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Identification of molecular heterogeneity in SNX27-retromer-mediated endosome-to-plasma-membrane recycling.

McGough IJ, Steinberg F, Gallon M, Yatsu A, Ohbayashi N, Heesom KJ, Fukuda M, Cullen PJ - J. Cell. Sci. (2014)

Bottom Line: Depletion of these proteins resulted in trafficking defects of retromer-dependent cargo, but differential and cargo-specific effects suggested a surprising degree of functional heterogeneity in retromer-mediated endosome-to-plasma-membrane sorting.Suppression of VPS35 led to mistrafficking of the melanogenic enzymes, tyrosinase and tryrosine-related protein 1 (Tyrp1), establishing that retromer acts in concert with VARP in this trafficking pathway.Overall, these data reveal hidden complexities in retromer-mediated sorting and open up new directions in our molecular understanding of this essential sorting complex.

View Article: PubMed Central - PubMed

Affiliation: The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.

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Identification of the VPS35 interactome using a quantitative SILAC proteomic approach reveals novel endosomally localized retromer interactors. (A) Lysates from control RPE1 cells and RPE1 cells with stable suppression of endogenous VPS35 (using an shRNA lentivirus targeting the 3′ UTR), prior to the stable re-expression of GFP–VPS35, were immunoblotted with anti-VPS35 and anti-tubulin antibodies. (B) Gene Ontology annotations revealed a preponderance of proteins involved in ‘establishment of localization’, ‘localization’ and ‘transport’ in the VPS35 interactome. DAVID was used to assign Gene Ontology annotations to proteins identified in the VPS35 SILAC proteomics with a >2.5-fold enrichment and with a minimum of two peptides. The larger the red node, the greater the number of proteins classified in that category; the thicker the edge between nodes, the greater the overlap of proteins within those classifications. (C) The majority of known retromer interactors were found in the VPS35 interactome. Network analysis of VPS35 interactome components identified in the SILAC proteomics was performed using the STRING database. Colours represent protein–protein interactions or protein complexes known to associate. (D) Novel interactions were confirmed by western blotting. Cell extracts derived from RPE1 cells lentivirally transduced with GFP or GFP–VPS35 were subjected to a GFP-nanotrap (GFP-IP) and subsequently analyzed for binding to the indicated proteins. The number of peptides and fold enrichment of the indicated proteins in the VPS35 SILAC proteomics are also indicated. (E) VARP, SDCCAG3 and RME-8 colocalize with VPS35. HeLa cells transiently transfected with VARP–Myc and untransfected HeLa cells were fixed and stained with antibodies raised against Myc, RME-8 or SDCCAG3 and co-stained with an antibody against endogenous VPS35 (red). Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm.
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f01: Identification of the VPS35 interactome using a quantitative SILAC proteomic approach reveals novel endosomally localized retromer interactors. (A) Lysates from control RPE1 cells and RPE1 cells with stable suppression of endogenous VPS35 (using an shRNA lentivirus targeting the 3′ UTR), prior to the stable re-expression of GFP–VPS35, were immunoblotted with anti-VPS35 and anti-tubulin antibodies. (B) Gene Ontology annotations revealed a preponderance of proteins involved in ‘establishment of localization’, ‘localization’ and ‘transport’ in the VPS35 interactome. DAVID was used to assign Gene Ontology annotations to proteins identified in the VPS35 SILAC proteomics with a >2.5-fold enrichment and with a minimum of two peptides. The larger the red node, the greater the number of proteins classified in that category; the thicker the edge between nodes, the greater the overlap of proteins within those classifications. (C) The majority of known retromer interactors were found in the VPS35 interactome. Network analysis of VPS35 interactome components identified in the SILAC proteomics was performed using the STRING database. Colours represent protein–protein interactions or protein complexes known to associate. (D) Novel interactions were confirmed by western blotting. Cell extracts derived from RPE1 cells lentivirally transduced with GFP or GFP–VPS35 were subjected to a GFP-nanotrap (GFP-IP) and subsequently analyzed for binding to the indicated proteins. The number of peptides and fold enrichment of the indicated proteins in the VPS35 SILAC proteomics are also indicated. (E) VARP, SDCCAG3 and RME-8 colocalize with VPS35. HeLa cells transiently transfected with VARP–Myc and untransfected HeLa cells were fixed and stained with antibodies raised against Myc, RME-8 or SDCCAG3 and co-stained with an antibody against endogenous VPS35 (red). Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm.

Mentions: To gain new and unbiased insight into the mechanistic basis of retromer-mediated endosomal sorting, we sought to obtain a quantitative high-resolution interactome of human VPS35, the core component of the retromer CSC (Seaman et al., 1998). To this end, we first generated an RPE1 cell line in which the expression of endogenous VPS35 was stably suppressed through lentiviral short hairpin (sh)RNA-mediated targeting of the 3′UTR. Into this cell line, we stably re-expressed GFP-tagged VPS35 at close to endogenous levels (Fig. 1A). The resultant cell line was cultured in heavy SILAC medium alongside an RPE1 cell line stably expressing GFP that was cultured in light SILAC medium. Following cellular lysis, GFP was precipitated using the highly efficient GFP-trap method (Trinkle-Mulcahy et al., 2008). Precipitates were combined, separated by SDS-PAGE and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-gel tryptic digestion. From 2530 quantified proteins (supplementary material Table S1), 63 proteins were considered to comprise the VPS35 interactome after filtering of the data by two criteria – protein quantification from two or more peptides and an enrichment of >2.5-fold over control. Gene ontology analysis revealed that the majority of these interactors had roles in ‘establishment of localization’, ‘localization’ and ‘transport’ (Fig. 1B). Network analysis (Fig. 1C) confirmed the presence of the known VPS35-interacting partners VPS26A and VPS26B, VPS29, TBC1D5 (Harbour et al., 2010), SNX27 (Steinberg et al., 2013) and all components of the actin polymerizing WASH complex (Derivery et al., 2009; Gomez and Billadeau, 2009; Harbour et al., 2010; Harbour et al., 2012; Jia et al., 2012; Hao et al., 2013). The lack of enrichment of the SNX-BAR components of the SNX-BAR–retromer is entirely consistent with the apparent low affinity of this interaction [the interaction of VPS29 with SNX1 has a Kd of >150 µM (Swarbrick et al., 2011)].


Identification of molecular heterogeneity in SNX27-retromer-mediated endosome-to-plasma-membrane recycling.

McGough IJ, Steinberg F, Gallon M, Yatsu A, Ohbayashi N, Heesom KJ, Fukuda M, Cullen PJ - J. Cell. Sci. (2014)

Identification of the VPS35 interactome using a quantitative SILAC proteomic approach reveals novel endosomally localized retromer interactors. (A) Lysates from control RPE1 cells and RPE1 cells with stable suppression of endogenous VPS35 (using an shRNA lentivirus targeting the 3′ UTR), prior to the stable re-expression of GFP–VPS35, were immunoblotted with anti-VPS35 and anti-tubulin antibodies. (B) Gene Ontology annotations revealed a preponderance of proteins involved in ‘establishment of localization’, ‘localization’ and ‘transport’ in the VPS35 interactome. DAVID was used to assign Gene Ontology annotations to proteins identified in the VPS35 SILAC proteomics with a >2.5-fold enrichment and with a minimum of two peptides. The larger the red node, the greater the number of proteins classified in that category; the thicker the edge between nodes, the greater the overlap of proteins within those classifications. (C) The majority of known retromer interactors were found in the VPS35 interactome. Network analysis of VPS35 interactome components identified in the SILAC proteomics was performed using the STRING database. Colours represent protein–protein interactions or protein complexes known to associate. (D) Novel interactions were confirmed by western blotting. Cell extracts derived from RPE1 cells lentivirally transduced with GFP or GFP–VPS35 were subjected to a GFP-nanotrap (GFP-IP) and subsequently analyzed for binding to the indicated proteins. The number of peptides and fold enrichment of the indicated proteins in the VPS35 SILAC proteomics are also indicated. (E) VARP, SDCCAG3 and RME-8 colocalize with VPS35. HeLa cells transiently transfected with VARP–Myc and untransfected HeLa cells were fixed and stained with antibodies raised against Myc, RME-8 or SDCCAG3 and co-stained with an antibody against endogenous VPS35 (red). Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm.
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f01: Identification of the VPS35 interactome using a quantitative SILAC proteomic approach reveals novel endosomally localized retromer interactors. (A) Lysates from control RPE1 cells and RPE1 cells with stable suppression of endogenous VPS35 (using an shRNA lentivirus targeting the 3′ UTR), prior to the stable re-expression of GFP–VPS35, were immunoblotted with anti-VPS35 and anti-tubulin antibodies. (B) Gene Ontology annotations revealed a preponderance of proteins involved in ‘establishment of localization’, ‘localization’ and ‘transport’ in the VPS35 interactome. DAVID was used to assign Gene Ontology annotations to proteins identified in the VPS35 SILAC proteomics with a >2.5-fold enrichment and with a minimum of two peptides. The larger the red node, the greater the number of proteins classified in that category; the thicker the edge between nodes, the greater the overlap of proteins within those classifications. (C) The majority of known retromer interactors were found in the VPS35 interactome. Network analysis of VPS35 interactome components identified in the SILAC proteomics was performed using the STRING database. Colours represent protein–protein interactions or protein complexes known to associate. (D) Novel interactions were confirmed by western blotting. Cell extracts derived from RPE1 cells lentivirally transduced with GFP or GFP–VPS35 were subjected to a GFP-nanotrap (GFP-IP) and subsequently analyzed for binding to the indicated proteins. The number of peptides and fold enrichment of the indicated proteins in the VPS35 SILAC proteomics are also indicated. (E) VARP, SDCCAG3 and RME-8 colocalize with VPS35. HeLa cells transiently transfected with VARP–Myc and untransfected HeLa cells were fixed and stained with antibodies raised against Myc, RME-8 or SDCCAG3 and co-stained with an antibody against endogenous VPS35 (red). Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm.
Mentions: To gain new and unbiased insight into the mechanistic basis of retromer-mediated endosomal sorting, we sought to obtain a quantitative high-resolution interactome of human VPS35, the core component of the retromer CSC (Seaman et al., 1998). To this end, we first generated an RPE1 cell line in which the expression of endogenous VPS35 was stably suppressed through lentiviral short hairpin (sh)RNA-mediated targeting of the 3′UTR. Into this cell line, we stably re-expressed GFP-tagged VPS35 at close to endogenous levels (Fig. 1A). The resultant cell line was cultured in heavy SILAC medium alongside an RPE1 cell line stably expressing GFP that was cultured in light SILAC medium. Following cellular lysis, GFP was precipitated using the highly efficient GFP-trap method (Trinkle-Mulcahy et al., 2008). Precipitates were combined, separated by SDS-PAGE and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-gel tryptic digestion. From 2530 quantified proteins (supplementary material Table S1), 63 proteins were considered to comprise the VPS35 interactome after filtering of the data by two criteria – protein quantification from two or more peptides and an enrichment of >2.5-fold over control. Gene ontology analysis revealed that the majority of these interactors had roles in ‘establishment of localization’, ‘localization’ and ‘transport’ (Fig. 1B). Network analysis (Fig. 1C) confirmed the presence of the known VPS35-interacting partners VPS26A and VPS26B, VPS29, TBC1D5 (Harbour et al., 2010), SNX27 (Steinberg et al., 2013) and all components of the actin polymerizing WASH complex (Derivery et al., 2009; Gomez and Billadeau, 2009; Harbour et al., 2010; Harbour et al., 2012; Jia et al., 2012; Hao et al., 2013). The lack of enrichment of the SNX-BAR components of the SNX-BAR–retromer is entirely consistent with the apparent low affinity of this interaction [the interaction of VPS29 with SNX1 has a Kd of >150 µM (Swarbrick et al., 2011)].

Bottom Line: Depletion of these proteins resulted in trafficking defects of retromer-dependent cargo, but differential and cargo-specific effects suggested a surprising degree of functional heterogeneity in retromer-mediated endosome-to-plasma-membrane sorting.Suppression of VPS35 led to mistrafficking of the melanogenic enzymes, tyrosinase and tryrosine-related protein 1 (Tyrp1), establishing that retromer acts in concert with VARP in this trafficking pathway.Overall, these data reveal hidden complexities in retromer-mediated sorting and open up new directions in our molecular understanding of this essential sorting complex.

View Article: PubMed Central - PubMed

Affiliation: The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.

Show MeSH
Related in: MedlinePlus