Identification of molecular heterogeneity in SNX27-retromer-mediated endosome-to-plasma-membrane recycling.
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.
Affiliation: The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.Show MeSH
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Mentions: In contrast to the subtle effects on GLUT1, subsequent examination of the additional SNX27–retromer cargos MCT1, CD97 and TRAILR1 revealed a functional link between retromer and VARP. Suppression of VARP and, as published previously, VPS35 (Steinberg et al., 2013) led to a pronounced reduction in cell-surface and total levels of MCT1, as defined by western blot analysis (Fig. 2D,E) and immunofluorescence (Fig. 4A). Biochemical analysis of the degradation kinetics of MCT1 established that the reduced abundance of this transporter was due to an enhanced degradation rate in VARP-suppressed cells (Fig. 4B). This was specific for MCT1, as GLUT1 and the transferrin receptor displayed normal degradation rates in parallel assays (Fig. 4B). Further analysis of VARP-suppressed cells, employing flow cytometry and imaging-based assays, also revealed greatly reduced cell-surface and total levels of CD97 (Fig. 5Aiii,B,Civ) and TRAILR1 (Fig. 6Aiii,B,Civ), and TRAILR1 uptake assays using an antibody against an exofacial epitope of this receptor also revealed an increase in the lysosomal accumulation of internalized TRAILR1 (supplementary material Fig. S1Aiv). All of these data are indicative of endosomal missorting leading to an enhanced degradation for these SNX27–retromer cargos under VARP-suppressed conditions.
Affiliation: The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.