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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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Localization of Rab24 deletion mutants and Rab24/Rab1B chimeras. 293 cells were transfected with expression vectors encoding (A,B) Rab24wt or Rab24(D123I), with deletions in the loop-8 insert region, or (C-F) a series of chimeric proteins composed of combinations of the N-terminal and C-terminal domains of Rab24 and Rab1B, with or without the D123I substitution (see Methods for detailed descriptions). After 24 h, the transfected cells were processed for immunofluorescence microscopy using the anti-myc monoclonal antibody and FITC-conjugated GAM IgG to localize the expressed proteins. Panels A-F show representative cells from cultures expressing the indicated proteins. The bar represents 10 microns.
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Figure 8: Localization of Rab24 deletion mutants and Rab24/Rab1B chimeras. 293 cells were transfected with expression vectors encoding (A,B) Rab24wt or Rab24(D123I), with deletions in the loop-8 insert region, or (C-F) a series of chimeric proteins composed of combinations of the N-terminal and C-terminal domains of Rab24 and Rab1B, with or without the D123I substitution (see Methods for detailed descriptions). After 24 h, the transfected cells were processed for immunofluorescence microscopy using the anti-myc monoclonal antibody and FITC-conjugated GAM IgG to localize the expressed proteins. Panels A-F show representative cells from cultures expressing the indicated proteins. The bar represents 10 microns.

Mentions: To learn more about the structural features of Rab24(D123I) that are responsible for triggering the formation of nuclear inclusions, we focused on regions of the protein that distinguish Rab24 from other members of the Rab family. We began by deleting a portion of the loop-8 insert, described in the preceding section. Four amino acids (ΔEDRR) were removed from both Rab24(wt) and Rab24(D123I). Immunofluorescent localization studies showed that this modification had no effect on distribution of the wild-type protein in the cytoplasm (Fig. 8A) and, contrary to expectations, did not interfere with the accumulation of the D123I mutant in nuclear inclusions (Fig. 8B).


Mutant Rab24 GTPase is targeted to nuclear inclusions.

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

Localization of Rab24 deletion mutants and Rab24/Rab1B chimeras. 293 cells were transfected with expression vectors encoding (A,B) Rab24wt or Rab24(D123I), with deletions in the loop-8 insert region, or (C-F) a series of chimeric proteins composed of combinations of the N-terminal and C-terminal domains of Rab24 and Rab1B, with or without the D123I substitution (see Methods for detailed descriptions). After 24 h, the transfected cells were processed for immunofluorescence microscopy using the anti-myc monoclonal antibody and FITC-conjugated GAM IgG to localize the expressed proteins. Panels A-F show representative cells from cultures expressing the indicated proteins. The bar represents 10 microns.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC130051&req=5

Figure 8: Localization of Rab24 deletion mutants and Rab24/Rab1B chimeras. 293 cells were transfected with expression vectors encoding (A,B) Rab24wt or Rab24(D123I), with deletions in the loop-8 insert region, or (C-F) a series of chimeric proteins composed of combinations of the N-terminal and C-terminal domains of Rab24 and Rab1B, with or without the D123I substitution (see Methods for detailed descriptions). After 24 h, the transfected cells were processed for immunofluorescence microscopy using the anti-myc monoclonal antibody and FITC-conjugated GAM IgG to localize the expressed proteins. Panels A-F show representative cells from cultures expressing the indicated proteins. The bar represents 10 microns.
Mentions: To learn more about the structural features of Rab24(D123I) that are responsible for triggering the formation of nuclear inclusions, we focused on regions of the protein that distinguish Rab24 from other members of the Rab family. We began by deleting a portion of the loop-8 insert, described in the preceding section. Four amino acids (ΔEDRR) were removed from both Rab24(wt) and Rab24(D123I). Immunofluorescent localization studies showed that this modification had no effect on distribution of the wild-type protein in the cytoplasm (Fig. 8A) and, contrary to expectations, did not interfere with the accumulation of the D123I mutant in nuclear inclusions (Fig. 8B).

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