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Toward a comprehensive map of the effectors of rab GTPases.

Gillingham AK, Sinka R, Torres IL, Lilley KS, Munro S - Dev. Cell (2014)

Bottom Line: However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear.For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors.In addition, we found numerous Rab-specific interactions with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function.

View Article: PubMed Central - PubMed

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

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Binding Partners of Rab6, Rab7, and Rab9(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab6 (detergent-free, lighter gray). The top 36 are shown (full list in Table S3). Known Rab6 effectors are marked in red, and other proteins with links to membrane traffic are indicated.(B) Predicted propensity for CG8578 to form coiled-coil along its length.(C) Anti-GFP immunoblot of affinity chromatography of S2 cell lysates expressing GFP-CG8578 using GDP- and GTP-locked versions of Rab6.(D) Interaction of Evi5 with Rab6 and Rab14 confirmed by affinity chromatography with lysates from cells expressing GFP-Evi5.(E) Summary of yeast two-hybrid assays with Evi5 truncations as prey and GDP- and GTP-locked version of Rab6 and Rab14 as bait.(F) Immunoblot of lysates and eluates from an affinity chromatography experiment of lysates prepared from GFP-Evi5 (315-621)-expressing cells using both Rab6- and Rab14-locked mutant proteins. The blot was probed with anti-GFP antibodies.(G) Live cells coexpressing GFP-Evi5 and RFP-Rab6 (left hand panels) and confocal micrographs of fixed cells coexpressing a C-terminal fragment of Evi5 (315-621aa) with RFP-Rab6 (right panels). The latter are immunolabeled with dGM130 antibodies. The N-terminal fragment (1–315) did not bind Rab6 in vitro and was diffuse in cells (data not shown).(H) Upper panels show confocal images of cells expressing GFP-CG12132, costained with anti-Rab7 antibodies (late endosomes) and anti-dGM130 antibodies (Golgi). Lower panels show GFP-CG12132 localization is distinct from RFP-Rab5 (early endosomes) in S2 cells.(I) Affinity chromatography of S2 cell lysates using nucleotide-locked versions of Rab9. Blots were probed with antibodies against golgins dGM130 and GMAP.Scale bars, 5 μm.See also Figure S5 and Table S3.
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fig5: Binding Partners of Rab6, Rab7, and Rab9(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab6 (detergent-free, lighter gray). The top 36 are shown (full list in Table S3). Known Rab6 effectors are marked in red, and other proteins with links to membrane traffic are indicated.(B) Predicted propensity for CG8578 to form coiled-coil along its length.(C) Anti-GFP immunoblot of affinity chromatography of S2 cell lysates expressing GFP-CG8578 using GDP- and GTP-locked versions of Rab6.(D) Interaction of Evi5 with Rab6 and Rab14 confirmed by affinity chromatography with lysates from cells expressing GFP-Evi5.(E) Summary of yeast two-hybrid assays with Evi5 truncations as prey and GDP- and GTP-locked version of Rab6 and Rab14 as bait.(F) Immunoblot of lysates and eluates from an affinity chromatography experiment of lysates prepared from GFP-Evi5 (315-621)-expressing cells using both Rab6- and Rab14-locked mutant proteins. The blot was probed with anti-GFP antibodies.(G) Live cells coexpressing GFP-Evi5 and RFP-Rab6 (left hand panels) and confocal micrographs of fixed cells coexpressing a C-terminal fragment of Evi5 (315-621aa) with RFP-Rab6 (right panels). The latter are immunolabeled with dGM130 antibodies. The N-terminal fragment (1–315) did not bind Rab6 in vitro and was diffuse in cells (data not shown).(H) Upper panels show confocal images of cells expressing GFP-CG12132, costained with anti-Rab7 antibodies (late endosomes) and anti-dGM130 antibodies (Golgi). Lower panels show GFP-CG12132 localization is distinct from RFP-Rab5 (early endosomes) in S2 cells.(I) Affinity chromatography of S2 cell lysates using nucleotide-locked versions of Rab9. Blots were probed with antibodies against golgins dGM130 and GMAP.Scale bars, 5 μm.See also Figure S5 and Table S3.

Mentions: Rab6 is widely conserved among eukaryotes. Localized to the trans-Golgi, it is involved in retrograde traffic from endosomes and possibly exit from the trans-Golgi network and retrograde trafficking from Golgi to ER. Known Rab6 effectors include the dynein adaptor BicaudalD; the lipid phosphatase OCRL1; the golgins TMF1, golgin-97, GCC88, and GCC185; the putative Rab GEF DENND5; and GORAB/SCYL1BP1 (Hutagalung and Novick, 2011). Drosophila orthologs of five of these proteins were found associated with high specificity to the Rab6 column, being BicD, CG3573 (OCRL1), CG33052 (GORAB), dGolgin-97, and dGCC88 (Figure 5A). The Rab6 column also bound specifically other components of membrane traffic and some proteins whose function is less clear.


Toward a comprehensive map of the effectors of rab GTPases.

Gillingham AK, Sinka R, Torres IL, Lilley KS, Munro S - Dev. Cell (2014)

Binding Partners of Rab6, Rab7, and Rab9(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab6 (detergent-free, lighter gray). The top 36 are shown (full list in Table S3). Known Rab6 effectors are marked in red, and other proteins with links to membrane traffic are indicated.(B) Predicted propensity for CG8578 to form coiled-coil along its length.(C) Anti-GFP immunoblot of affinity chromatography of S2 cell lysates expressing GFP-CG8578 using GDP- and GTP-locked versions of Rab6.(D) Interaction of Evi5 with Rab6 and Rab14 confirmed by affinity chromatography with lysates from cells expressing GFP-Evi5.(E) Summary of yeast two-hybrid assays with Evi5 truncations as prey and GDP- and GTP-locked version of Rab6 and Rab14 as bait.(F) Immunoblot of lysates and eluates from an affinity chromatography experiment of lysates prepared from GFP-Evi5 (315-621)-expressing cells using both Rab6- and Rab14-locked mutant proteins. The blot was probed with anti-GFP antibodies.(G) Live cells coexpressing GFP-Evi5 and RFP-Rab6 (left hand panels) and confocal micrographs of fixed cells coexpressing a C-terminal fragment of Evi5 (315-621aa) with RFP-Rab6 (right panels). The latter are immunolabeled with dGM130 antibodies. The N-terminal fragment (1–315) did not bind Rab6 in vitro and was diffuse in cells (data not shown).(H) Upper panels show confocal images of cells expressing GFP-CG12132, costained with anti-Rab7 antibodies (late endosomes) and anti-dGM130 antibodies (Golgi). Lower panels show GFP-CG12132 localization is distinct from RFP-Rab5 (early endosomes) in S2 cells.(I) Affinity chromatography of S2 cell lysates using nucleotide-locked versions of Rab9. Blots were probed with antibodies against golgins dGM130 and GMAP.Scale bars, 5 μm.See also Figure S5 and Table S3.
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Related In: Results  -  Collection

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fig5: Binding Partners of Rab6, Rab7, and Rab9(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab6 (detergent-free, lighter gray). The top 36 are shown (full list in Table S3). Known Rab6 effectors are marked in red, and other proteins with links to membrane traffic are indicated.(B) Predicted propensity for CG8578 to form coiled-coil along its length.(C) Anti-GFP immunoblot of affinity chromatography of S2 cell lysates expressing GFP-CG8578 using GDP- and GTP-locked versions of Rab6.(D) Interaction of Evi5 with Rab6 and Rab14 confirmed by affinity chromatography with lysates from cells expressing GFP-Evi5.(E) Summary of yeast two-hybrid assays with Evi5 truncations as prey and GDP- and GTP-locked version of Rab6 and Rab14 as bait.(F) Immunoblot of lysates and eluates from an affinity chromatography experiment of lysates prepared from GFP-Evi5 (315-621)-expressing cells using both Rab6- and Rab14-locked mutant proteins. The blot was probed with anti-GFP antibodies.(G) Live cells coexpressing GFP-Evi5 and RFP-Rab6 (left hand panels) and confocal micrographs of fixed cells coexpressing a C-terminal fragment of Evi5 (315-621aa) with RFP-Rab6 (right panels). The latter are immunolabeled with dGM130 antibodies. The N-terminal fragment (1–315) did not bind Rab6 in vitro and was diffuse in cells (data not shown).(H) Upper panels show confocal images of cells expressing GFP-CG12132, costained with anti-Rab7 antibodies (late endosomes) and anti-dGM130 antibodies (Golgi). Lower panels show GFP-CG12132 localization is distinct from RFP-Rab5 (early endosomes) in S2 cells.(I) Affinity chromatography of S2 cell lysates using nucleotide-locked versions of Rab9. Blots were probed with antibodies against golgins dGM130 and GMAP.Scale bars, 5 μm.See also Figure S5 and Table S3.
Mentions: Rab6 is widely conserved among eukaryotes. Localized to the trans-Golgi, it is involved in retrograde traffic from endosomes and possibly exit from the trans-Golgi network and retrograde trafficking from Golgi to ER. Known Rab6 effectors include the dynein adaptor BicaudalD; the lipid phosphatase OCRL1; the golgins TMF1, golgin-97, GCC88, and GCC185; the putative Rab GEF DENND5; and GORAB/SCYL1BP1 (Hutagalung and Novick, 2011). Drosophila orthologs of five of these proteins were found associated with high specificity to the Rab6 column, being BicD, CG3573 (OCRL1), CG33052 (GORAB), dGolgin-97, and dGCC88 (Figure 5A). The Rab6 column also bound specifically other components of membrane traffic and some proteins whose function is less clear.

Bottom Line: However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear.For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors.In addition, we found numerous Rab-specific interactions with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus