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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.

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Endocytosis and exocytosis is not significantly altered in RabX1- and RabX2-2KO mutants.(A) Uptake of Alexa 488-conjugated transferrin in wild type, RabX1- and Rab X2-2KO mutants. Parasites grown to log phase were washed and incubated with Alexa 488-conjugated transferrin in their growth medium. Aliquots were taken at 0, 5, 10 and 20 minutes of incubation, cells washed to remove unbound transferrin and levels of intracellular transferrin determined by FACS. Uptake of Alexa 488-conjugated transferrin reached a maximum by 10 minutes in wild type, RabX1-2KO and RabX2-2KO parasites. Further, there was no significant difference in uptake between the wild type and the knockout lines. (B) Export of newly synthesized VSG in wild type, RabX1- and RabX2-2KO mutants. Parasites grown to log phase were pulse labeled with 35S-methionine. Samples were taken at 0, 20, 40 and 60 minutes of incubation and soluble VSG was hydrolyzed by GPI-PLC after hypotonic lysis of the parasites. Soluble VSG was quantified by analyzing VSG intensity using NIH ImageJ. Results are shown as percentage of total recovered soluble VSG after background subtraction. Note that there is no significant difference in the export of VSG by RabX1- and RabX2-2KO mutants compared to wild type parasites. Each analysis was performed twice with highly similar results.
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pone-0007217-g007: Endocytosis and exocytosis is not significantly altered in RabX1- and RabX2-2KO mutants.(A) Uptake of Alexa 488-conjugated transferrin in wild type, RabX1- and Rab X2-2KO mutants. Parasites grown to log phase were washed and incubated with Alexa 488-conjugated transferrin in their growth medium. Aliquots were taken at 0, 5, 10 and 20 minutes of incubation, cells washed to remove unbound transferrin and levels of intracellular transferrin determined by FACS. Uptake of Alexa 488-conjugated transferrin reached a maximum by 10 minutes in wild type, RabX1-2KO and RabX2-2KO parasites. Further, there was no significant difference in uptake between the wild type and the knockout lines. (B) Export of newly synthesized VSG in wild type, RabX1- and RabX2-2KO mutants. Parasites grown to log phase were pulse labeled with 35S-methionine. Samples were taken at 0, 20, 40 and 60 minutes of incubation and soluble VSG was hydrolyzed by GPI-PLC after hypotonic lysis of the parasites. Soluble VSG was quantified by analyzing VSG intensity using NIH ImageJ. Results are shown as percentage of total recovered soluble VSG after background subtraction. Note that there is no significant difference in the export of VSG by RabX1- and RabX2-2KO mutants compared to wild type parasites. Each analysis was performed twice with highly similar results.

Mentions: To investigate the roles of RabX1 and RabX2 in endocytosis we analyzed the ability of the knockout cells to endocytose Alexa 488-conjugated transferrin, quantifying uptake by fluorescence-activated cell sorting (FACS). In wild type parasites transferrin accumulation reached a maximum by ten minutes (Figure 7A). Similarly RabX1-2KO cells and RabX2-2KO cells reached maximum uptake after ten minutes. There was also no major significant difference in the rate of transferrin uptake between the wild type and both of the knockout lines (Figure 7A). Both 2KO lines accumulated slightly less transferrin, and for the RabX2 knockout this was ∼20% less than wild type. However, the absence of any significant defect to uptake kinetics suggests that RabX1 and RabX2 do not play a major role in receptor-mediated endocytosis, and is consistent with the absence of any morphological effect on endocytic markers in the knockout cells and the absence of an enlarged flagellar pocket, both of which are associated with knockdown of early endocytic Rab proteins [42], [43].


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)

Endocytosis and exocytosis is not significantly altered in RabX1- and RabX2-2KO mutants.(A) Uptake of Alexa 488-conjugated transferrin in wild type, RabX1- and Rab X2-2KO mutants. Parasites grown to log phase were washed and incubated with Alexa 488-conjugated transferrin in their growth medium. Aliquots were taken at 0, 5, 10 and 20 minutes of incubation, cells washed to remove unbound transferrin and levels of intracellular transferrin determined by FACS. Uptake of Alexa 488-conjugated transferrin reached a maximum by 10 minutes in wild type, RabX1-2KO and RabX2-2KO parasites. Further, there was no significant difference in uptake between the wild type and the knockout lines. (B) Export of newly synthesized VSG in wild type, RabX1- and RabX2-2KO mutants. Parasites grown to log phase were pulse labeled with 35S-methionine. Samples were taken at 0, 20, 40 and 60 minutes of incubation and soluble VSG was hydrolyzed by GPI-PLC after hypotonic lysis of the parasites. Soluble VSG was quantified by analyzing VSG intensity using NIH ImageJ. Results are shown as percentage of total recovered soluble VSG after background subtraction. Note that there is no significant difference in the export of VSG by RabX1- and RabX2-2KO mutants compared to wild type parasites. Each analysis was performed twice with highly similar results.
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Related In: Results  -  Collection

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pone-0007217-g007: Endocytosis and exocytosis is not significantly altered in RabX1- and RabX2-2KO mutants.(A) Uptake of Alexa 488-conjugated transferrin in wild type, RabX1- and Rab X2-2KO mutants. Parasites grown to log phase were washed and incubated with Alexa 488-conjugated transferrin in their growth medium. Aliquots were taken at 0, 5, 10 and 20 minutes of incubation, cells washed to remove unbound transferrin and levels of intracellular transferrin determined by FACS. Uptake of Alexa 488-conjugated transferrin reached a maximum by 10 minutes in wild type, RabX1-2KO and RabX2-2KO parasites. Further, there was no significant difference in uptake between the wild type and the knockout lines. (B) Export of newly synthesized VSG in wild type, RabX1- and RabX2-2KO mutants. Parasites grown to log phase were pulse labeled with 35S-methionine. Samples were taken at 0, 20, 40 and 60 minutes of incubation and soluble VSG was hydrolyzed by GPI-PLC after hypotonic lysis of the parasites. Soluble VSG was quantified by analyzing VSG intensity using NIH ImageJ. Results are shown as percentage of total recovered soluble VSG after background subtraction. Note that there is no significant difference in the export of VSG by RabX1- and RabX2-2KO mutants compared to wild type parasites. Each analysis was performed twice with highly similar results.
Mentions: To investigate the roles of RabX1 and RabX2 in endocytosis we analyzed the ability of the knockout cells to endocytose Alexa 488-conjugated transferrin, quantifying uptake by fluorescence-activated cell sorting (FACS). In wild type parasites transferrin accumulation reached a maximum by ten minutes (Figure 7A). Similarly RabX1-2KO cells and RabX2-2KO cells reached maximum uptake after ten minutes. There was also no major significant difference in the rate of transferrin uptake between the wild type and both of the knockout lines (Figure 7A). Both 2KO lines accumulated slightly less transferrin, and for the RabX2 knockout this was ∼20% less than wild type. However, the absence of any significant defect to uptake kinetics suggests that RabX1 and RabX2 do not play a major role in receptor-mediated endocytosis, and is consistent with the absence of any morphological effect on endocytic markers in the knockout cells and the absence of an enlarged flagellar pocket, both of which are associated with knockdown of early endocytic Rab proteins [42], [43].

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