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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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Rab18 Has Roles at the ER and the Golgi(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab18 (detergent-free, lighter gray). Subunits of the NRZ/Dsl1 complex (yellow dots), and other proteins with links to membrane traffic are indicated. The NRZ/Dsl1 complex contains two membrane-spanning proteins and so would not be isolated by the detergent-free approach (white circles). The top 42 S scores are shown with the full list, including the Drosophila orthologs of Rab3GAP, in Table S3.(B) Affinity chromatography of S2 cell lysate with Rab18. Blots were probed with either anti-ZW10 antibodies or anti-Rod antibodies as indicated.(C) Confocal micrographs of COS cells expressing GFP-HsRab18 and stained with antibodies against endogenous ZW10. The ZW10 accumulates around the Rab18-positive lipid droplets.(D) Affinity chromatography by Rab6 and Rab18 of lysates prepared from S2 cells expressing GFP-TBC1D5.(E) Fluorescent images of GFP-TBC1D5 expressed in S2 cells alone (top panels) or with myc-Rab6 (middle panels) or RFP-Rab18 (lower panels). Cells were stained with the Golgi antibodies dGM130 or dGolgin-245 as indicated. Expression of Rab18 enhances recruitment of TBC1D5 to the Golgi.(F) S2 cells expressing LRRK2-myc or Spartin-GFP were subjected to affinity chromatography with locked forms of Rab18. The resulting blots were probed for the relevant tag.(G) Fluorescent images of RFP-Rab19 and GFP-Plx expressed in S2 cells. Cells were stained for the Golgi marker dGM130.(H) Affinity chromatography by Rab19 of lysates prepared from S2 cells expressing GFP-Plx.Scale bars, 5 μm (10 μm in C).See also Figure S6 and Table S3.
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fig6: Rab18 Has Roles at the ER and the Golgi(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab18 (detergent-free, lighter gray). Subunits of the NRZ/Dsl1 complex (yellow dots), and other proteins with links to membrane traffic are indicated. The NRZ/Dsl1 complex contains two membrane-spanning proteins and so would not be isolated by the detergent-free approach (white circles). The top 42 S scores are shown with the full list, including the Drosophila orthologs of Rab3GAP, in Table S3.(B) Affinity chromatography of S2 cell lysate with Rab18. Blots were probed with either anti-ZW10 antibodies or anti-Rod antibodies as indicated.(C) Confocal micrographs of COS cells expressing GFP-HsRab18 and stained with antibodies against endogenous ZW10. The ZW10 accumulates around the Rab18-positive lipid droplets.(D) Affinity chromatography by Rab6 and Rab18 of lysates prepared from S2 cells expressing GFP-TBC1D5.(E) Fluorescent images of GFP-TBC1D5 expressed in S2 cells alone (top panels) or with myc-Rab6 (middle panels) or RFP-Rab18 (lower panels). Cells were stained with the Golgi antibodies dGM130 or dGolgin-245 as indicated. Expression of Rab18 enhances recruitment of TBC1D5 to the Golgi.(F) S2 cells expressing LRRK2-myc or Spartin-GFP were subjected to affinity chromatography with locked forms of Rab18. The resulting blots were probed for the relevant tag.(G) Fluorescent images of RFP-Rab19 and GFP-Plx expressed in S2 cells. Cells were stained for the Golgi marker dGM130.(H) Affinity chromatography by Rab19 of lysates prepared from S2 cells expressing GFP-Plx.Scale bars, 5 μm (10 μm in C).See also Figure S6 and Table S3.

Mentions: Several proteins bound with high specificity to Rab18 (Figure 6A). These include subunits of the Dsl1 complex (or NRZ in mammals), which is localized to ER membranes and tethers and then fuses vesicles returning from the Golgi (Civril et al., 2010; Wainman et al., 2012). These subunits (Zw10, Rod, Zwilch, RINT1, and syntaxin-18) were only recovered from the detergent lysate, consistent with syntaxin-18 being a SNARE with a transmembrane domain. The only subunits not detected were two further SNAREs, which are below the ∼45 kDa minimum of this data set. Antisera against two NRZ subunits confirmed the interaction with Rab18 and showed it to be GTP specific (Figure 6B). In S2 cells, red fluorescent protein (RFP)-Rab18 colocalized with ER and early Golgi, and with GFP-tagged ZW10 (Figures S6A and S6B). These results suggest that Rab18 may assist the tethering of COPI-coated vesicles to the ER. In mammalian cells, Rab18 localizes to the ER and Golgi, but when overexpressed, it accumulates on lipid droplets (Ozeki et al., 2005). When we overexpressed Rab18 in mammalian cells, the endogenous NRZ subunits shifted from a diffuse distribution to being clustered around lipid droplets (Figures 6C and S6C). In addition, when human Rab18 was used for affinity chromatography of human cell lysates, subunits of NRZ were among the most abundant proteins showing GTP-specific binding (Figure S6D). The physiological relevance of overexpressed Rab18 being on lipid droplets is unclear, but these data at least indicate that its interaction with NRZ is relevant to mammalian cells and would explain why overexpression of Rab18 induces an association between lipid droplets and ER (Ozeki et al., 2005).


Toward a comprehensive map of the effectors of rab GTPases.

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

Rab18 Has Roles at the ER and the Golgi(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab18 (detergent-free, lighter gray). Subunits of the NRZ/Dsl1 complex (yellow dots), and other proteins with links to membrane traffic are indicated. The NRZ/Dsl1 complex contains two membrane-spanning proteins and so would not be isolated by the detergent-free approach (white circles). The top 42 S scores are shown with the full list, including the Drosophila orthologs of Rab3GAP, in Table S3.(B) Affinity chromatography of S2 cell lysate with Rab18. Blots were probed with either anti-ZW10 antibodies or anti-Rod antibodies as indicated.(C) Confocal micrographs of COS cells expressing GFP-HsRab18 and stained with antibodies against endogenous ZW10. The ZW10 accumulates around the Rab18-positive lipid droplets.(D) Affinity chromatography by Rab6 and Rab18 of lysates prepared from S2 cells expressing GFP-TBC1D5.(E) Fluorescent images of GFP-TBC1D5 expressed in S2 cells alone (top panels) or with myc-Rab6 (middle panels) or RFP-Rab18 (lower panels). Cells were stained with the Golgi antibodies dGM130 or dGolgin-245 as indicated. Expression of Rab18 enhances recruitment of TBC1D5 to the Golgi.(F) S2 cells expressing LRRK2-myc or Spartin-GFP were subjected to affinity chromatography with locked forms of Rab18. The resulting blots were probed for the relevant tag.(G) Fluorescent images of RFP-Rab19 and GFP-Plx expressed in S2 cells. Cells were stained for the Golgi marker dGM130.(H) Affinity chromatography by Rab19 of lysates prepared from S2 cells expressing GFP-Plx.Scale bars, 5 μm (10 μm in C).See also Figure S6 and Table S3.
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fig6: Rab18 Has Roles at the ER and the Golgi(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab18 (detergent-free, lighter gray). Subunits of the NRZ/Dsl1 complex (yellow dots), and other proteins with links to membrane traffic are indicated. The NRZ/Dsl1 complex contains two membrane-spanning proteins and so would not be isolated by the detergent-free approach (white circles). The top 42 S scores are shown with the full list, including the Drosophila orthologs of Rab3GAP, in Table S3.(B) Affinity chromatography of S2 cell lysate with Rab18. Blots were probed with either anti-ZW10 antibodies or anti-Rod antibodies as indicated.(C) Confocal micrographs of COS cells expressing GFP-HsRab18 and stained with antibodies against endogenous ZW10. The ZW10 accumulates around the Rab18-positive lipid droplets.(D) Affinity chromatography by Rab6 and Rab18 of lysates prepared from S2 cells expressing GFP-TBC1D5.(E) Fluorescent images of GFP-TBC1D5 expressed in S2 cells alone (top panels) or with myc-Rab6 (middle panels) or RFP-Rab18 (lower panels). Cells were stained with the Golgi antibodies dGM130 or dGolgin-245 as indicated. Expression of Rab18 enhances recruitment of TBC1D5 to the Golgi.(F) S2 cells expressing LRRK2-myc or Spartin-GFP were subjected to affinity chromatography with locked forms of Rab18. The resulting blots were probed for the relevant tag.(G) Fluorescent images of RFP-Rab19 and GFP-Plx expressed in S2 cells. Cells were stained for the Golgi marker dGM130.(H) Affinity chromatography by Rab19 of lysates prepared from S2 cells expressing GFP-Plx.Scale bars, 5 μm (10 μm in C).See also Figure S6 and Table S3.
Mentions: Several proteins bound with high specificity to Rab18 (Figure 6A). These include subunits of the Dsl1 complex (or NRZ in mammals), which is localized to ER membranes and tethers and then fuses vesicles returning from the Golgi (Civril et al., 2010; Wainman et al., 2012). These subunits (Zw10, Rod, Zwilch, RINT1, and syntaxin-18) were only recovered from the detergent lysate, consistent with syntaxin-18 being a SNARE with a transmembrane domain. The only subunits not detected were two further SNAREs, which are below the ∼45 kDa minimum of this data set. Antisera against two NRZ subunits confirmed the interaction with Rab18 and showed it to be GTP specific (Figure 6B). In S2 cells, red fluorescent protein (RFP)-Rab18 colocalized with ER and early Golgi, and with GFP-tagged ZW10 (Figures S6A and S6B). These results suggest that Rab18 may assist the tethering of COPI-coated vesicles to the ER. In mammalian cells, Rab18 localizes to the ER and Golgi, but when overexpressed, it accumulates on lipid droplets (Ozeki et al., 2005). When we overexpressed Rab18 in mammalian cells, the endogenous NRZ subunits shifted from a diffuse distribution to being clustered around lipid droplets (Figures 6C and S6C). In addition, when human Rab18 was used for affinity chromatography of human cell lysates, subunits of NRZ were among the most abundant proteins showing GTP-specific binding (Figure S6D). The physiological relevance of overexpressed Rab18 being on lipid droplets is unclear, but these data at least indicate that its interaction with NRZ is relevant to mammalian cells and would explain why overexpression of Rab18 induces an association between lipid droplets and ER (Ozeki et al., 2005).

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