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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Electron microscopy of inclusion bodies in 293 cells expressing mycRab24(D123I). Cells were harvested 24 h after transfection. Expressed protein was detected with the mouse monoclonal antibody against the myc epitope, followed by secondary antibody conjugated to 10 nm gold particles. Cells were processed for electron microscopy as described in the Methods. Panel A shows a cell where the majority of immunoreactive inclusions accumulated within nucleus (N). Panel B shows the intranuclear inclusions at higher magnification, revealing the individual gold particles concentrated in dense spherical particles of nearly uniform diameter. Panel C shows an example of a cell where disruption of the nuclear envelope was observed (between arrows) in the region near the accumulated intranuclear inclusions. Panel D shows a cell where inclusions were observed in the perinuclear cytoplasm. The dimension bars in each panel are as follows: A, 0.5 micron; B, 0.1 micron; C, 0.5 micron; D, 0.5 micron. The solid arrowheads in panels A, C and D indicate the nuclear membrane.
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Figure 3: Electron microscopy of inclusion bodies in 293 cells expressing mycRab24(D123I). Cells were harvested 24 h after transfection. Expressed protein was detected with the mouse monoclonal antibody against the myc epitope, followed by secondary antibody conjugated to 10 nm gold particles. Cells were processed for electron microscopy as described in the Methods. Panel A shows a cell where the majority of immunoreactive inclusions accumulated within nucleus (N). Panel B shows the intranuclear inclusions at higher magnification, revealing the individual gold particles concentrated in dense spherical particles of nearly uniform diameter. Panel C shows an example of a cell where disruption of the nuclear envelope was observed (between arrows) in the region near the accumulated intranuclear inclusions. Panel D shows a cell where inclusions were observed in the perinuclear cytoplasm. The dimension bars in each panel are as follows: A, 0.5 micron; B, 0.1 micron; C, 0.5 micron; D, 0.5 micron. The solid arrowheads in panels A, C and D indicate the nuclear membrane.

Mentions: Several follow-up studies supported the notion that the punctate structures containing the Rab24 mutants were inclusion bodies rather than membrane-bound organelles. For example, immunofluorescence microscopy revealed little or no overlap between these structures and markers for Golgi (Rab6, Lens culinaris lectin), endosome (EEA1) or lysosome (LAMP-1) membranes (data not shown). Although Rab24 has been proposed as a possible regulator of autophagic processes [24,43], the accumulation of punctate bodies in cells expressing Rab24(D123I) was not prevented by 3-methyadenine, a known inhibitor of autophagosome formation [44]. Most importantly, electron microscopy with immunogold labeling of mycRab24(D123I) in ultra-thin sections confirmed the initial impression that the inclusions are in fact localized within the nucleus. The expressed protein was concentrated in clusters of dense granules of fairly uniform diameter (80–150 nm) with no evidence of a surrounding membrane (Fig. 3A,3B). The cytoplasmic inclusions containing mycRab24(D123I) were similar to those observed inside the nucleus, except that they were larger, with a diameter 2–3 times that of the nuclear particles (Fig. 3D). Such structures were never observed in cells expressing mycRab24wt or any other over-expressed Rab GTPase.


Mutant Rab24 GTPase is targeted to nuclear inclusions.

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

Electron microscopy of inclusion bodies in 293 cells expressing mycRab24(D123I). Cells were harvested 24 h after transfection. Expressed protein was detected with the mouse monoclonal antibody against the myc epitope, followed by secondary antibody conjugated to 10 nm gold particles. Cells were processed for electron microscopy as described in the Methods. Panel A shows a cell where the majority of immunoreactive inclusions accumulated within nucleus (N). Panel B shows the intranuclear inclusions at higher magnification, revealing the individual gold particles concentrated in dense spherical particles of nearly uniform diameter. Panel C shows an example of a cell where disruption of the nuclear envelope was observed (between arrows) in the region near the accumulated intranuclear inclusions. Panel D shows a cell where inclusions were observed in the perinuclear cytoplasm. The dimension bars in each panel are as follows: A, 0.5 micron; B, 0.1 micron; C, 0.5 micron; D, 0.5 micron. The solid arrowheads in panels A, C and D indicate the nuclear membrane.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC130051&req=5

Figure 3: Electron microscopy of inclusion bodies in 293 cells expressing mycRab24(D123I). Cells were harvested 24 h after transfection. Expressed protein was detected with the mouse monoclonal antibody against the myc epitope, followed by secondary antibody conjugated to 10 nm gold particles. Cells were processed for electron microscopy as described in the Methods. Panel A shows a cell where the majority of immunoreactive inclusions accumulated within nucleus (N). Panel B shows the intranuclear inclusions at higher magnification, revealing the individual gold particles concentrated in dense spherical particles of nearly uniform diameter. Panel C shows an example of a cell where disruption of the nuclear envelope was observed (between arrows) in the region near the accumulated intranuclear inclusions. Panel D shows a cell where inclusions were observed in the perinuclear cytoplasm. The dimension bars in each panel are as follows: A, 0.5 micron; B, 0.1 micron; C, 0.5 micron; D, 0.5 micron. The solid arrowheads in panels A, C and D indicate the nuclear membrane.
Mentions: Several follow-up studies supported the notion that the punctate structures containing the Rab24 mutants were inclusion bodies rather than membrane-bound organelles. For example, immunofluorescence microscopy revealed little or no overlap between these structures and markers for Golgi (Rab6, Lens culinaris lectin), endosome (EEA1) or lysosome (LAMP-1) membranes (data not shown). Although Rab24 has been proposed as a possible regulator of autophagic processes [24,43], the accumulation of punctate bodies in cells expressing Rab24(D123I) was not prevented by 3-methyadenine, a known inhibitor of autophagosome formation [44]. Most importantly, electron microscopy with immunogold labeling of mycRab24(D123I) in ultra-thin sections confirmed the initial impression that the inclusions are in fact localized within the nucleus. The expressed protein was concentrated in clusters of dense granules of fairly uniform diameter (80–150 nm) with no evidence of a surrounding membrane (Fig. 3A,3B). The cytoplasmic inclusions containing mycRab24(D123I) were similar to those observed inside the nucleus, except that they were larger, with a diameter 2–3 times that of the nuclear particles (Fig. 3D). Such structures were never observed in cells expressing mycRab24wt or any other over-expressed Rab GTPase.

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