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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 enhanced green fluorescent protein (EGFP) Rab24 fusion proteins. HEK293 cells were transfected with pCMV5 expression vectors encoding: (A) EGFP fused to full-length (fl) Rab24(D123I), (B) EGFP fused to the Rab24 C-terminal hypervariable domain (ct), or (C) EGFP alone. Cells were processed for immunofluorescence microscopy 24 h after transfection. The EGFP proteins were localized with a monoclonal antibody against green fluorescent protein (GFP) followed by FITC-conjugated GAM IgG. The Rab24 sequence was detected in the same cells with a rabbit polyclonal antibody against the C-terminal domain, followed by rhodamine-conjugated GAR IgG. The bar represents 10 microns.
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Figure 9: Localization of enhanced green fluorescent protein (EGFP) Rab24 fusion proteins. HEK293 cells were transfected with pCMV5 expression vectors encoding: (A) EGFP fused to full-length (fl) Rab24(D123I), (B) EGFP fused to the Rab24 C-terminal hypervariable domain (ct), or (C) EGFP alone. Cells were processed for immunofluorescence microscopy 24 h after transfection. The EGFP proteins were localized with a monoclonal antibody against green fluorescent protein (GFP) followed by FITC-conjugated GAM IgG. The Rab24 sequence was detected in the same cells with a rabbit polyclonal antibody against the C-terminal domain, followed by rhodamine-conjugated GAR IgG. The bar represents 10 microns.

Mentions: To determine whether the hypervariable domain of Rab24 was sufficient to promote the formation of inclusion bodies when fused to a heterologous protein, we prepared fusion constructs with enhanced green fluorescent protein (EGFP) containing either full-length Rab24(D123I) or just the Rab24 C-terminal domain. As shown in Fig. 9A, EGFP-Rab24(D123I) accumulated in inclusion bodies similar to those described earlier for mycRab24(D123I). In contrast, the EGFP fusion protein containing only the hypervariable domain of Rab24 exhibited a diffuse cytoplasmic and nuclear localization that was essentially identical to the pattern observed with EGFP alone (Fig. 9B &9C). Therefore, it appears that although the C-terminal domain of Rab24 is required to promote its incorporation into inclusion bodies, it does so only when expressed in the context of a Rab GTPase with a mutation in the N(T)KxD nucleotide binding motif.


Mutant Rab24 GTPase is targeted to nuclear inclusions.

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

Localization of enhanced green fluorescent protein (EGFP) Rab24 fusion proteins. HEK293 cells were transfected with pCMV5 expression vectors encoding: (A) EGFP fused to full-length (fl) Rab24(D123I), (B) EGFP fused to the Rab24 C-terminal hypervariable domain (ct), or (C) EGFP alone. Cells were processed for immunofluorescence microscopy 24 h after transfection. The EGFP proteins were localized with a monoclonal antibody against green fluorescent protein (GFP) followed by FITC-conjugated GAM IgG. The Rab24 sequence was detected in the same cells with a rabbit polyclonal antibody against the C-terminal domain, followed by rhodamine-conjugated GAR IgG. The bar represents 10 microns.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Localization of enhanced green fluorescent protein (EGFP) Rab24 fusion proteins. HEK293 cells were transfected with pCMV5 expression vectors encoding: (A) EGFP fused to full-length (fl) Rab24(D123I), (B) EGFP fused to the Rab24 C-terminal hypervariable domain (ct), or (C) EGFP alone. Cells were processed for immunofluorescence microscopy 24 h after transfection. The EGFP proteins were localized with a monoclonal antibody against green fluorescent protein (GFP) followed by FITC-conjugated GAM IgG. The Rab24 sequence was detected in the same cells with a rabbit polyclonal antibody against the C-terminal domain, followed by rhodamine-conjugated GAR IgG. The bar represents 10 microns.
Mentions: To determine whether the hypervariable domain of Rab24 was sufficient to promote the formation of inclusion bodies when fused to a heterologous protein, we prepared fusion constructs with enhanced green fluorescent protein (EGFP) containing either full-length Rab24(D123I) or just the Rab24 C-terminal domain. As shown in Fig. 9A, EGFP-Rab24(D123I) accumulated in inclusion bodies similar to those described earlier for mycRab24(D123I). In contrast, the EGFP fusion protein containing only the hypervariable domain of Rab24 exhibited a diffuse cytoplasmic and nuclear localization that was essentially identical to the pattern observed with EGFP alone (Fig. 9B &9C). Therefore, it appears that although the C-terminal domain of Rab24 is required to promote its incorporation into inclusion bodies, it does so only when expressed in the context of a Rab GTPase with a mutation in the N(T)KxD nucleotide binding motif.

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