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 ubiquitin and Hsp70 in Rab24(D123I)-induced inclusions. HEK293 cells expressing mycRab24(D123I) were processed for immunofluorescence microscopy 24 h after transfection. In all panels, the myc-tagged Rab protein was detected with polyclonal antibody against the myc epitope followed by FITC-conjugated GAR IgG. Cells were co-stained with mouse monoclonal antibodies against (A) ubiquitin, (B) Hsp70/Hsc70 or (C) Hsp90, followed by rhodamine-conjugated GAM IgG. Non-transfected cells (no myc staining above background) are marked with asterisks in each panel. The bar represents 10 microns.
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Figure 10: Localization of ubiquitin and Hsp70 in Rab24(D123I)-induced inclusions. HEK293 cells expressing mycRab24(D123I) were processed for immunofluorescence microscopy 24 h after transfection. In all panels, the myc-tagged Rab protein was detected with polyclonal antibody against the myc epitope followed by FITC-conjugated GAR IgG. Cells were co-stained with mouse monoclonal antibodies against (A) ubiquitin, (B) Hsp70/Hsc70 or (C) Hsp90, followed by rhodamine-conjugated GAM IgG. Non-transfected cells (no myc staining above background) are marked with asterisks in each panel. The bar represents 10 microns.

Mentions: Although the formation of intranuclear inclusions has not been observed in previous studies of Ras-related GTPases, it is a common feature of several neurodegenerative disorders where abnormal proteins containing poly-Gln tracts are synthesized as a result of CAG codon expansions [52]. Examples include Huntington's disease (huntingtin) [53,54], spinocerebellar ataxia (SCA) types 1 and 3 (ataxin-1 and ataxin-3) [55,56] and dentatorubral-pallidoluysian atrophy (atrophin-1) [57]. These poly-Gln protein aggregates typically contain ubiquitin [58] and members of the Hsp70 chaperone family [59], consistent with a proposed pathway for cellular clearance of toxic misfolded proteins. We noted that the inclusions caused by expression of Rab24(D123I) were similar morphologically to those described in some of the poly-Gln disorders and hypothesized that they might arise via a similar pathway. An immunofluorescence study was performed to determine whether or not the intranuclear Rab24(D123I) aggregates might sequester ubiquitin and Hsp70. Both ubiquitin (Fig. 10A) and Hsp70 (Fig. 10B) showed substantial overlap with nuclear and cytoplasmic aggregates containing mycRab24(D123I). In contrast, the aggregates did not contain Hsp90 (Fig. 10C), suggesting that the sequestration of Hsp70 was specific.


Mutant Rab24 GTPase is targeted to nuclear inclusions.

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

Localization of ubiquitin and Hsp70 in Rab24(D123I)-induced inclusions. HEK293 cells expressing mycRab24(D123I) were processed for immunofluorescence microscopy 24 h after transfection. In all panels, the myc-tagged Rab protein was detected with polyclonal antibody against the myc epitope followed by FITC-conjugated GAR IgG. Cells were co-stained with mouse monoclonal antibodies against (A) ubiquitin, (B) Hsp70/Hsc70 or (C) Hsp90, followed by rhodamine-conjugated GAM IgG. Non-transfected cells (no myc staining above background) are marked with asterisks in each panel. The bar represents 10 microns.
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Related In: Results  -  Collection

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Figure 10: Localization of ubiquitin and Hsp70 in Rab24(D123I)-induced inclusions. HEK293 cells expressing mycRab24(D123I) were processed for immunofluorescence microscopy 24 h after transfection. In all panels, the myc-tagged Rab protein was detected with polyclonal antibody against the myc epitope followed by FITC-conjugated GAR IgG. Cells were co-stained with mouse monoclonal antibodies against (A) ubiquitin, (B) Hsp70/Hsc70 or (C) Hsp90, followed by rhodamine-conjugated GAM IgG. Non-transfected cells (no myc staining above background) are marked with asterisks in each panel. The bar represents 10 microns.
Mentions: Although the formation of intranuclear inclusions has not been observed in previous studies of Ras-related GTPases, it is a common feature of several neurodegenerative disorders where abnormal proteins containing poly-Gln tracts are synthesized as a result of CAG codon expansions [52]. Examples include Huntington's disease (huntingtin) [53,54], spinocerebellar ataxia (SCA) types 1 and 3 (ataxin-1 and ataxin-3) [55,56] and dentatorubral-pallidoluysian atrophy (atrophin-1) [57]. These poly-Gln protein aggregates typically contain ubiquitin [58] and members of the Hsp70 chaperone family [59], consistent with a proposed pathway for cellular clearance of toxic misfolded proteins. We noted that the inclusions caused by expression of Rab24(D123I) were similar morphologically to those described in some of the poly-Gln disorders and hypothesized that they might arise via a similar pathway. An immunofluorescence study was performed to determine whether or not the intranuclear Rab24(D123I) aggregates might sequester ubiquitin and Hsp70. Both ubiquitin (Fig. 10A) and Hsp70 (Fig. 10B) showed substantial overlap with nuclear and cytoplasmic aggregates containing mycRab24(D123I). In contrast, the aggregates did not contain Hsp90 (Fig. 10C), suggesting that the sequestration of Hsp70 was specific.

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