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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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Accumulation of Rab24(D123I)-positive intranuclear inclusions is not accompanied by chromosomal DNA fragmentation. 293 cells expressing mycRab24(D123I) were fixed 24 h after transfection and subjected to the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling reaction (TUNEL). The transfected cells were identified by co-staining with anti-myc monoclonal antibody followed by rhodamine-conjugated GAM IgG. (A) Representative cells with mycRab24(D123I)-positive nuclear inclusions exhibit varying degrees of nuclear disruption but are uniformly negative when subjected to the TUNEL reaction. (B) Parallel cultures of non-transfected 293 cells were treated with 1 μM staurosporine for 24 h to induce apoptosis. Examples of TUNEL-positive cells are shown at the same exposure setting used to photograph the TUNEL-negative cells in panel A. Nuclei were visualized by staining with propidium iodide (PI).
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Figure 5: Accumulation of Rab24(D123I)-positive intranuclear inclusions is not accompanied by chromosomal DNA fragmentation. 293 cells expressing mycRab24(D123I) were fixed 24 h after transfection and subjected to the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling reaction (TUNEL). The transfected cells were identified by co-staining with anti-myc monoclonal antibody followed by rhodamine-conjugated GAM IgG. (A) Representative cells with mycRab24(D123I)-positive nuclear inclusions exhibit varying degrees of nuclear disruption but are uniformly negative when subjected to the TUNEL reaction. (B) Parallel cultures of non-transfected 293 cells were treated with 1 μM staurosporine for 24 h to induce apoptosis. Examples of TUNEL-positive cells are shown at the same exposure setting used to photograph the TUNEL-negative cells in panel A. Nuclei were visualized by staining with propidium iodide (PI).

Mentions: The morphological changes occurring in cells with accumulated Rab24(D123I) inclusions did not match the hallmark features of apoptosis (e.g., membrane blebbing, condensation of chromatin at the nuclear periphery). This was confirmed by TUNEL assays where cells at various stages of nuclear inclusion body accumulation showed no evidence of DNA fragmentation, either before or after disruption of the nuclear envelope (Fig. 5A). In contrast, TUNEL-positive cells were readily detected in parallel cultures where apoptosis was induced by treatment with staurosporine (Fig. 5B).


Mutant Rab24 GTPase is targeted to nuclear inclusions.

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

Accumulation of Rab24(D123I)-positive intranuclear inclusions is not accompanied by chromosomal DNA fragmentation. 293 cells expressing mycRab24(D123I) were fixed 24 h after transfection and subjected to the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling reaction (TUNEL). The transfected cells were identified by co-staining with anti-myc monoclonal antibody followed by rhodamine-conjugated GAM IgG. (A) Representative cells with mycRab24(D123I)-positive nuclear inclusions exhibit varying degrees of nuclear disruption but are uniformly negative when subjected to the TUNEL reaction. (B) Parallel cultures of non-transfected 293 cells were treated with 1 μM staurosporine for 24 h to induce apoptosis. Examples of TUNEL-positive cells are shown at the same exposure setting used to photograph the TUNEL-negative cells in panel A. Nuclei were visualized by staining with propidium iodide (PI).
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Related In: Results  -  Collection

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Figure 5: Accumulation of Rab24(D123I)-positive intranuclear inclusions is not accompanied by chromosomal DNA fragmentation. 293 cells expressing mycRab24(D123I) were fixed 24 h after transfection and subjected to the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling reaction (TUNEL). The transfected cells were identified by co-staining with anti-myc monoclonal antibody followed by rhodamine-conjugated GAM IgG. (A) Representative cells with mycRab24(D123I)-positive nuclear inclusions exhibit varying degrees of nuclear disruption but are uniformly negative when subjected to the TUNEL reaction. (B) Parallel cultures of non-transfected 293 cells were treated with 1 μM staurosporine for 24 h to induce apoptosis. Examples of TUNEL-positive cells are shown at the same exposure setting used to photograph the TUNEL-negative cells in panel A. Nuclei were visualized by staining with propidium iodide (PI).
Mentions: The morphological changes occurring in cells with accumulated Rab24(D123I) inclusions did not match the hallmark features of apoptosis (e.g., membrane blebbing, condensation of chromatin at the nuclear periphery). This was confirmed by TUNEL assays where cells at various stages of nuclear inclusion body accumulation showed no evidence of DNA fragmentation, either before or after disruption of the nuclear envelope (Fig. 5A). In contrast, TUNEL-positive cells were readily detected in parallel cultures where apoptosis was induced by treatment with staurosporine (Fig. 5B).

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