Limits...
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 different Rab GTPases with amino acid substitutions in the N(T)KxD nucleotide binding motif. (A & B) NIH 3T3 cells were transfected with pCMV5 vectors encoding different myc-tagged Rab proteins with the indicated amino acid substitutions. Twenty-four hours after transfection, cells were processed for immunofluorescence microscopy, using the anti-myc monoclonal antibody, followed by FITC-conjugated GAM IgG. Non-transfected cells cannot be seen at the exposure setting used for the photographs. The bar in the upper left panel is 10 microns. (C) Autoradiogram of a thin-layer plate showing 32P-labeled GDP and GTP eluted from the indicated immunoprecipitated proteins. The immunoblot in the lower panel compares the relative amount of mycRab24 collected in 1/10 of each immunoprecipitate.
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Figure 2: Localization of different Rab GTPases with amino acid substitutions in the N(T)KxD nucleotide binding motif. (A & B) NIH 3T3 cells were transfected with pCMV5 vectors encoding different myc-tagged Rab proteins with the indicated amino acid substitutions. Twenty-four hours after transfection, cells were processed for immunofluorescence microscopy, using the anti-myc monoclonal antibody, followed by FITC-conjugated GAM IgG. Non-transfected cells cannot be seen at the exposure setting used for the photographs. The bar in the upper left panel is 10 microns. (C) Autoradiogram of a thin-layer plate showing 32P-labeled GDP and GTP eluted from the indicated immunoprecipitated proteins. The immunoblot in the lower panel compares the relative amount of mycRab24 collected in 1/10 of each immunoprecipitate.

Mentions: The nuclear inclusions containing Rab24(D123I) were not cell-type specific. Fig. 2A shows that similar punctate structures were formed when the D123I mutant was expressed in NIH 3T3 fibroblasts. We also verified that the unique behavior of the Rab24 mutant was not related specifically to the substitution at the aspartate position in the N(T)KxD motif. Thus, similar inclusions were observed when Rab24 was expressed with substitutions at the N/T position; i.e., T120A (Fig. 2A) or T120I (not shown). To test the possibility that protein aggregates might be formed when any Rab GTPase is overexpressed with a mutation that impairs nucleotide binding, we examined the localization of four additional members of the Rab family containing amino acid substitutions in the N(T)KxD motif. In all cases, these proteins exhibited a general cytoplasmic localization with no evidence of nuclear inclusions (Fig. 2A).


Mutant Rab24 GTPase is targeted to nuclear inclusions.

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

Localization of different Rab GTPases with amino acid substitutions in the N(T)KxD nucleotide binding motif. (A & B) NIH 3T3 cells were transfected with pCMV5 vectors encoding different myc-tagged Rab proteins with the indicated amino acid substitutions. Twenty-four hours after transfection, cells were processed for immunofluorescence microscopy, using the anti-myc monoclonal antibody, followed by FITC-conjugated GAM IgG. Non-transfected cells cannot be seen at the exposure setting used for the photographs. The bar in the upper left panel is 10 microns. (C) Autoradiogram of a thin-layer plate showing 32P-labeled GDP and GTP eluted from the indicated immunoprecipitated proteins. The immunoblot in the lower panel compares the relative amount of mycRab24 collected in 1/10 of each immunoprecipitate.
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Related In: Results  -  Collection

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

Figure 2: Localization of different Rab GTPases with amino acid substitutions in the N(T)KxD nucleotide binding motif. (A & B) NIH 3T3 cells were transfected with pCMV5 vectors encoding different myc-tagged Rab proteins with the indicated amino acid substitutions. Twenty-four hours after transfection, cells were processed for immunofluorescence microscopy, using the anti-myc monoclonal antibody, followed by FITC-conjugated GAM IgG. Non-transfected cells cannot be seen at the exposure setting used for the photographs. The bar in the upper left panel is 10 microns. (C) Autoradiogram of a thin-layer plate showing 32P-labeled GDP and GTP eluted from the indicated immunoprecipitated proteins. The immunoblot in the lower panel compares the relative amount of mycRab24 collected in 1/10 of each immunoprecipitate.
Mentions: The nuclear inclusions containing Rab24(D123I) were not cell-type specific. Fig. 2A shows that similar punctate structures were formed when the D123I mutant was expressed in NIH 3T3 fibroblasts. We also verified that the unique behavior of the Rab24 mutant was not related specifically to the substitution at the aspartate position in the N(T)KxD motif. Thus, similar inclusions were observed when Rab24 was expressed with substitutions at the N/T position; i.e., T120A (Fig. 2A) or T120I (not shown). To test the possibility that protein aggregates might be formed when any Rab GTPase is overexpressed with a mutation that impairs nucleotide binding, we examined the localization of four additional members of the Rab family containing amino acid substitutions in the N(T)KxD motif. In all cases, these proteins exhibited a general cytoplasmic localization with no evidence of nuclear inclusions (Fig. 2A).

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