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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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RNAi indicates that RabX1 and RabX2 are non-essential for normal growth of T. brucei.Growth curves of BSF SMB and PCF PTT parasites after tetracycline-induced RNAi for RabX1 and RabX2. Top left panel, Growth curve for BSF SMB parasites transfected with p2T7TAblue-RabX1 RNAi construct. Bottom left panel, growth curve for BSF SMB parasites transfected with p2T7TAblue-RabX2. Top right panel, growth curve for PCF PTT parasites transfected with p2T7-177-RabX1. Bottom right panel, growth curve for PCF PTT parasites transfected with p2T7-177-RabX2. RNAi was induced in BSF SMB parasites by the addition of 1 µg/ml and in PCF PTT lines by the addition of 10 µg/ml of tetracycline. BSF or PCF cells were cultured in the absence (open symbol) or presence (closed symbol) of tetracycline. Insets in the respective growth curves show Western blots demonstrating the RNAi-mediated down regulation of RabX1 or RabX2 along with BiP, an ER marker, as a loading control. Western blot for RabX1 and RabX2 was performed with 1×107 cells after two days of incubation with 1 µg/ml tetracycline in BSF and four days of incubation with 10 µg/ml tetracycline in PCF. NI, non-induced; I, induced. The experiments have been repeated at least twice.
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pone-0007217-g002: RNAi indicates that RabX1 and RabX2 are non-essential for normal growth of T. brucei.Growth curves of BSF SMB and PCF PTT parasites after tetracycline-induced RNAi for RabX1 and RabX2. Top left panel, Growth curve for BSF SMB parasites transfected with p2T7TAblue-RabX1 RNAi construct. Bottom left panel, growth curve for BSF SMB parasites transfected with p2T7TAblue-RabX2. Top right panel, growth curve for PCF PTT parasites transfected with p2T7-177-RabX1. Bottom right panel, growth curve for PCF PTT parasites transfected with p2T7-177-RabX2. RNAi was induced in BSF SMB parasites by the addition of 1 µg/ml and in PCF PTT lines by the addition of 10 µg/ml of tetracycline. BSF or PCF cells were cultured in the absence (open symbol) or presence (closed symbol) of tetracycline. Insets in the respective growth curves show Western blots demonstrating the RNAi-mediated down regulation of RabX1 or RabX2 along with BiP, an ER marker, as a loading control. Western blot for RabX1 and RabX2 was performed with 1×107 cells after two days of incubation with 1 µg/ml tetracycline in BSF and four days of incubation with 10 µg/ml tetracycline in PCF. NI, non-induced; I, induced. The experiments have been repeated at least twice.

Mentions: To investigate if RabX1 and RabX2 are essential proteins, we generated RNAi cell lines in both BSF and PCF stages. In BSF, RabX1 and RabX2 RNAi lines were induced with tetracycline to express the respective double-stranded RNA and parasite replication monitored until eight days post-induction. There was no significant growth defect in either the RabX1 and RabX2 RNAi lines when compared to the non-induced parasites (Figure 2). The level of RabX1 protein has been previously reported to be down-regulated by >95% by RNAi-mediated suppression [23]. A similar down-regulation was observed for RabX2 protein after two days of RNAi-mediated suppression (Figure 2). Similarly, RNAi-mediated down-regulation of RabX1 and RabX2 in PCF parasites resulted in no significant growth defect over nine days post-induction. The levels of RabX1 and RabX2 proteins were down-regulated by >80% and ∼60% respectively after four days post-induction demonstrating efficient suppression (Figure 2). Overall these data suggest that neither RabX1 nor RabX2 gene products are required for normal proliferation in BSF and PCF parasites.


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)

RNAi indicates that RabX1 and RabX2 are non-essential for normal growth of T. brucei.Growth curves of BSF SMB and PCF PTT parasites after tetracycline-induced RNAi for RabX1 and RabX2. Top left panel, Growth curve for BSF SMB parasites transfected with p2T7TAblue-RabX1 RNAi construct. Bottom left panel, growth curve for BSF SMB parasites transfected with p2T7TAblue-RabX2. Top right panel, growth curve for PCF PTT parasites transfected with p2T7-177-RabX1. Bottom right panel, growth curve for PCF PTT parasites transfected with p2T7-177-RabX2. RNAi was induced in BSF SMB parasites by the addition of 1 µg/ml and in PCF PTT lines by the addition of 10 µg/ml of tetracycline. BSF or PCF cells were cultured in the absence (open symbol) or presence (closed symbol) of tetracycline. Insets in the respective growth curves show Western blots demonstrating the RNAi-mediated down regulation of RabX1 or RabX2 along with BiP, an ER marker, as a loading control. Western blot for RabX1 and RabX2 was performed with 1×107 cells after two days of incubation with 1 µg/ml tetracycline in BSF and four days of incubation with 10 µg/ml tetracycline in PCF. NI, non-induced; I, induced. The experiments have been repeated at least twice.
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pone-0007217-g002: RNAi indicates that RabX1 and RabX2 are non-essential for normal growth of T. brucei.Growth curves of BSF SMB and PCF PTT parasites after tetracycline-induced RNAi for RabX1 and RabX2. Top left panel, Growth curve for BSF SMB parasites transfected with p2T7TAblue-RabX1 RNAi construct. Bottom left panel, growth curve for BSF SMB parasites transfected with p2T7TAblue-RabX2. Top right panel, growth curve for PCF PTT parasites transfected with p2T7-177-RabX1. Bottom right panel, growth curve for PCF PTT parasites transfected with p2T7-177-RabX2. RNAi was induced in BSF SMB parasites by the addition of 1 µg/ml and in PCF PTT lines by the addition of 10 µg/ml of tetracycline. BSF or PCF cells were cultured in the absence (open symbol) or presence (closed symbol) of tetracycline. Insets in the respective growth curves show Western blots demonstrating the RNAi-mediated down regulation of RabX1 or RabX2 along with BiP, an ER marker, as a loading control. Western blot for RabX1 and RabX2 was performed with 1×107 cells after two days of incubation with 1 µg/ml tetracycline in BSF and four days of incubation with 10 µg/ml tetracycline in PCF. NI, non-induced; I, induced. The experiments have been repeated at least twice.
Mentions: To investigate if RabX1 and RabX2 are essential proteins, we generated RNAi cell lines in both BSF and PCF stages. In BSF, RabX1 and RabX2 RNAi lines were induced with tetracycline to express the respective double-stranded RNA and parasite replication monitored until eight days post-induction. There was no significant growth defect in either the RabX1 and RabX2 RNAi lines when compared to the non-induced parasites (Figure 2). The level of RabX1 protein has been previously reported to be down-regulated by >95% by RNAi-mediated suppression [23]. A similar down-regulation was observed for RabX2 protein after two days of RNAi-mediated suppression (Figure 2). Similarly, RNAi-mediated down-regulation of RabX1 and RabX2 in PCF parasites resulted in no significant growth defect over nine days post-induction. The levels of RabX1 and RabX2 proteins were down-regulated by >80% and ∼60% respectively after four days post-induction demonstrating efficient suppression (Figure 2). Overall these data suggest that neither RabX1 nor RabX2 gene products are required for normal proliferation in BSF and PCF parasites.

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