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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

Affinity chromatography of cell extracts with Arl5.(A) Comparison of the spectral counts for proteins isolated from lysates prepared from Drosophila heads by affinity chromatography with GST tagged forms of GTP-locked or GDP-locked Arl5. Abundant GTP-specific interactors are labeled, ochre dots indicating GARP subunits. Full list of bound proteins is in supplementary material Table S1. (B) Comparison of the spectral counts for the top twenty proteins found exclusively with the GTP-form of Arl5 in the affinity purification shown in (A) versus the same from an affinity purification performed with Arl5 bound to liposomes. Proteins in both datasets are indicated. See supplementary material Table S2 for the full list of proteins from the liposome purification.
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f03: Affinity chromatography of cell extracts with Arl5.(A) Comparison of the spectral counts for proteins isolated from lysates prepared from Drosophila heads by affinity chromatography with GST tagged forms of GTP-locked or GDP-locked Arl5. Abundant GTP-specific interactors are labeled, ochre dots indicating GARP subunits. Full list of bound proteins is in supplementary material Table S1. (B) Comparison of the spectral counts for the top twenty proteins found exclusively with the GTP-form of Arl5 in the affinity purification shown in (A) versus the same from an affinity purification performed with Arl5 bound to liposomes. Proteins in both datasets are indicated. See supplementary material Table S2 for the full list of proteins from the liposome purification.

Mentions: To identify effectors that Arl5 recruits to the Golgi we used two different affinity chromatography methods based on versions of Arl5 carrying mutations that have been shown in other members of the Arf family to lock the protein in the GDP- (inactive) or GTP-bound (active) state (Christis and Munro, 2012; Dascher and Balch, 1994; Houghton et al., 2012). In the first approach GST fusions to the Arl5 forms were immobilized on Sepharose beads and used to isolate proteins from lysates prepared from adult heads. After washing, the bound proteins were eluted with high salt, separated on a protein gel, digested with trypsin, and the resulting peptides sequenced by tandem mass spectrometry. The number of spectra obtained for each protein was used as an approximate measure of abundance and used to compare binding to the GTP- and GDP-bound forms of Arl5 (Fig. 3A). Amongst the more abundant proteins that bound specifically to Arl5-GTP were the Drosophila orthologues of the four subunits of the GARP complex (CG15087/Vps51, CG7371/Vps52, CG3338/Vps53, and CG3766/Vps54 or Scat)). The other abundant interactors were mostly proteins that interact with the actin cytoskeleton but these are relatively common contaminants in affinity purifications (Mellacheruvu et al., 2013).


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)

Affinity chromatography of cell extracts with Arl5.(A) Comparison of the spectral counts for proteins isolated from lysates prepared from Drosophila heads by affinity chromatography with GST tagged forms of GTP-locked or GDP-locked Arl5. Abundant GTP-specific interactors are labeled, ochre dots indicating GARP subunits. Full list of bound proteins is in supplementary material Table S1. (B) Comparison of the spectral counts for the top twenty proteins found exclusively with the GTP-form of Arl5 in the affinity purification shown in (A) versus the same from an affinity purification performed with Arl5 bound to liposomes. Proteins in both datasets are indicated. See supplementary material Table S2 for the full list of proteins from the liposome purification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400590&req=5

f03: Affinity chromatography of cell extracts with Arl5.(A) Comparison of the spectral counts for proteins isolated from lysates prepared from Drosophila heads by affinity chromatography with GST tagged forms of GTP-locked or GDP-locked Arl5. Abundant GTP-specific interactors are labeled, ochre dots indicating GARP subunits. Full list of bound proteins is in supplementary material Table S1. (B) Comparison of the spectral counts for the top twenty proteins found exclusively with the GTP-form of Arl5 in the affinity purification shown in (A) versus the same from an affinity purification performed with Arl5 bound to liposomes. Proteins in both datasets are indicated. See supplementary material Table S2 for the full list of proteins from the liposome purification.
Mentions: To identify effectors that Arl5 recruits to the Golgi we used two different affinity chromatography methods based on versions of Arl5 carrying mutations that have been shown in other members of the Arf family to lock the protein in the GDP- (inactive) or GTP-bound (active) state (Christis and Munro, 2012; Dascher and Balch, 1994; Houghton et al., 2012). In the first approach GST fusions to the Arl5 forms were immobilized on Sepharose beads and used to isolate proteins from lysates prepared from adult heads. After washing, the bound proteins were eluted with high salt, separated on a protein gel, digested with trypsin, and the resulting peptides sequenced by tandem mass spectrometry. The number of spectra obtained for each protein was used as an approximate measure of abundance and used to compare binding to the GTP- and GDP-bound forms of Arl5 (Fig. 3A). Amongst the more abundant proteins that bound specifically to Arl5-GTP were the Drosophila orthologues of the four subunits of the GARP complex (CG15087/Vps51, CG7371/Vps52, CG3338/Vps53, and CG3766/Vps54 or Scat)). The other abundant interactors were mostly proteins that interact with the actin cytoskeleton but these are relatively common contaminants in affinity purifications (Mellacheruvu et al., 2013).

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