the Rab24 C-terminal hypervariable domain, as indicated above each panel. At the exposure setting used for the photograph, the Rab24 antibody did not give a detectable signal in the non-transfected cells (indicated by asterisk). The bar equals10 microns. (B) Cells were transfected with a vector encoding Rab24(D123I) without the myc epitope, and the expressed protein was localized with the Rab24 antibody. (C) Transfected cells expressing mycRab24wt or mycRab24(D123I) were lysed in buffer without detergent and fractionated by centrifugation as described in the Methods. The cytosol and particulate fractions were subjected to SDS-PAGE and immunoblot analysis, using the anti-myc monoclonal antibody to detect the expressed proteins. (D) Transfected cells expressing mycRab24wt or mycRab24(D123I) were lysed in high-detergent buffer (see Methods) and the proteins recovered in the detergent-soluble and insoluble fractions were assayed by SDS-PAGE and immunoblot analysis, using the anti-myc monoclonal antibody.

Mentions: When mycRab24(wt) was transiently expressed in 293 cells, immunofluorescence microscopy revealed a diffuse reticular distribution throughout the cytoplasm, with a concentration in the perinuclear region (Fig. 1A). In contrast, when the same studies were performed with cells expressing mycRab24(D123I), we observed a striking accumulation of the mutant protein in punctate structures localized in both cytoplasmic and nuclear compartments. In most cells the inclusions appeared to be concentrated predominantly within the nucleus (Fig. 1A). Identical structures were seen when immunofluorescence microscopy was performed with antibodies against the N-terminal myc epitope or the C-terminal hypervariable domain of Rab24, indicating that the immunofluorescence pattern reflected the localization of full-length mycRab24(D123I). Finally, the localization pattern of Rab24(D123I) remained the same when the protein was expressed without the myc tag (Fig. 1B).


Mutant Rab24 GTPase is targeted to nuclear inclusions.

Maltese WA, Soule G, Gunning W, Calomeni E, Alexander B - BMC Cell Biol. (2002)

Subcellular distribution of wild-type and mutant Rab24 expressed in 293 cells. (A) Cells were transfected with vectors encoding mycRab24wt or mycRab24(D123I) and 24 h later the expressed proteins were localized by immunofluorescence microscopy, using primary antibodies against the myc epitope or the Rab24 C-terminal hypervariable domain, as indicated above each panel. At the exposure setting used for the photograph, the Rab24 antibody did not give a detectable signal in the non-transfected cells (indicated by asterisk). The bar equals10 microns. (B) Cells were transfected with a vector encoding Rab24(D123I) without the myc epitope, and the expressed protein was localized with the Rab24 antibody. (C) Transfected cells expressing mycRab24wt or mycRab24(D123I) were lysed in buffer without detergent and fractionated by centrifugation as described in the Methods. The cytosol and particulate fractions were subjected to SDS-PAGE and immunoblot analysis, using the anti-myc monoclonal antibody to detect the expressed proteins. (D) Transfected cells expressing mycRab24wt or mycRab24(D123I) were lysed in high-detergent buffer (see Methods) and the proteins recovered in the detergent-soluble and insoluble fractions were assayed by SDS-PAGE and immunoblot analysis, using the anti-myc monoclonal antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC130051&req=5

Figure 1: Subcellular distribution of wild-type and mutant Rab24 expressed in 293 cells. (A) Cells were transfected with vectors encoding mycRab24wt or mycRab24(D123I) and 24 h later the expressed proteins were localized by immunofluorescence microscopy, using primary antibodies against the myc epitope or the Rab24 C-terminal hypervariable domain, as indicated above each panel. At the exposure setting used for the photograph, the Rab24 antibody did not give a detectable signal in the non-transfected cells (indicated by asterisk). The bar equals10 microns. (B) Cells were transfected with a vector encoding Rab24(D123I) without the myc epitope, and the expressed protein was localized with the Rab24 antibody. (C) Transfected cells expressing mycRab24wt or mycRab24(D123I) were lysed in buffer without detergent and fractionated by centrifugation as described in the Methods. The cytosol and particulate fractions were subjected to SDS-PAGE and immunoblot analysis, using the anti-myc monoclonal antibody to detect the expressed proteins. (D) Transfected cells expressing mycRab24wt or mycRab24(D123I) were lysed in high-detergent buffer (see Methods) and the proteins recovered in the detergent-soluble and insoluble fractions were assayed by SDS-PAGE and immunoblot analysis, using the anti-myc monoclonal antibody.
Mentions: When mycRab24(wt) was transiently expressed in 293 cells, immunofluorescence microscopy revealed a diffuse reticular distribution throughout the cytoplasm, with a concentration in the perinuclear region (Fig. 1A). In contrast, when the same studies were performed with cells expressing mycRab24(D123I), we observed a striking accumulation of the mutant protein in punctate structures localized in both cytoplasmic and nuclear compartments. In most cells the inclusions appeared to be concentrated predominantly within the nucleus (Fig. 1A). Identical structures were seen when immunofluorescence microscopy was performed with antibodies against the N-terminal myc epitope or the C-terminal hypervariable domain of Rab24, indicating that the immunofluorescence pattern reflected the localization of full-length mycRab24(D123I). Finally, the localization pattern of Rab24(D123I) remained the same when the protein was expressed without the myc tag (Fig. 1B).

Bottom Line: Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form.If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope.However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, OH 43614, USA. wmaltese@mco.edu

ABSTRACT

Background: Members of the Rab GTPase family regulate intracellular protein trafficking, but the specific function of Rab24 remains unknown. Several attributes distinguish this protein from other members of the Rab family, including a low intrinsic GTPase activity.

Results: The functions of other Rab proteins have been defined through the use of dominant-negative mutants with amino acid substitutions in the conserved N(T)KxD nucleotide binding motif. Surprisingly, when such Rab24 constructs were expressed in cultured cells, they accumulated in nuclear inclusions which disrupted the integrity of the nuclear envelope. The inclusions reacted positively with antibodies against ubiquitin and Hsp70, similar to protein aggregates observed in polyglutamine disorders. They also appeared to sequester importin-beta and GFP-coupled glucocorticoid receptor. Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form. Studies with Rab24/Rab1B chimeras indicated that targeting of the mutant protein to inclusions requires the unique C-terminal domain of Rab24.

Conclusion: These studies demonstrate that mutations in Rab24 can trigger a cytopathic cellular response involving accumulation of nuclear inclusions. If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope. However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.

Show MeSH
Related in: MedlinePlus