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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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Rab4 Interacts with a Second Form of the GARP Complex(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab4 (detergent-free, lighter gray). The top 24 are shown (full list in Table S3). Known Rab4 effector marked in red, and other proteins with links to membrane traffic are indicated.(B) Anti-GFP immunoblot of affinity chromatography of lysates from S2 cells expressing GFP-CG31064 (FYVE domain form) or CG7371-GFP using GDP- and GTP-locked Rabs.(C) Confocal micrographs of COS cells coexpressing VPS51-GFP and VPS53-myc (upper panels) and GFP-Rab4A and VPS53-myc (lower panels). The proteins are human, and cells were stained for the early endosomal marker EEA1.(D) Fluorescent images of S2 cells coexpressing GFP-CG4996 and RFP-Rab4. Cells were immunolabeled with Hrs antibodies.(E) Immunoblots with anti-myc of GFP-Trap precipitations from extracts of S2 cells cotransfected with GFP-CG4996 or GFP, and with CG7371-Myc (Vps52) or CG3338-Myc (Vps53). GFP-Trap (ChromoTek) was used according to manufacturer’s instructions. Inputs are 10%. Both Vps51 and Vps53 associate with CG4996. ip, immunoprecipitation.Scale bars, 5 μm (D) or 10 μm (C).See also Figure S4 and Table S3.
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fig4: Rab4 Interacts with a Second Form of the GARP Complex(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab4 (detergent-free, lighter gray). The top 24 are shown (full list in Table S3). Known Rab4 effector marked in red, and other proteins with links to membrane traffic are indicated.(B) Anti-GFP immunoblot of affinity chromatography of lysates from S2 cells expressing GFP-CG31064 (FYVE domain form) or CG7371-GFP using GDP- and GTP-locked Rabs.(C) Confocal micrographs of COS cells coexpressing VPS51-GFP and VPS53-myc (upper panels) and GFP-Rab4A and VPS53-myc (lower panels). The proteins are human, and cells were stained for the early endosomal marker EEA1.(D) Fluorescent images of S2 cells coexpressing GFP-CG4996 and RFP-Rab4. Cells were immunolabeled with Hrs antibodies.(E) Immunoblots with anti-myc of GFP-Trap precipitations from extracts of S2 cells cotransfected with GFP-CG4996 or GFP, and with CG7371-Myc (Vps52) or CG3338-Myc (Vps53). GFP-Trap (ChromoTek) was used according to manufacturer’s instructions. Inputs are 10%. Both Vps51 and Vps53 associate with CG4996. ip, immunoprecipitation.Scale bars, 5 μm (D) or 10 μm (C).See also Figure S4 and Table S3.

Mentions: Rab4 is widely conserved in evolution but has been lost in various lineages including budding yeasts. It is localized to endosomes and is proposed to play a role in recycling back to the surface. The best characterized Rab4 effector is RUFY1 (or Rabip4), one of three closely related paralogs (Cormont et al., 2001). CG31064, the Drosophila ortholog of these proteins, showed a strong and highly specific interaction with Rab4 under both lysis conditions (which we confirmed to be GTP-specific using a GFP-tagged CG31064), indicating that the Rab4-GTP was functional (Figures 4A and 4B). In addition to CG31064, there were several other proteins that were specific to Rab4 that have not previously been reported to be effectors. Most striking were Vps51, Vps52, and Vps53, three subunits of the GARP tethering complex that is found on the Golgi and involved in retrograde traffic from endosomes (Bonifacino and Hierro, 2011). These were found with or without detergent, and we were able to confirm that GARP subunit binding is GTP specific (Figure 4B). We also examined a GARP subunit (Vps53) in mammalian cells and found that it could be relocated to endosomes by overexpression of Rab4 (Figure 4C).


Toward a comprehensive map of the effectors of rab GTPases.

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

Rab4 Interacts with a Second Form of the GARP Complex(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab4 (detergent-free, lighter gray). The top 24 are shown (full list in Table S3). Known Rab4 effector marked in red, and other proteins with links to membrane traffic are indicated.(B) Anti-GFP immunoblot of affinity chromatography of lysates from S2 cells expressing GFP-CG31064 (FYVE domain form) or CG7371-GFP using GDP- and GTP-locked Rabs.(C) Confocal micrographs of COS cells coexpressing VPS51-GFP and VPS53-myc (upper panels) and GFP-Rab4A and VPS53-myc (lower panels). The proteins are human, and cells were stained for the early endosomal marker EEA1.(D) Fluorescent images of S2 cells coexpressing GFP-CG4996 and RFP-Rab4. Cells were immunolabeled with Hrs antibodies.(E) Immunoblots with anti-myc of GFP-Trap precipitations from extracts of S2 cells cotransfected with GFP-CG4996 or GFP, and with CG7371-Myc (Vps52) or CG3338-Myc (Vps53). GFP-Trap (ChromoTek) was used according to manufacturer’s instructions. Inputs are 10%. Both Vps51 and Vps53 associate with CG4996. ip, immunoprecipitation.Scale bars, 5 μm (D) or 10 μm (C).See also Figure S4 and Table S3.
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fig4: Rab4 Interacts with a Second Form of the GARP Complex(A) Proteins isolated from cell lysates ranked by S score for interaction with GST-Rab4 (detergent-free, lighter gray). The top 24 are shown (full list in Table S3). Known Rab4 effector marked in red, and other proteins with links to membrane traffic are indicated.(B) Anti-GFP immunoblot of affinity chromatography of lysates from S2 cells expressing GFP-CG31064 (FYVE domain form) or CG7371-GFP using GDP- and GTP-locked Rabs.(C) Confocal micrographs of COS cells coexpressing VPS51-GFP and VPS53-myc (upper panels) and GFP-Rab4A and VPS53-myc (lower panels). The proteins are human, and cells were stained for the early endosomal marker EEA1.(D) Fluorescent images of S2 cells coexpressing GFP-CG4996 and RFP-Rab4. Cells were immunolabeled with Hrs antibodies.(E) Immunoblots with anti-myc of GFP-Trap precipitations from extracts of S2 cells cotransfected with GFP-CG4996 or GFP, and with CG7371-Myc (Vps52) or CG3338-Myc (Vps53). GFP-Trap (ChromoTek) was used according to manufacturer’s instructions. Inputs are 10%. Both Vps51 and Vps53 associate with CG4996. ip, immunoprecipitation.Scale bars, 5 μm (D) or 10 μm (C).See also Figure S4 and Table S3.
Mentions: Rab4 is widely conserved in evolution but has been lost in various lineages including budding yeasts. It is localized to endosomes and is proposed to play a role in recycling back to the surface. The best characterized Rab4 effector is RUFY1 (or Rabip4), one of three closely related paralogs (Cormont et al., 2001). CG31064, the Drosophila ortholog of these proteins, showed a strong and highly specific interaction with Rab4 under both lysis conditions (which we confirmed to be GTP-specific using a GFP-tagged CG31064), indicating that the Rab4-GTP was functional (Figures 4A and 4B). In addition to CG31064, there were several other proteins that were specific to Rab4 that have not previously been reported to be effectors. Most striking were Vps51, Vps52, and Vps53, three subunits of the GARP tethering complex that is found on the Golgi and involved in retrograde traffic from endosomes (Bonifacino and Hierro, 2011). These were found with or without detergent, and we were able to confirm that GARP subunit binding is GTP specific (Figure 4B). We also examined a GARP subunit (Vps53) in mammalian cells and found that it could be relocated to endosomes by overexpression of Rab4 (Figure 4C).

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