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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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Suppression of the novel interactors identified in the VPS35 SILAC proteomics screen affects retromer-mediated endosome-to-plasma-membrane transport. (A) Western blot analysis of lysates derived from HeLa cells that were transfected with siRNAs against the indicated targets. Tubulin is shown as a loading control. (B,C) VPS35, ANKRD50 and FAM21 suppression leads to an increase in the lysosomal accumulation of GLUT1. Immunofluorescent staining of endogenous GLUT1 and the lysosomal marker LAMP1 in HeLa cells deficient for VPS35, FAM21, RME-8, SDCCAG3, VARP or ANKRD50 (B). Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm. (C) The data show the mean±s.e.m. (150 cells acquired in three independent experiments, n = 3); *P<0.05 (unpaired Student's t-test). (D,E) Surface levels of MCT1 and GLUT1 are decreased upon suppression of certain retromer interactors. HeLa cells were transfected with the indicated siRNAs and the surface abundance of GLUT1 and MCT1 was determined by quantitative western blotting (D). The abundance of GLUT1 in total cell lysates is also shown. (E) Graphical representation of the loss of GLUT1 and MCT1 from the surface of HeLa cells transfected with the indicated siRNAs. Data show the mean±s.e.m. [three (MCT1) and six (GLUT1) independent experiments]; *P<0.05 (unpaired Student's t-test).
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f02: Suppression of the novel interactors identified in the VPS35 SILAC proteomics screen affects retromer-mediated endosome-to-plasma-membrane transport. (A) Western blot analysis of lysates derived from HeLa cells that were transfected with siRNAs against the indicated targets. Tubulin is shown as a loading control. (B,C) VPS35, ANKRD50 and FAM21 suppression leads to an increase in the lysosomal accumulation of GLUT1. Immunofluorescent staining of endogenous GLUT1 and the lysosomal marker LAMP1 in HeLa cells deficient for VPS35, FAM21, RME-8, SDCCAG3, VARP or ANKRD50 (B). Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm. (C) The data show the mean±s.e.m. (150 cells acquired in three independent experiments, n = 3); *P<0.05 (unpaired Student's t-test). (D,E) Surface levels of MCT1 and GLUT1 are decreased upon suppression of certain retromer interactors. HeLa cells were transfected with the indicated siRNAs and the surface abundance of GLUT1 and MCT1 was determined by quantitative western blotting (D). The abundance of GLUT1 in total cell lysates is also shown. (E) Graphical representation of the loss of GLUT1 and MCT1 from the surface of HeLa cells transfected with the indicated siRNAs. Data show the mean±s.e.m. [three (MCT1) and six (GLUT1) independent experiments]; *P<0.05 (unpaired Student's t-test).

Mentions: In HeLa cells, RNAi-mediated suppression of VPS35, FAM21, VARP, SDCCAG3, RME-8 and ANKRD50 was highly efficient (Fig. 2A). In control cells, GLUT1 was mostly localized to the plasma membrane, indicating efficient recycling (Fig. 2B). Like suppression of VPS35, suppression of ANKRD50 resulted in a loss of GLUT1 expression at the cell surface, which corresponded to an increase in GLUT1 localization to a LAMP1-labeled lysosomal compartment (Fig. 2B,C). Moreover, suppression of ANKRD50 led to a decrease in surface expression of GLUT1, as determined through a surface biotinylation protocol, and a decrease in the whole-cell level of the transporter (Fig. 2D,E). Entirely consistent with a missorting of internalized GLUT1 into the lysosomal-degradative pathway, the observed decrease in the whole-cell level of the GLUT1 was reversed by treating ANKRD50-suppressed cells with bafilomycin to inhibit lysosomal degradation (Fig. 3B). Finally, and to be discussed in more detail later, suppression of ANKRD50 also phenocopied a loss of SNX27–retromer function in the endosome-to-plasma-membrane sorting of MCT1, CD97 and TRAILR1. Taken together, these data identify a role for ANKRD50 in the endosome-to-plasma-membrane sorting of multiple cargos and provide evidence that, mechanistically, its role in this process is linked with the SNX27–retromer sorting complex.


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)

Suppression of the novel interactors identified in the VPS35 SILAC proteomics screen affects retromer-mediated endosome-to-plasma-membrane transport. (A) Western blot analysis of lysates derived from HeLa cells that were transfected with siRNAs against the indicated targets. Tubulin is shown as a loading control. (B,C) VPS35, ANKRD50 and FAM21 suppression leads to an increase in the lysosomal accumulation of GLUT1. Immunofluorescent staining of endogenous GLUT1 and the lysosomal marker LAMP1 in HeLa cells deficient for VPS35, FAM21, RME-8, SDCCAG3, VARP or ANKRD50 (B). Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm. (C) The data show the mean±s.e.m. (150 cells acquired in three independent experiments, n = 3); *P<0.05 (unpaired Student's t-test). (D,E) Surface levels of MCT1 and GLUT1 are decreased upon suppression of certain retromer interactors. HeLa cells were transfected with the indicated siRNAs and the surface abundance of GLUT1 and MCT1 was determined by quantitative western blotting (D). The abundance of GLUT1 in total cell lysates is also shown. (E) Graphical representation of the loss of GLUT1 and MCT1 from the surface of HeLa cells transfected with the indicated siRNAs. Data show the mean±s.e.m. [three (MCT1) and six (GLUT1) independent experiments]; *P<0.05 (unpaired Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231307&req=5

f02: Suppression of the novel interactors identified in the VPS35 SILAC proteomics screen affects retromer-mediated endosome-to-plasma-membrane transport. (A) Western blot analysis of lysates derived from HeLa cells that were transfected with siRNAs against the indicated targets. Tubulin is shown as a loading control. (B,C) VPS35, ANKRD50 and FAM21 suppression leads to an increase in the lysosomal accumulation of GLUT1. Immunofluorescent staining of endogenous GLUT1 and the lysosomal marker LAMP1 in HeLa cells deficient for VPS35, FAM21, RME-8, SDCCAG3, VARP or ANKRD50 (B). Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm. (C) The data show the mean±s.e.m. (150 cells acquired in three independent experiments, n = 3); *P<0.05 (unpaired Student's t-test). (D,E) Surface levels of MCT1 and GLUT1 are decreased upon suppression of certain retromer interactors. HeLa cells were transfected with the indicated siRNAs and the surface abundance of GLUT1 and MCT1 was determined by quantitative western blotting (D). The abundance of GLUT1 in total cell lysates is also shown. (E) Graphical representation of the loss of GLUT1 and MCT1 from the surface of HeLa cells transfected with the indicated siRNAs. Data show the mean±s.e.m. [three (MCT1) and six (GLUT1) independent experiments]; *P<0.05 (unpaired Student's t-test).
Mentions: In HeLa cells, RNAi-mediated suppression of VPS35, FAM21, VARP, SDCCAG3, RME-8 and ANKRD50 was highly efficient (Fig. 2A). In control cells, GLUT1 was mostly localized to the plasma membrane, indicating efficient recycling (Fig. 2B). Like suppression of VPS35, suppression of ANKRD50 resulted in a loss of GLUT1 expression at the cell surface, which corresponded to an increase in GLUT1 localization to a LAMP1-labeled lysosomal compartment (Fig. 2B,C). Moreover, suppression of ANKRD50 led to a decrease in surface expression of GLUT1, as determined through a surface biotinylation protocol, and a decrease in the whole-cell level of the transporter (Fig. 2D,E). Entirely consistent with a missorting of internalized GLUT1 into the lysosomal-degradative pathway, the observed decrease in the whole-cell level of the GLUT1 was reversed by treating ANKRD50-suppressed cells with bafilomycin to inhibit lysosomal degradation (Fig. 3B). Finally, and to be discussed in more detail later, suppression of ANKRD50 also phenocopied a loss of SNX27–retromer function in the endosome-to-plasma-membrane sorting of MCT1, CD97 and TRAILR1. Taken together, these data identify a role for ANKRD50 in the endosome-to-plasma-membrane sorting of multiple cargos and provide evidence that, mechanistically, its role in this process is linked with the SNX27–retromer sorting complex.

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