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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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Related in: MedlinePlus

Analysis of the Rab5 Interactome to Test the Affinity Chromatography Approach(A) Schematic of the Rab effector isolation protocol.(B) Comparison of the spectral counts for proteins isolated by affinity chromatography with GST alone or GST-tagged GTP-locked Rab5. Colors indicate known Rab5 effectors, endosomal proteins not previously reported to bind Rab5, or typical nonspecific binders in affinity purifications (chaperones, cytoskeletal proteins, etc).(C) Plot of spectral counts versus mRNA levels for each of the Rab5 binding proteins. Coloring is as in (B).(D) Proteins ranked by the total number of spectral counts obtained with GST-Rab5 using detergent lysis, along with their spectral counts in other GST-Rab eluates. Circle area is proportional to the spectral counts. Only the top 70 proteins are shown, with the full list in Table S1A. Known Rab5 effectors (red), other proteins associated with endosomes (red frame), and typical nonspecific binders (blue) are highlighted.(E) Same as in (D), except that the data are not spectral counts but S scores as determined using the CompPASS method (Sowa et al., 2009). Only the top 70 proteins are shown (full list in Table S1B). The S score gives greater weight to proteins binding fewer Rabs and promotes known effectors.(F) Same as in (E), except S score for Rab effectors isolated using detergent-free lysis.(G) Affinity chromatography of lysates from S2 cells expressing GFP-CG6607 or GFP-CG11490 (dTBC1D15) using GDP- and GTP-locked Rab5. Blots were probed with antibodies against GFP.(H) Confocal micrographs of Drosophila S2 cells expressing both GFP-CG6607 and RFP-Rab5. Cells were stained with antibodies against Rab7.Scale bar, 5 μm.See also Figure S1 and Tables S1A and S1B.
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fig1: Analysis of the Rab5 Interactome to Test the Affinity Chromatography Approach(A) Schematic of the Rab effector isolation protocol.(B) Comparison of the spectral counts for proteins isolated by affinity chromatography with GST alone or GST-tagged GTP-locked Rab5. Colors indicate known Rab5 effectors, endosomal proteins not previously reported to bind Rab5, or typical nonspecific binders in affinity purifications (chaperones, cytoskeletal proteins, etc).(C) Plot of spectral counts versus mRNA levels for each of the Rab5 binding proteins. Coloring is as in (B).(D) Proteins ranked by the total number of spectral counts obtained with GST-Rab5 using detergent lysis, along with their spectral counts in other GST-Rab eluates. Circle area is proportional to the spectral counts. Only the top 70 proteins are shown, with the full list in Table S1A. Known Rab5 effectors (red), other proteins associated with endosomes (red frame), and typical nonspecific binders (blue) are highlighted.(E) Same as in (D), except that the data are not spectral counts but S scores as determined using the CompPASS method (Sowa et al., 2009). Only the top 70 proteins are shown (full list in Table S1B). The S score gives greater weight to proteins binding fewer Rabs and promotes known effectors.(F) Same as in (E), except S score for Rab effectors isolated using detergent-free lysis.(G) Affinity chromatography of lysates from S2 cells expressing GFP-CG6607 or GFP-CG11490 (dTBC1D15) using GDP- and GTP-locked Rab5. Blots were probed with antibodies against GFP.(H) Confocal micrographs of Drosophila S2 cells expressing both GFP-CG6607 and RFP-Rab5. Cells were stained with antibodies against Rab7.Scale bar, 5 μm.See also Figure S1 and Tables S1A and S1B.

Mentions: Drosophila melanogaster has 27 Rabs, of which 23 have at least one mammalian ortholog, with these mammalian orthologs representing 50 of the 66 human Rabs (Figures S1A and S1B available online). All 23 Drosophila Rabs that have a mammalian ortholog were expressed as fusions to glutathione S-transferase (GST) with the Q→L mutation that is known in several Rabs to stabilize the GTP-bound form (Hyvola et al., 2006; Li and Stahl, 1993). They were coupled to glutathione Sepharose for affinity chromatography of lysates of Drosophila S2 cells, a widely used cell line thought to be from a macrophage-like lineage (Figure 1A). S2 cells were lysed in the detergent CHAPS, and the clarified lysate was applied to each GST-Rab column. After washing, proteins were eluted and separated on SDS gels. The gel lanes above the GST-Rab (∼45–50 kDa) were cut into sections and digested with trypsin, and peptides were sequenced by tandem mass spectrometry (Table S1).


Toward a comprehensive map of the effectors of rab GTPases.

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

Analysis of the Rab5 Interactome to Test the Affinity Chromatography Approach(A) Schematic of the Rab effector isolation protocol.(B) Comparison of the spectral counts for proteins isolated by affinity chromatography with GST alone or GST-tagged GTP-locked Rab5. Colors indicate known Rab5 effectors, endosomal proteins not previously reported to bind Rab5, or typical nonspecific binders in affinity purifications (chaperones, cytoskeletal proteins, etc).(C) Plot of spectral counts versus mRNA levels for each of the Rab5 binding proteins. Coloring is as in (B).(D) Proteins ranked by the total number of spectral counts obtained with GST-Rab5 using detergent lysis, along with their spectral counts in other GST-Rab eluates. Circle area is proportional to the spectral counts. Only the top 70 proteins are shown, with the full list in Table S1A. Known Rab5 effectors (red), other proteins associated with endosomes (red frame), and typical nonspecific binders (blue) are highlighted.(E) Same as in (D), except that the data are not spectral counts but S scores as determined using the CompPASS method (Sowa et al., 2009). Only the top 70 proteins are shown (full list in Table S1B). The S score gives greater weight to proteins binding fewer Rabs and promotes known effectors.(F) Same as in (E), except S score for Rab effectors isolated using detergent-free lysis.(G) Affinity chromatography of lysates from S2 cells expressing GFP-CG6607 or GFP-CG11490 (dTBC1D15) using GDP- and GTP-locked Rab5. Blots were probed with antibodies against GFP.(H) Confocal micrographs of Drosophila S2 cells expressing both GFP-CG6607 and RFP-Rab5. Cells were stained with antibodies against Rab7.Scale bar, 5 μm.See also Figure S1 and Tables S1A and S1B.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4232348&req=5

fig1: Analysis of the Rab5 Interactome to Test the Affinity Chromatography Approach(A) Schematic of the Rab effector isolation protocol.(B) Comparison of the spectral counts for proteins isolated by affinity chromatography with GST alone or GST-tagged GTP-locked Rab5. Colors indicate known Rab5 effectors, endosomal proteins not previously reported to bind Rab5, or typical nonspecific binders in affinity purifications (chaperones, cytoskeletal proteins, etc).(C) Plot of spectral counts versus mRNA levels for each of the Rab5 binding proteins. Coloring is as in (B).(D) Proteins ranked by the total number of spectral counts obtained with GST-Rab5 using detergent lysis, along with their spectral counts in other GST-Rab eluates. Circle area is proportional to the spectral counts. Only the top 70 proteins are shown, with the full list in Table S1A. Known Rab5 effectors (red), other proteins associated with endosomes (red frame), and typical nonspecific binders (blue) are highlighted.(E) Same as in (D), except that the data are not spectral counts but S scores as determined using the CompPASS method (Sowa et al., 2009). Only the top 70 proteins are shown (full list in Table S1B). The S score gives greater weight to proteins binding fewer Rabs and promotes known effectors.(F) Same as in (E), except S score for Rab effectors isolated using detergent-free lysis.(G) Affinity chromatography of lysates from S2 cells expressing GFP-CG6607 or GFP-CG11490 (dTBC1D15) using GDP- and GTP-locked Rab5. Blots were probed with antibodies against GFP.(H) Confocal micrographs of Drosophila S2 cells expressing both GFP-CG6607 and RFP-Rab5. Cells were stained with antibodies against Rab7.Scale bar, 5 μm.See also Figure S1 and Tables S1A and S1B.
Mentions: Drosophila melanogaster has 27 Rabs, of which 23 have at least one mammalian ortholog, with these mammalian orthologs representing 50 of the 66 human Rabs (Figures S1A and S1B available online). All 23 Drosophila Rabs that have a mammalian ortholog were expressed as fusions to glutathione S-transferase (GST) with the Q→L mutation that is known in several Rabs to stabilize the GTP-bound form (Hyvola et al., 2006; Li and Stahl, 1993). They were coupled to glutathione Sepharose for affinity chromatography of lysates of Drosophila S2 cells, a widely used cell line thought to be from a macrophage-like lineage (Figure 1A). S2 cells were lysed in the detergent CHAPS, and the clarified lysate was applied to each GST-Rab column. After washing, proteins were eluted and separated on SDS gels. The gel lanes above the GST-Rab (∼45–50 kDa) were cut into sections and digested with trypsin, and peptides were sequenced by tandem mass spectrometry (Table S1).

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