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The small G protein Arl5 contributes to endosome-to-Golgi traffic by aiding the recruitment of the GARP complex to the Golgi.

Rosa-Ferreira C, Christis C, Torres IL, Munro S - Biol Open (2015)

Bottom Line: In addition, in HeLa cells GARP also becomes cytosolic upon depletion of Arl5b.These phenotypes are consistent with a role in endosome-to-Golgi traffic, but are less severe than loss of GARP itself.Thus it appears that Arl5 is one of the factors that directs the recruitment of the GARP complex to the trans-Golgi, and this function is conserved in both flies and humans.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

No MeSH data available.


Related in: MedlinePlus

Loss of Arl5 leads to the accumulation of swollen endosomal compartments.(A) Cross-section view of epithelial follicle cells of control (w1118) and Arl5KO1 mutant (Arl5KO1/Arl5KO1) shows that the loss of Arl5 results in the accumulation of enlarged late endosomal and lysosomal structures, marked by the exogenously expressed marker YFP-Rab7 (green). The trans-Golgi marker dGolgin-245 remains largely unaltered between mutant and control follicle cells. (B) Quantification of the extent of accumulation of YFP-Rab7-containing structures. A single image such as those in (A) was obtained from each of 13 flies for each stock, and a mean determined of average intensity of the cytoplasmic puncta in each image. Error bars show standard error of mean. The average fluorescence relative to that of cytoplasm being approximately 1.5× higher in the Arl5KO1 homozygous mutant. (C) Confocal micrographs of L3 larval salivary glands from control (w1118) and Arl5KO1 mutant (Arl5KO1/Arl5KO1), expressing a GFP-tagged form of the Drosophila receptor of precursor of lysosomal hydrolases (LERP), revealed that absence of Arl5 leads to the enlargement of structures positive for GFP-LERP (green), which only localized moderately with dGolgin-245 (red), both in control and in the Arl5KO1 mutant. (D) Quantification of the extent of accumulation of GFP-LERP- containing structures. A single image such as those in (C) was obtained from each of 9 flies for each stock, and a mean determined of the average intensity of the cytoplasmic puncta in each image. Error bars show standard error of mean. The average fluorescence of GFP-LERP positive structures versus that of the cytoplasm is 1.6× higher in the Arl5KO1 mutant than in control L3 salivary gland cells, suggesting its altered retrograde traffic from endosomes to the TGN. Scale bars are 10 µm. (E) Anti-GFP immunoblots of extracts from ovaries of flies that express YFP-Rab7 and either have, or lack, Arl5. (F) Anti-GFP immunoblots of extracts from salivary glands of flies that express GFP-LERP and either have, or lack, Arl5.
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f05: Loss of Arl5 leads to the accumulation of swollen endosomal compartments.(A) Cross-section view of epithelial follicle cells of control (w1118) and Arl5KO1 mutant (Arl5KO1/Arl5KO1) shows that the loss of Arl5 results in the accumulation of enlarged late endosomal and lysosomal structures, marked by the exogenously expressed marker YFP-Rab7 (green). The trans-Golgi marker dGolgin-245 remains largely unaltered between mutant and control follicle cells. (B) Quantification of the extent of accumulation of YFP-Rab7-containing structures. A single image such as those in (A) was obtained from each of 13 flies for each stock, and a mean determined of average intensity of the cytoplasmic puncta in each image. Error bars show standard error of mean. The average fluorescence relative to that of cytoplasm being approximately 1.5× higher in the Arl5KO1 homozygous mutant. (C) Confocal micrographs of L3 larval salivary glands from control (w1118) and Arl5KO1 mutant (Arl5KO1/Arl5KO1), expressing a GFP-tagged form of the Drosophila receptor of precursor of lysosomal hydrolases (LERP), revealed that absence of Arl5 leads to the enlargement of structures positive for GFP-LERP (green), which only localized moderately with dGolgin-245 (red), both in control and in the Arl5KO1 mutant. (D) Quantification of the extent of accumulation of GFP-LERP- containing structures. A single image such as those in (C) was obtained from each of 9 flies for each stock, and a mean determined of the average intensity of the cytoplasmic puncta in each image. Error bars show standard error of mean. The average fluorescence of GFP-LERP positive structures versus that of the cytoplasm is 1.6× higher in the Arl5KO1 mutant than in control L3 salivary gland cells, suggesting its altered retrograde traffic from endosomes to the TGN. Scale bars are 10 µm. (E) Anti-GFP immunoblots of extracts from ovaries of flies that express YFP-Rab7 and either have, or lack, Arl5. (F) Anti-GFP immunoblots of extracts from salivary glands of flies that express GFP-LERP and either have, or lack, Arl5.

Mentions: To further examine the consequences of the loss of Arl5 we analyzed the morphology of several compartments from the endocytic pathway by expressing in follicle cells the late endosomal marker Rab7 tagged with a fluorescent protein. We observed a clear enlargement of late endosomal and lysosomal structures in Arl5KO1 mutant follicle cells as marked by YFP-Rab7 (Fig. 5A). Indeed, the average fluorescence of structures containing YFP-Rab7 is approximately 1.5 fold higher in the Arl5KO1 mutant follicle cells (Fig. 5B). This phenotype was also observed in salivary glands after expression of YFP-Rab7 (supplementary material Fig. S2). To determine whether the enlargement of the YFP-Rab7 compartment was due to defects in retrograde transport from endosomes to the TGN we examined the distribution of GFP-LERP (lysosomal enzyme receptor protein), the Drosophila ortholog of the mammalian MPRs (Burgess et al., 2012; Hirst et al., 2009). In L3 larval salivary glands, structures positive for GFP-LERP were enlarged in the Arl5KO1 mutant compared to the control (Fig. 5C), with an average fluorescence 1.6 fold higher relative to wild-type (Fig. 5D). Immunoblotting indicated that the altered distribution of Rab7 and LERP proteins was not a consequence of increased levels as these appeared unaffected by loss of Arl5 (Fig. 5E,F). Taken together, these results suggest that Arl5 loss leads to altered retrograde transport from endosomes and an enlargement of endosomal structures.


The small G protein Arl5 contributes to endosome-to-Golgi traffic by aiding the recruitment of the GARP complex to the Golgi.

Rosa-Ferreira C, Christis C, Torres IL, Munro S - Biol Open (2015)

Loss of Arl5 leads to the accumulation of swollen endosomal compartments.(A) Cross-section view of epithelial follicle cells of control (w1118) and Arl5KO1 mutant (Arl5KO1/Arl5KO1) shows that the loss of Arl5 results in the accumulation of enlarged late endosomal and lysosomal structures, marked by the exogenously expressed marker YFP-Rab7 (green). The trans-Golgi marker dGolgin-245 remains largely unaltered between mutant and control follicle cells. (B) Quantification of the extent of accumulation of YFP-Rab7-containing structures. A single image such as those in (A) was obtained from each of 13 flies for each stock, and a mean determined of average intensity of the cytoplasmic puncta in each image. Error bars show standard error of mean. The average fluorescence relative to that of cytoplasm being approximately 1.5× higher in the Arl5KO1 homozygous mutant. (C) Confocal micrographs of L3 larval salivary glands from control (w1118) and Arl5KO1 mutant (Arl5KO1/Arl5KO1), expressing a GFP-tagged form of the Drosophila receptor of precursor of lysosomal hydrolases (LERP), revealed that absence of Arl5 leads to the enlargement of structures positive for GFP-LERP (green), which only localized moderately with dGolgin-245 (red), both in control and in the Arl5KO1 mutant. (D) Quantification of the extent of accumulation of GFP-LERP- containing structures. A single image such as those in (C) was obtained from each of 9 flies for each stock, and a mean determined of the average intensity of the cytoplasmic puncta in each image. Error bars show standard error of mean. The average fluorescence of GFP-LERP positive structures versus that of the cytoplasm is 1.6× higher in the Arl5KO1 mutant than in control L3 salivary gland cells, suggesting its altered retrograde traffic from endosomes to the TGN. Scale bars are 10 µm. (E) Anti-GFP immunoblots of extracts from ovaries of flies that express YFP-Rab7 and either have, or lack, Arl5. (F) Anti-GFP immunoblots of extracts from salivary glands of flies that express GFP-LERP and either have, or lack, Arl5.
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Related In: Results  -  Collection

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f05: Loss of Arl5 leads to the accumulation of swollen endosomal compartments.(A) Cross-section view of epithelial follicle cells of control (w1118) and Arl5KO1 mutant (Arl5KO1/Arl5KO1) shows that the loss of Arl5 results in the accumulation of enlarged late endosomal and lysosomal structures, marked by the exogenously expressed marker YFP-Rab7 (green). The trans-Golgi marker dGolgin-245 remains largely unaltered between mutant and control follicle cells. (B) Quantification of the extent of accumulation of YFP-Rab7-containing structures. A single image such as those in (A) was obtained from each of 13 flies for each stock, and a mean determined of average intensity of the cytoplasmic puncta in each image. Error bars show standard error of mean. The average fluorescence relative to that of cytoplasm being approximately 1.5× higher in the Arl5KO1 homozygous mutant. (C) Confocal micrographs of L3 larval salivary glands from control (w1118) and Arl5KO1 mutant (Arl5KO1/Arl5KO1), expressing a GFP-tagged form of the Drosophila receptor of precursor of lysosomal hydrolases (LERP), revealed that absence of Arl5 leads to the enlargement of structures positive for GFP-LERP (green), which only localized moderately with dGolgin-245 (red), both in control and in the Arl5KO1 mutant. (D) Quantification of the extent of accumulation of GFP-LERP- containing structures. A single image such as those in (C) was obtained from each of 9 flies for each stock, and a mean determined of the average intensity of the cytoplasmic puncta in each image. Error bars show standard error of mean. The average fluorescence of GFP-LERP positive structures versus that of the cytoplasm is 1.6× higher in the Arl5KO1 mutant than in control L3 salivary gland cells, suggesting its altered retrograde traffic from endosomes to the TGN. Scale bars are 10 µm. (E) Anti-GFP immunoblots of extracts from ovaries of flies that express YFP-Rab7 and either have, or lack, Arl5. (F) Anti-GFP immunoblots of extracts from salivary glands of flies that express GFP-LERP and either have, or lack, Arl5.
Mentions: To further examine the consequences of the loss of Arl5 we analyzed the morphology of several compartments from the endocytic pathway by expressing in follicle cells the late endosomal marker Rab7 tagged with a fluorescent protein. We observed a clear enlargement of late endosomal and lysosomal structures in Arl5KO1 mutant follicle cells as marked by YFP-Rab7 (Fig. 5A). Indeed, the average fluorescence of structures containing YFP-Rab7 is approximately 1.5 fold higher in the Arl5KO1 mutant follicle cells (Fig. 5B). This phenotype was also observed in salivary glands after expression of YFP-Rab7 (supplementary material Fig. S2). To determine whether the enlargement of the YFP-Rab7 compartment was due to defects in retrograde transport from endosomes to the TGN we examined the distribution of GFP-LERP (lysosomal enzyme receptor protein), the Drosophila ortholog of the mammalian MPRs (Burgess et al., 2012; Hirst et al., 2009). In L3 larval salivary glands, structures positive for GFP-LERP were enlarged in the Arl5KO1 mutant compared to the control (Fig. 5C), with an average fluorescence 1.6 fold higher relative to wild-type (Fig. 5D). Immunoblotting indicated that the altered distribution of Rab7 and LERP proteins was not a consequence of increased levels as these appeared unaffected by loss of Arl5 (Fig. 5E,F). Taken together, these results suggest that Arl5 loss leads to altered retrograde transport from endosomes and an enlargement of endosomal structures.

Bottom Line: In addition, in HeLa cells GARP also becomes cytosolic upon depletion of Arl5b.These phenotypes are consistent with a role in endosome-to-Golgi traffic, but are less severe than loss of GARP itself.Thus it appears that Arl5 is one of the factors that directs the recruitment of the GARP complex to the trans-Golgi, and this function is conserved in both flies and humans.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

No MeSH data available.


Related in: MedlinePlus