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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

Arl5 is required for the normal localization of the GARP complex subunit Vps52 to the Golgi.(A) Drosophila Vps52 localizes to the TGN. Larval L3 salivary gland duct cells and epithelial follicle cells expressing Vps52-GFP under the control of Act5C promoter were stained for cis-Golgi and/or trans-Golgi markers. In duct cells, Vps52-GFP (green) was found adjacent to the trans-Golgi marker dGolgin-245 (blue) and partially overlapped with AP-1 (red) at the trans-Golgi network. In follicle cells, Vps52-GFP (green) was located next to dGM130 (blue) and juxtaposed to dGolgin-245 (red). The insets are magnifications of the indicated boxed areas. (B) Cross-section and side view of epithelial follicle cells from flies that were either homozygous for Arl5KO1 mutation (Arl5KO1/Arl5KO1) or were w1118 (control), expressing Vps52-GFP and stained for dGolgin-245. There is a decrease in the abundance and intensity of Vps52-GFP puncta in the Arl5KO1 mutant, with a concomitant increase in the cytosolic pool. (C) Quantification of the effect of the Arl5 deletion as the ratio of the average fluorescence of Vps52-GFP at the Golgi in relation to the cytoplasmic pool of Vps52-GFP. A single image such as those in (B) was obtained from each of 11 flies for each stock, and the mean determined of the overall ratios of Golgi/cytoplasmic intensity in each image. Error bars show standard error of mean. The Golgi fraction of Vps52-GFP is 1.5 fold higher in wild-type follicle cells than in the Arl5KO1/Arl5KO1 mutant, indicating that Arl5 aids the recruitment of the GARP complex to the Golgi. (D) The total amount of Vps52-GFP is comparable between control and the Arl5KO1 mutant. Immunoblot of ovaries expressing Vps52-GFP in the presence (Vps52GFP/act5C GAL4) or absence of Arl5 (Vps52GFP/act5C GAL4; Arl5KO1/Arl5KO1) and probed for GFP and β-Actin, used as a loading control. Scale bars are 10 µm.
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f04: Arl5 is required for the normal localization of the GARP complex subunit Vps52 to the Golgi.(A) Drosophila Vps52 localizes to the TGN. Larval L3 salivary gland duct cells and epithelial follicle cells expressing Vps52-GFP under the control of Act5C promoter were stained for cis-Golgi and/or trans-Golgi markers. In duct cells, Vps52-GFP (green) was found adjacent to the trans-Golgi marker dGolgin-245 (blue) and partially overlapped with AP-1 (red) at the trans-Golgi network. In follicle cells, Vps52-GFP (green) was located next to dGM130 (blue) and juxtaposed to dGolgin-245 (red). The insets are magnifications of the indicated boxed areas. (B) Cross-section and side view of epithelial follicle cells from flies that were either homozygous for Arl5KO1 mutation (Arl5KO1/Arl5KO1) or were w1118 (control), expressing Vps52-GFP and stained for dGolgin-245. There is a decrease in the abundance and intensity of Vps52-GFP puncta in the Arl5KO1 mutant, with a concomitant increase in the cytosolic pool. (C) Quantification of the effect of the Arl5 deletion as the ratio of the average fluorescence of Vps52-GFP at the Golgi in relation to the cytoplasmic pool of Vps52-GFP. A single image such as those in (B) was obtained from each of 11 flies for each stock, and the mean determined of the overall ratios of Golgi/cytoplasmic intensity in each image. Error bars show standard error of mean. The Golgi fraction of Vps52-GFP is 1.5 fold higher in wild-type follicle cells than in the Arl5KO1/Arl5KO1 mutant, indicating that Arl5 aids the recruitment of the GARP complex to the Golgi. (D) The total amount of Vps52-GFP is comparable between control and the Arl5KO1 mutant. Immunoblot of ovaries expressing Vps52-GFP in the presence (Vps52GFP/act5C GAL4) or absence of Arl5 (Vps52GFP/act5C GAL4; Arl5KO1/Arl5KO1) and probed for GFP and β-Actin, used as a loading control. Scale bars are 10 µm.

Mentions: To verify whether Arl5 has a role in recruiting the GARP complex to the Golgi in vivo, we first generated a fly line expressing under UAS control a GFP-tagged form of the GARP subunit Vps52 (CG7371). In both salivary gland and follicle cells, Vps52-GFP was found in a punctate pattern typical of the Golgi, with the protein partially overlapping the trans-Golgi markers dGolgin-245 and AP-1, but adjacent to, and further from, the cis-Golgi marker dGM130. (Fig. 4A; supplementary material Fig. S1C). This implies that the exogenous Vps52-GFP is being incorporated into the GARP complex and that this tagged form of the complex is still recruited to the TGN. To test the requirement for Arl5 in the recruitment of Vps52-GFP we examined its distribution in control and in Arl5KO1 mutant follicle cells. Removal of Arl5 reduced the intensity of Vps52-GFP on the Golgi and there was a substantial increase in the amount of GFP in the cytoplasm (Fig. 4B). This was confirmed by quantitation of the average fluorescence of the Golgi fraction of Vps52-GFP versus the cytoplasmic fraction (Fig. 4C), whilst protein blotting confirmed that the total amount of Vps52-GFP is similar between wild-type and Arl5KO1 mutant ovaries (Fig. 4D). This indicates a deficit in the recruitment of the GARP complex to the trans-Golgi in the absence of Arl5.


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)

Arl5 is required for the normal localization of the GARP complex subunit Vps52 to the Golgi.(A) Drosophila Vps52 localizes to the TGN. Larval L3 salivary gland duct cells and epithelial follicle cells expressing Vps52-GFP under the control of Act5C promoter were stained for cis-Golgi and/or trans-Golgi markers. In duct cells, Vps52-GFP (green) was found adjacent to the trans-Golgi marker dGolgin-245 (blue) and partially overlapped with AP-1 (red) at the trans-Golgi network. In follicle cells, Vps52-GFP (green) was located next to dGM130 (blue) and juxtaposed to dGolgin-245 (red). The insets are magnifications of the indicated boxed areas. (B) Cross-section and side view of epithelial follicle cells from flies that were either homozygous for Arl5KO1 mutation (Arl5KO1/Arl5KO1) or were w1118 (control), expressing Vps52-GFP and stained for dGolgin-245. There is a decrease in the abundance and intensity of Vps52-GFP puncta in the Arl5KO1 mutant, with a concomitant increase in the cytosolic pool. (C) Quantification of the effect of the Arl5 deletion as the ratio of the average fluorescence of Vps52-GFP at the Golgi in relation to the cytoplasmic pool of Vps52-GFP. A single image such as those in (B) was obtained from each of 11 flies for each stock, and the mean determined of the overall ratios of Golgi/cytoplasmic intensity in each image. Error bars show standard error of mean. The Golgi fraction of Vps52-GFP is 1.5 fold higher in wild-type follicle cells than in the Arl5KO1/Arl5KO1 mutant, indicating that Arl5 aids the recruitment of the GARP complex to the Golgi. (D) The total amount of Vps52-GFP is comparable between control and the Arl5KO1 mutant. Immunoblot of ovaries expressing Vps52-GFP in the presence (Vps52GFP/act5C GAL4) or absence of Arl5 (Vps52GFP/act5C GAL4; Arl5KO1/Arl5KO1) and probed for GFP and β-Actin, used as a loading control. Scale bars are 10 µm.
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Related In: Results  -  Collection

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f04: Arl5 is required for the normal localization of the GARP complex subunit Vps52 to the Golgi.(A) Drosophila Vps52 localizes to the TGN. Larval L3 salivary gland duct cells and epithelial follicle cells expressing Vps52-GFP under the control of Act5C promoter were stained for cis-Golgi and/or trans-Golgi markers. In duct cells, Vps52-GFP (green) was found adjacent to the trans-Golgi marker dGolgin-245 (blue) and partially overlapped with AP-1 (red) at the trans-Golgi network. In follicle cells, Vps52-GFP (green) was located next to dGM130 (blue) and juxtaposed to dGolgin-245 (red). The insets are magnifications of the indicated boxed areas. (B) Cross-section and side view of epithelial follicle cells from flies that were either homozygous for Arl5KO1 mutation (Arl5KO1/Arl5KO1) or were w1118 (control), expressing Vps52-GFP and stained for dGolgin-245. There is a decrease in the abundance and intensity of Vps52-GFP puncta in the Arl5KO1 mutant, with a concomitant increase in the cytosolic pool. (C) Quantification of the effect of the Arl5 deletion as the ratio of the average fluorescence of Vps52-GFP at the Golgi in relation to the cytoplasmic pool of Vps52-GFP. A single image such as those in (B) was obtained from each of 11 flies for each stock, and the mean determined of the overall ratios of Golgi/cytoplasmic intensity in each image. Error bars show standard error of mean. The Golgi fraction of Vps52-GFP is 1.5 fold higher in wild-type follicle cells than in the Arl5KO1/Arl5KO1 mutant, indicating that Arl5 aids the recruitment of the GARP complex to the Golgi. (D) The total amount of Vps52-GFP is comparable between control and the Arl5KO1 mutant. Immunoblot of ovaries expressing Vps52-GFP in the presence (Vps52GFP/act5C GAL4) or absence of Arl5 (Vps52GFP/act5C GAL4; Arl5KO1/Arl5KO1) and probed for GFP and β-Actin, used as a loading control. Scale bars are 10 µm.
Mentions: To verify whether Arl5 has a role in recruiting the GARP complex to the Golgi in vivo, we first generated a fly line expressing under UAS control a GFP-tagged form of the GARP subunit Vps52 (CG7371). In both salivary gland and follicle cells, Vps52-GFP was found in a punctate pattern typical of the Golgi, with the protein partially overlapping the trans-Golgi markers dGolgin-245 and AP-1, but adjacent to, and further from, the cis-Golgi marker dGM130. (Fig. 4A; supplementary material Fig. S1C). This implies that the exogenous Vps52-GFP is being incorporated into the GARP complex and that this tagged form of the complex is still recruited to the TGN. To test the requirement for Arl5 in the recruitment of Vps52-GFP we examined its distribution in control and in Arl5KO1 mutant follicle cells. Removal of Arl5 reduced the intensity of Vps52-GFP on the Golgi and there was a substantial increase in the amount of GFP in the cytoplasm (Fig. 4B). This was confirmed by quantitation of the average fluorescence of the Golgi fraction of Vps52-GFP versus the cytoplasmic fraction (Fig. 4C), whilst protein blotting confirmed that the total amount of Vps52-GFP is similar between wild-type and Arl5KO1 mutant ovaries (Fig. 4D). This indicates a deficit in the recruitment of the GARP complex to the trans-Golgi in the absence of Arl5.

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