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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

Arl5b is required for the recruitment of Vps54 to the TGN.(A) Confocal micrographs of HeLa cells expressing myc-Vps54 (red) and depleted of mock (control) or the indicated proteins with siRNAs and stained for TGN46 (blue). Knockdown of Arl5b or of Arl5a and Arl5b, simultaneously resulted in the striking redistribution of myc-Vps54 from the TGN to the cytoplasm. Expression of a siRNA-resistant form of Arl5b-GFP (Arl5b-GFPRes) was sufficient to rescue the mislocalization of myc-Vps54 upon depletion of either Arl5b or of Arl5a and Arl5b. Scale bars are 10 µm. (B) RT-PCR from HeLa cells silenced for the indicated proteins with siRNAs showed the efficient depletion of the corresponding mRNAs. Amplification of the housekeeping gene GAPDH and of Rab4a was used as a control for the total amount of mRNA across all samples. (C) Quantification of the effect of depletion of the proteins indicated in (A) on the localization of myc-Vps54. Transfected cells were examined for detectable myc-Vps54 on the Golgi (n = 29, 28, 47, 52, 32, 18 respectively). Strikingly, myc-Vps54 was redistributed from the TGN to the cytoplasm in 70% of cells silenced for Arl5b and in 52% of cells depleted for Arl5a and Arl5b. Expression of a siRNA-resistant form of Arl5b-GFP in cells depleted for Arl5b alone or for Arl5a and Arl5b increased the number of cells with myc-Vps54 at the TGN to levels comparable to mock silenced cells (control).
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f06: Arl5b is required for the recruitment of Vps54 to the TGN.(A) Confocal micrographs of HeLa cells expressing myc-Vps54 (red) and depleted of mock (control) or the indicated proteins with siRNAs and stained for TGN46 (blue). Knockdown of Arl5b or of Arl5a and Arl5b, simultaneously resulted in the striking redistribution of myc-Vps54 from the TGN to the cytoplasm. Expression of a siRNA-resistant form of Arl5b-GFP (Arl5b-GFPRes) was sufficient to rescue the mislocalization of myc-Vps54 upon depletion of either Arl5b or of Arl5a and Arl5b. Scale bars are 10 µm. (B) RT-PCR from HeLa cells silenced for the indicated proteins with siRNAs showed the efficient depletion of the corresponding mRNAs. Amplification of the housekeeping gene GAPDH and of Rab4a was used as a control for the total amount of mRNA across all samples. (C) Quantification of the effect of depletion of the proteins indicated in (A) on the localization of myc-Vps54. Transfected cells were examined for detectable myc-Vps54 on the Golgi (n = 29, 28, 47, 52, 32, 18 respectively). Strikingly, myc-Vps54 was redistributed from the TGN to the cytoplasm in 70% of cells silenced for Arl5b and in 52% of cells depleted for Arl5a and Arl5b. Expression of a siRNA-resistant form of Arl5b-GFP in cells depleted for Arl5b alone or for Arl5a and Arl5b increased the number of cells with myc-Vps54 at the TGN to levels comparable to mock silenced cells (control).

Mentions: To monitor targeting of the GARP complex we used a myc-tagged version of Vps54, and consistent with previous studies (Pérez-Victoria et al., 2008), this was efficiently recruited to the Golgi in HeLa cells (Fig. 6A). RT-PCR from HeLa cells showed that both Arl5a and Arl5b are expressed, and confirmed efficient depletion of endogenous mRNA for either, or both simultaneously (Fig. 6B). Examining the distribution of myc-Vps54 showed that the knockdown of Arl5b, but not of Arl5a, resulted in a substantial loss of myc-Vps54 from the TGN and redistribution to the cytoplasm, with only 30% of cells analyzed maintaining myc-Vps54 at the TGN in contrast to 94% in the case of Arl5a depletion (Fig. 6A,C).


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)

Arl5b is required for the recruitment of Vps54 to the TGN.(A) Confocal micrographs of HeLa cells expressing myc-Vps54 (red) and depleted of mock (control) or the indicated proteins with siRNAs and stained for TGN46 (blue). Knockdown of Arl5b or of Arl5a and Arl5b, simultaneously resulted in the striking redistribution of myc-Vps54 from the TGN to the cytoplasm. Expression of a siRNA-resistant form of Arl5b-GFP (Arl5b-GFPRes) was sufficient to rescue the mislocalization of myc-Vps54 upon depletion of either Arl5b or of Arl5a and Arl5b. Scale bars are 10 µm. (B) RT-PCR from HeLa cells silenced for the indicated proteins with siRNAs showed the efficient depletion of the corresponding mRNAs. Amplification of the housekeeping gene GAPDH and of Rab4a was used as a control for the total amount of mRNA across all samples. (C) Quantification of the effect of depletion of the proteins indicated in (A) on the localization of myc-Vps54. Transfected cells were examined for detectable myc-Vps54 on the Golgi (n = 29, 28, 47, 52, 32, 18 respectively). Strikingly, myc-Vps54 was redistributed from the TGN to the cytoplasm in 70% of cells silenced for Arl5b and in 52% of cells depleted for Arl5a and Arl5b. Expression of a siRNA-resistant form of Arl5b-GFP in cells depleted for Arl5b alone or for Arl5a and Arl5b increased the number of cells with myc-Vps54 at the TGN to levels comparable to mock silenced cells (control).
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Related In: Results  -  Collection

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f06: Arl5b is required for the recruitment of Vps54 to the TGN.(A) Confocal micrographs of HeLa cells expressing myc-Vps54 (red) and depleted of mock (control) or the indicated proteins with siRNAs and stained for TGN46 (blue). Knockdown of Arl5b or of Arl5a and Arl5b, simultaneously resulted in the striking redistribution of myc-Vps54 from the TGN to the cytoplasm. Expression of a siRNA-resistant form of Arl5b-GFP (Arl5b-GFPRes) was sufficient to rescue the mislocalization of myc-Vps54 upon depletion of either Arl5b or of Arl5a and Arl5b. Scale bars are 10 µm. (B) RT-PCR from HeLa cells silenced for the indicated proteins with siRNAs showed the efficient depletion of the corresponding mRNAs. Amplification of the housekeeping gene GAPDH and of Rab4a was used as a control for the total amount of mRNA across all samples. (C) Quantification of the effect of depletion of the proteins indicated in (A) on the localization of myc-Vps54. Transfected cells were examined for detectable myc-Vps54 on the Golgi (n = 29, 28, 47, 52, 32, 18 respectively). Strikingly, myc-Vps54 was redistributed from the TGN to the cytoplasm in 70% of cells silenced for Arl5b and in 52% of cells depleted for Arl5a and Arl5b. Expression of a siRNA-resistant form of Arl5b-GFP in cells depleted for Arl5b alone or for Arl5a and Arl5b increased the number of cells with myc-Vps54 at the TGN to levels comparable to mock silenced cells (control).
Mentions: To monitor targeting of the GARP complex we used a myc-tagged version of Vps54, and consistent with previous studies (Pérez-Victoria et al., 2008), this was efficiently recruited to the Golgi in HeLa cells (Fig. 6A). RT-PCR from HeLa cells showed that both Arl5a and Arl5b are expressed, and confirmed efficient depletion of endogenous mRNA for either, or both simultaneously (Fig. 6B). Examining the distribution of myc-Vps54 showed that the knockdown of Arl5b, but not of Arl5a, resulted in a substantial loss of myc-Vps54 from the TGN and redistribution to the cytoplasm, with only 30% of cells analyzed maintaining myc-Vps54 at the TGN in contrast to 94% in the case of Arl5a depletion (Fig. 6A,C).

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