Limits...
The trypanosome Rab-related proteins RabX1 and RabX2 play no role in intracellular trafficking but may be involved in fly infectivity.

Natesan SK, Peacock L, Leung KF, Matthews KR, Gibson W, Field MC - PLoS ONE (2009)

Bottom Line: Analysis of the knockout cell lines unexpectedly failed to uncover a defect in exocytosis, endocytosis or in the morphology or location of multiple markers for the endomembrane system, suggesting that neither RabX1 nor RabX2 has a major role in intracellular transport.RabX1 and RabX2, two members of the trypanosome Rab subfamily, were shown to have no major detectable role in intracellular transport, despite the localization of each gene product to highly specific endomembrane compartments.These data extend the functional scope of Rab proteins in trypanosomes to include non-canonical roles in differentiation-associated processes in protozoa.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Background: Rab GTPases constitute the largest subgroup of the Ras superfamily and are primarily involved in vesicle targeting. The full extent of Rab family function is unexplored. Several divergent Rab-like proteins are known but few have been characterized. In Trypanosoma brucei there are sixteen Rab genes, but RabX1, RabX2 and RabX3 are divergent within canonical sequence regions. Where known, trypanosome Rab functions are broadly conserved when orthologous relationships may be robustly established, but specific functions for RabX1, X2 and X3 have yet to be determined. RabX1 and RabX2 originated via tandem duplication and subcellular localization places RabX1 at the endoplasmic reticulum, while RabX2 is at the Golgi complex, suggesting distinct functions. We wished to determine whether RabX1 and RabX2 are involved in vesicle transport or other cellular processes.

Methodology/principal findings: Using comparative genomics we find that RabX1 and RabX2 are restricted to trypanosomatids. Gene knockout indicates that RabX1 and RabX2 are non-essential. Simultaneous RNAi knockdown of both RabX1 and RabX2, while partial, was also non-lethal and may suggest non-redundant function, consistent with the distinct locations of the proteins. Analysis of the knockout cell lines unexpectedly failed to uncover a defect in exocytosis, endocytosis or in the morphology or location of multiple markers for the endomembrane system, suggesting that neither RabX1 nor RabX2 has a major role in intracellular transport. However, it was apparent that RabX1 and RabX2 knockout cells displayed somewhat enhanced survival within flies.

Conclusions/significance: RabX1 and RabX2, two members of the trypanosome Rab subfamily, were shown to have no major detectable role in intracellular transport, despite the localization of each gene product to highly specific endomembrane compartments. These data extend the functional scope of Rab proteins in trypanosomes to include non-canonical roles in differentiation-associated processes in protozoa.

Show MeSH

Related in: MedlinePlus

Knockout of RabX1 and RabX2 does not affect the location of endomembrane compartment markers.Immunofluorescence demonstrating the locations of BiP, clathrin, ISG65, p67, Rab1 and Rab2 (red) in both RabX1- and RabX2-2KO parasites. Parasites were counterstained with DAPI (blue) for DNA. Scale bar 2 µm. Note that the location of BiP and Rab2 with the ER, Rab1 with the Golgi complex, clathrin and ISG65 with endosomes and p67 with lysosomes is unchanged in both RabX1- and RabX2-2KO in comparison to wild type parasites. Multiple cells were analyzed and representative examples shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2748683&req=5

pone-0007217-g006: Knockout of RabX1 and RabX2 does not affect the location of endomembrane compartment markers.Immunofluorescence demonstrating the locations of BiP, clathrin, ISG65, p67, Rab1 and Rab2 (red) in both RabX1- and RabX2-2KO parasites. Parasites were counterstained with DAPI (blue) for DNA. Scale bar 2 µm. Note that the location of BiP and Rab2 with the ER, Rab1 with the Golgi complex, clathrin and ISG65 with endosomes and p67 with lysosomes is unchanged in both RabX1- and RabX2-2KO in comparison to wild type parasites. Multiple cells were analyzed and representative examples shown.

Mentions: To investigate the effects of RabX1 or RabX2 knockout on the trafficking machinery, we first performed immunofluorescence analysis with established markers for various membrane-bound compartments. Immunofluorescence staining of cells with BiP or Rab2, both ER markers, indicated no changes to their localization at the ER in both RabX1-2KO or RabX2-2KO cells (Figure 6). Rab1, a Golgi complex marker, was normally localized in both the knockout lines indicating no changes to the morphology of the Golgi apparatus (Figure 6). Similarly, there was no change in the location of clathrin and ISG65, both endocytic markers, or p67, a lysosomal marker, in RabX1-2KO or RabX2-2KO cells (Figure 6). Thus the results from various immunofluorescence analyses indicate that RabX1 and RabX2 do not contribute to the structural integrity of the major compartments involved in exocytosis or endocytosis.


The trypanosome Rab-related proteins RabX1 and RabX2 play no role in intracellular trafficking but may be involved in fly infectivity.

Natesan SK, Peacock L, Leung KF, Matthews KR, Gibson W, Field MC - PLoS ONE (2009)

Knockout of RabX1 and RabX2 does not affect the location of endomembrane compartment markers.Immunofluorescence demonstrating the locations of BiP, clathrin, ISG65, p67, Rab1 and Rab2 (red) in both RabX1- and RabX2-2KO parasites. Parasites were counterstained with DAPI (blue) for DNA. Scale bar 2 µm. Note that the location of BiP and Rab2 with the ER, Rab1 with the Golgi complex, clathrin and ISG65 with endosomes and p67 with lysosomes is unchanged in both RabX1- and RabX2-2KO in comparison to wild type parasites. Multiple cells were analyzed and representative examples shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2748683&req=5

pone-0007217-g006: Knockout of RabX1 and RabX2 does not affect the location of endomembrane compartment markers.Immunofluorescence demonstrating the locations of BiP, clathrin, ISG65, p67, Rab1 and Rab2 (red) in both RabX1- and RabX2-2KO parasites. Parasites were counterstained with DAPI (blue) for DNA. Scale bar 2 µm. Note that the location of BiP and Rab2 with the ER, Rab1 with the Golgi complex, clathrin and ISG65 with endosomes and p67 with lysosomes is unchanged in both RabX1- and RabX2-2KO in comparison to wild type parasites. Multiple cells were analyzed and representative examples shown.
Mentions: To investigate the effects of RabX1 or RabX2 knockout on the trafficking machinery, we first performed immunofluorescence analysis with established markers for various membrane-bound compartments. Immunofluorescence staining of cells with BiP or Rab2, both ER markers, indicated no changes to their localization at the ER in both RabX1-2KO or RabX2-2KO cells (Figure 6). Rab1, a Golgi complex marker, was normally localized in both the knockout lines indicating no changes to the morphology of the Golgi apparatus (Figure 6). Similarly, there was no change in the location of clathrin and ISG65, both endocytic markers, or p67, a lysosomal marker, in RabX1-2KO or RabX2-2KO cells (Figure 6). Thus the results from various immunofluorescence analyses indicate that RabX1 and RabX2 do not contribute to the structural integrity of the major compartments involved in exocytosis or endocytosis.

Bottom Line: Analysis of the knockout cell lines unexpectedly failed to uncover a defect in exocytosis, endocytosis or in the morphology or location of multiple markers for the endomembrane system, suggesting that neither RabX1 nor RabX2 has a major role in intracellular transport.RabX1 and RabX2, two members of the trypanosome Rab subfamily, were shown to have no major detectable role in intracellular transport, despite the localization of each gene product to highly specific endomembrane compartments.These data extend the functional scope of Rab proteins in trypanosomes to include non-canonical roles in differentiation-associated processes in protozoa.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Background: Rab GTPases constitute the largest subgroup of the Ras superfamily and are primarily involved in vesicle targeting. The full extent of Rab family function is unexplored. Several divergent Rab-like proteins are known but few have been characterized. In Trypanosoma brucei there are sixteen Rab genes, but RabX1, RabX2 and RabX3 are divergent within canonical sequence regions. Where known, trypanosome Rab functions are broadly conserved when orthologous relationships may be robustly established, but specific functions for RabX1, X2 and X3 have yet to be determined. RabX1 and RabX2 originated via tandem duplication and subcellular localization places RabX1 at the endoplasmic reticulum, while RabX2 is at the Golgi complex, suggesting distinct functions. We wished to determine whether RabX1 and RabX2 are involved in vesicle transport or other cellular processes.

Methodology/principal findings: Using comparative genomics we find that RabX1 and RabX2 are restricted to trypanosomatids. Gene knockout indicates that RabX1 and RabX2 are non-essential. Simultaneous RNAi knockdown of both RabX1 and RabX2, while partial, was also non-lethal and may suggest non-redundant function, consistent with the distinct locations of the proteins. Analysis of the knockout cell lines unexpectedly failed to uncover a defect in exocytosis, endocytosis or in the morphology or location of multiple markers for the endomembrane system, suggesting that neither RabX1 nor RabX2 has a major role in intracellular transport. However, it was apparent that RabX1 and RabX2 knockout cells displayed somewhat enhanced survival within flies.

Conclusions/significance: RabX1 and RabX2, two members of the trypanosome Rab subfamily, were shown to have no major detectable role in intracellular transport, despite the localization of each gene product to highly specific endomembrane compartments. These data extend the functional scope of Rab proteins in trypanosomes to include non-canonical roles in differentiation-associated processes in protozoa.

Show MeSH
Related in: MedlinePlus