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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Effects of Rab24 wt and Rab24(D123I) on nuclear translocation of glucocorticoid receptor. (A) 3T3 cells expressing GR-GFP with mycRab24wt show predominant cytoplasmic localization of both proteins in the absence of Dex (left panels). After 10 min incubation with 1 μM Dex, most of the GC-GFP is within the nucleus in cells expressing Rab24wt (center panels). When GR-GFP was co-expressed with mycRab24(D123I) in the absence of steroid, much of the GR-GFP was localized to inclusion bodies containing the Rab24 mutant (right panel). Identical results were observed when 1 μM Dex was added (not shown). (B). Higher magnification shows extensive co-localization of myc-Rab24(D123I) (red) with GR-GFP (green) in nuclear inclusion bodies. The bar represents 10 microns.
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Figure 6: Effects of Rab24 wt and Rab24(D123I) on nuclear translocation of glucocorticoid receptor. (A) 3T3 cells expressing GR-GFP with mycRab24wt show predominant cytoplasmic localization of both proteins in the absence of Dex (left panels). After 10 min incubation with 1 μM Dex, most of the GC-GFP is within the nucleus in cells expressing Rab24wt (center panels). When GR-GFP was co-expressed with mycRab24(D123I) in the absence of steroid, much of the GR-GFP was localized to inclusion bodies containing the Rab24 mutant (right panel). Identical results were observed when 1 μM Dex was added (not shown). (B). Higher magnification shows extensive co-localization of myc-Rab24(D123I) (red) with GR-GFP (green) in nuclear inclusion bodies. The bar represents 10 microns.

Mentions: Rab24 contains a unique arginine-rich insert, 126EEDRRRRR133[25] in the domain cognate to loop-8 in H-Ras [34]. The latter sequence is reminiscent of unusual arginine-rich nuclear localization signals that allow proteins like HIV tet and rev to undergo nuclear translocation by association with importin-β, in the absence of importin-α [45,46]. Since the loop-8 insert in Rab24 lies adjacent to the conserved N(T)KxD nucleotide binding cassette, we hypothesized that conformational changes caused by the D123I substitution might render the Rab24 mutant capable of disrupting the nuclear import machinery [47]. To test this possibility, we co-expressed mycRab24wt or mycRab24(D123I) with GR-GFP, a fusion between the glucocorticoid receptor and green fluorescent protein. This protein contains a prototypical nuclear localization signal but remains in the cytoplasm when expressed in cultured cells in the absence of steroids. Upon addition of 1 μM dexamethasone (Dex) the receptor is translocated into the nucleus within 10 min [48]. When GR-GFP was co-expressed with the wild-type Rab24 in NIH3T3 cells, we observed normal ligand-dependent translocation of GR-GFP into the nucleus when Dex was applied (Fig. 6A). However, when GR-GFP was coexpressed with mycRab24(D123I) there was a striking perturbation of receptor localization. Instead of remaining in the cytoplasm in the absence of steroid, nearly all of the GR-GFP was found in the cytoplasmic and nuclear inclusion bodies containing the Rab24 mutant (Fig. 6A). The latter point is clearly illustrated by the overlap of GR-GFP (green) fluorescence with rhodamine-IgG staining for mycRab24(D123I) (red) in Fig. 6B. The possibility that the inclusions induced by mycRab24(D123I) contain aggregated nuclear import complexes was further supported by immuno-staining with an antibody against importin-β (karyopherin-β), which revealed focal concentrations of importin-β in these structures (Fig. 7A). However, other proteins such as Ran binding protein (Fig. 7B) and Ran GAP (not shown), which are recruited to the nuclear import complex through interactions with the Ran GTPase, did not appear to be present in the inclusion bodies.


Mutant Rab24 GTPase is targeted to nuclear inclusions.

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

Effects of Rab24 wt and Rab24(D123I) on nuclear translocation of glucocorticoid receptor. (A) 3T3 cells expressing GR-GFP with mycRab24wt show predominant cytoplasmic localization of both proteins in the absence of Dex (left panels). After 10 min incubation with 1 μM Dex, most of the GC-GFP is within the nucleus in cells expressing Rab24wt (center panels). When GR-GFP was co-expressed with mycRab24(D123I) in the absence of steroid, much of the GR-GFP was localized to inclusion bodies containing the Rab24 mutant (right panel). Identical results were observed when 1 μM Dex was added (not shown). (B). Higher magnification shows extensive co-localization of myc-Rab24(D123I) (red) with GR-GFP (green) in nuclear inclusion bodies. The bar represents 10 microns.
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Figure 6: Effects of Rab24 wt and Rab24(D123I) on nuclear translocation of glucocorticoid receptor. (A) 3T3 cells expressing GR-GFP with mycRab24wt show predominant cytoplasmic localization of both proteins in the absence of Dex (left panels). After 10 min incubation with 1 μM Dex, most of the GC-GFP is within the nucleus in cells expressing Rab24wt (center panels). When GR-GFP was co-expressed with mycRab24(D123I) in the absence of steroid, much of the GR-GFP was localized to inclusion bodies containing the Rab24 mutant (right panel). Identical results were observed when 1 μM Dex was added (not shown). (B). Higher magnification shows extensive co-localization of myc-Rab24(D123I) (red) with GR-GFP (green) in nuclear inclusion bodies. The bar represents 10 microns.
Mentions: Rab24 contains a unique arginine-rich insert, 126EEDRRRRR133[25] in the domain cognate to loop-8 in H-Ras [34]. The latter sequence is reminiscent of unusual arginine-rich nuclear localization signals that allow proteins like HIV tet and rev to undergo nuclear translocation by association with importin-β, in the absence of importin-α [45,46]. Since the loop-8 insert in Rab24 lies adjacent to the conserved N(T)KxD nucleotide binding cassette, we hypothesized that conformational changes caused by the D123I substitution might render the Rab24 mutant capable of disrupting the nuclear import machinery [47]. To test this possibility, we co-expressed mycRab24wt or mycRab24(D123I) with GR-GFP, a fusion between the glucocorticoid receptor and green fluorescent protein. This protein contains a prototypical nuclear localization signal but remains in the cytoplasm when expressed in cultured cells in the absence of steroids. Upon addition of 1 μM dexamethasone (Dex) the receptor is translocated into the nucleus within 10 min [48]. When GR-GFP was co-expressed with the wild-type Rab24 in NIH3T3 cells, we observed normal ligand-dependent translocation of GR-GFP into the nucleus when Dex was applied (Fig. 6A). However, when GR-GFP was coexpressed with mycRab24(D123I) there was a striking perturbation of receptor localization. Instead of remaining in the cytoplasm in the absence of steroid, nearly all of the GR-GFP was found in the cytoplasmic and nuclear inclusion bodies containing the Rab24 mutant (Fig. 6A). The latter point is clearly illustrated by the overlap of GR-GFP (green) fluorescence with rhodamine-IgG staining for mycRab24(D123I) (red) in Fig. 6B. The possibility that the inclusions induced by mycRab24(D123I) contain aggregated nuclear import complexes was further supported by immuno-staining with an antibody against importin-β (karyopherin-β), which revealed focal concentrations of importin-β in these structures (Fig. 7A). However, other proteins such as Ran binding protein (Fig. 7B) and Ran GAP (not shown), which are recruited to the nuclear import complex through interactions with the Ran GTPase, did not appear to be present in the inclusion bodies.

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