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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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Rab24 is not conjugated to ubiquitin. HEK293 cells were transfected with vectors encoding His6-tagged Rab24wt or Rab24(D123). (A) The expressed proteins were extracted in high-detergent buffer, purified on Ni++ spin columns, and subjected to SDS-PAGE and immunoblot analysis using a monoclonal antibody against the His6 epitope. Cells were incubated with (+) or without (-) 20 μM lactacystin for 4 h prior to harvest. (B) Separate aliquots of the proteins collected from the Ni++ columns (cells treated with lactacystin in panel A) were immunoblotted with a monoclonal antibody against ubiquitin. To confirm the reactivity of the antibody, it was also applied to a blot containing an aliquot of total protein from untransfected cells treated with lactacystin for 4 h (cell lysate). The exposure time was the same for all blots.
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Figure 11: Rab24 is not conjugated to ubiquitin. HEK293 cells were transfected with vectors encoding His6-tagged Rab24wt or Rab24(D123). (A) The expressed proteins were extracted in high-detergent buffer, purified on Ni++ spin columns, and subjected to SDS-PAGE and immunoblot analysis using a monoclonal antibody against the His6 epitope. Cells were incubated with (+) or without (-) 20 μM lactacystin for 4 h prior to harvest. (B) Separate aliquots of the proteins collected from the Ni++ columns (cells treated with lactacystin in panel A) were immunoblotted with a monoclonal antibody against ubiquitin. To confirm the reactivity of the antibody, it was also applied to a blot containing an aliquot of total protein from untransfected cells treated with lactacystin for 4 h (cell lysate). The exposure time was the same for all blots.

Mentions: To determine whether ubiquitin was conjugated directly to Rab24(D123I), it was necessary to solubilize the protein aggregates in a high-detergent buffer that was incompatible with standard immunoprecipitation methods. Therefore, we generated a mammalian expression vector encoding a His6-tagged version of Rab24(D123I) and used Ni2+ affinity resin to isolate the expressed protein from detergent extracts. When expressed in 293 cells the His6Rab24(D123I) accumulated in inclusion bodies that were morphologically identical to those formed by the myc-tagged and untagged proteins (not shown). When the His6-tagged protein was extracted from the inclusions and subjected to SDS-PAGE, we saw no evidence of higher molecular mass forms that would correspond to poly-ubiquitinated species, using either anti-His6 (Fig. 11A) or anti-Rab24 (not shown) antibodies. Moreover, immunoblot analysis of the His6Rab24(D123I) isolated from cells treated with lactacystin (to block degradation of poly-ubiquitinated proteins by the 26S proteasome) showed no detectable ubiquitin associated with the protein, although the same antibody detected multiple ubiquitinated polypeptides in the whole-cell lysate (Fig. 11B). Again, these findings agree with recent studies of poly-Gln protein aggregates, which indicate that although ubiquitinated proteins are present in the inclusions, they are not conjugated directly to the initiating poly-Gln polypeptide [60].


Mutant Rab24 GTPase is targeted to nuclear inclusions.

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

Rab24 is not conjugated to ubiquitin. HEK293 cells were transfected with vectors encoding His6-tagged Rab24wt or Rab24(D123). (A) The expressed proteins were extracted in high-detergent buffer, purified on Ni++ spin columns, and subjected to SDS-PAGE and immunoblot analysis using a monoclonal antibody against the His6 epitope. Cells were incubated with (+) or without (-) 20 μM lactacystin for 4 h prior to harvest. (B) Separate aliquots of the proteins collected from the Ni++ columns (cells treated with lactacystin in panel A) were immunoblotted with a monoclonal antibody against ubiquitin. To confirm the reactivity of the antibody, it was also applied to a blot containing an aliquot of total protein from untransfected cells treated with lactacystin for 4 h (cell lysate). The exposure time was the same for all blots.
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Related In: Results  -  Collection

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

Figure 11: Rab24 is not conjugated to ubiquitin. HEK293 cells were transfected with vectors encoding His6-tagged Rab24wt or Rab24(D123). (A) The expressed proteins were extracted in high-detergent buffer, purified on Ni++ spin columns, and subjected to SDS-PAGE and immunoblot analysis using a monoclonal antibody against the His6 epitope. Cells were incubated with (+) or without (-) 20 μM lactacystin for 4 h prior to harvest. (B) Separate aliquots of the proteins collected from the Ni++ columns (cells treated with lactacystin in panel A) were immunoblotted with a monoclonal antibody against ubiquitin. To confirm the reactivity of the antibody, it was also applied to a blot containing an aliquot of total protein from untransfected cells treated with lactacystin for 4 h (cell lysate). The exposure time was the same for all blots.
Mentions: To determine whether ubiquitin was conjugated directly to Rab24(D123I), it was necessary to solubilize the protein aggregates in a high-detergent buffer that was incompatible with standard immunoprecipitation methods. Therefore, we generated a mammalian expression vector encoding a His6-tagged version of Rab24(D123I) and used Ni2+ affinity resin to isolate the expressed protein from detergent extracts. When expressed in 293 cells the His6Rab24(D123I) accumulated in inclusion bodies that were morphologically identical to those formed by the myc-tagged and untagged proteins (not shown). When the His6-tagged protein was extracted from the inclusions and subjected to SDS-PAGE, we saw no evidence of higher molecular mass forms that would correspond to poly-ubiquitinated species, using either anti-His6 (Fig. 11A) or anti-Rab24 (not shown) antibodies. Moreover, immunoblot analysis of the His6Rab24(D123I) isolated from cells treated with lactacystin (to block degradation of poly-ubiquitinated proteins by the 26S proteasome) showed no detectable ubiquitin associated with the protein, although the same antibody detected multiple ubiquitinated polypeptides in the whole-cell lysate (Fig. 11B). Again, these findings agree with recent studies of poly-Gln protein aggregates, which indicate that although ubiquitinated proteins are present in the inclusions, they are not conjugated directly to the initiating poly-Gln polypeptide [60].

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