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

Stumpy and procyclic markers are not expressed in RabX1 or RabX2 gene knockouts.(A) Cell morphology comparison of knockout lines against wild type background cells. Cells were stained with DAPI and morphology was observed by phase contrast. Scale bar 2 µm. (B) Lysates from 427 PCFs, 427 BSFs, RabX1-2KO BSFs, RabX2-2KO BSFs and stumpy cells were probed with either anti-PAD1 or anti-procyclin antibodies. Molecular weight marker is shown on the left. BiP was used as loading control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2748683&req=5

pone-0007217-g010: Stumpy and procyclic markers are not expressed in RabX1 or RabX2 gene knockouts.(A) Cell morphology comparison of knockout lines against wild type background cells. Cells were stained with DAPI and morphology was observed by phase contrast. Scale bar 2 µm. (B) Lysates from 427 PCFs, 427 BSFs, RabX1-2KO BSFs, RabX2-2KO BSFs and stumpy cells were probed with either anti-PAD1 or anti-procyclin antibodies. Molecular weight marker is shown on the left. BiP was used as loading control.

Mentions: To determine whether RabX1-2KO and RabX2-2KO knockout lines are involved in established pathways for differentiation through the cell cycle, we checked for any morphological changes of the knockout lines versus the wild type 427 BSF. If a block to cell cycle progression was removed, we should witness the appearance of stumpy form cells. However, no observable change in cellular morphology was noted between wild type and knockout lines (Figure 10A), and is also consistent with the absence of obvious growth defects in the knockout cultures. This was confirmed by probing lysates from wild type 427 BSF, RabX1-2KO, RabX2-2KO, 427 PCFs and short stumpy cells with either antibodies against procyclin (PCF marker) or PAD1 (a stumpy-specific marker) [44]. We only detected procyclin in PCFs and PAD1 was exclusively detected in stumpy cells, suggesting that knockout of RabX1 or RabX2 did not lead to expression of either of these differentiation markers (Figure 10B).


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)

Stumpy and procyclic markers are not expressed in RabX1 or RabX2 gene knockouts.(A) Cell morphology comparison of knockout lines against wild type background cells. Cells were stained with DAPI and morphology was observed by phase contrast. Scale bar 2 µm. (B) Lysates from 427 PCFs, 427 BSFs, RabX1-2KO BSFs, RabX2-2KO BSFs and stumpy cells were probed with either anti-PAD1 or anti-procyclin antibodies. Molecular weight marker is shown on the left. BiP was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007217-g010: Stumpy and procyclic markers are not expressed in RabX1 or RabX2 gene knockouts.(A) Cell morphology comparison of knockout lines against wild type background cells. Cells were stained with DAPI and morphology was observed by phase contrast. Scale bar 2 µm. (B) Lysates from 427 PCFs, 427 BSFs, RabX1-2KO BSFs, RabX2-2KO BSFs and stumpy cells were probed with either anti-PAD1 or anti-procyclin antibodies. Molecular weight marker is shown on the left. BiP was used as loading control.
Mentions: To determine whether RabX1-2KO and RabX2-2KO knockout lines are involved in established pathways for differentiation through the cell cycle, we checked for any morphological changes of the knockout lines versus the wild type 427 BSF. If a block to cell cycle progression was removed, we should witness the appearance of stumpy form cells. However, no observable change in cellular morphology was noted between wild type and knockout lines (Figure 10A), and is also consistent with the absence of obvious growth defects in the knockout cultures. This was confirmed by probing lysates from wild type 427 BSF, RabX1-2KO, RabX2-2KO, 427 PCFs and short stumpy cells with either antibodies against procyclin (PCF marker) or PAD1 (a stumpy-specific marker) [44]. We only detected procyclin in PCFs and PAD1 was exclusively detected in stumpy cells, suggesting that knockout of RabX1 or RabX2 did not lead to expression of either of these differentiation markers (Figure 10B).

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