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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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FAM21 suppression leads to an intracellular accumulation of GLUT1 in a SNX1- and transferrin-positive compartment. (A) Immunofluorescent staining of endogenous GLUT1 and SNX1, or GLUT1 and dsRed–transferrin after 1 h uptake in FAM21-deficient HeLa cells. Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm. (B) HeLa cells were transfected with the indicated siRNAs and, 48 h later, were treated with either DMSO or 0.1 µM bafilomycin in DMSO for a further 12 h prior to analysis of GLUT1 levels in total-cell lysates. (C) 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. (D) Immunofluorescent staining of endogenous MCT1 in control and FAM21-deficient HeLa cells. Scale bars: 10 µm.
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f03: FAM21 suppression leads to an intracellular accumulation of GLUT1 in a SNX1- and transferrin-positive compartment. (A) Immunofluorescent staining of endogenous GLUT1 and SNX1, or GLUT1 and dsRed–transferrin after 1 h uptake in FAM21-deficient HeLa cells. Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm. (B) HeLa cells were transfected with the indicated siRNAs and, 48 h later, were treated with either DMSO or 0.1 µM bafilomycin in DMSO for a further 12 h prior to analysis of GLUT1 levels in total-cell lysates. (C) 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. (D) Immunofluorescent staining of endogenous MCT1 in control and FAM21-deficient HeLa cells. Scale bars: 10 µm.

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)

FAM21 suppression leads to an intracellular accumulation of GLUT1 in a SNX1- and transferrin-positive compartment. (A) Immunofluorescent staining of endogenous GLUT1 and SNX1, or GLUT1 and dsRed–transferrin after 1 h uptake in FAM21-deficient HeLa cells. Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm. (B) HeLa cells were transfected with the indicated siRNAs and, 48 h later, were treated with either DMSO or 0.1 µM bafilomycin in DMSO for a further 12 h prior to analysis of GLUT1 levels in total-cell lysates. (C) 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. (D) Immunofluorescent staining of endogenous MCT1 in control and FAM21-deficient HeLa cells. Scale bars: 10 µm.
© Copyright Policy - open-access
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f03: FAM21 suppression leads to an intracellular accumulation of GLUT1 in a SNX1- and transferrin-positive compartment. (A) Immunofluorescent staining of endogenous GLUT1 and SNX1, or GLUT1 and dsRed–transferrin after 1 h uptake in FAM21-deficient HeLa cells. Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm. (B) HeLa cells were transfected with the indicated siRNAs and, 48 h later, were treated with either DMSO or 0.1 µM bafilomycin in DMSO for a further 12 h prior to analysis of GLUT1 levels in total-cell lysates. (C) 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. (D) Immunofluorescent staining of endogenous MCT1 in control and FAM21-deficient HeLa cells. Scale bars: 10 µm.
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