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. 2021 Feb 19;220(4):e202010048. doi: 10.1083/jcb.202010048

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© 2021 Mao et al.

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Figure 6. — Phosphorylation of SNX27 at Ser51 specifically inhibits endocytic recycling of PDZbm-containing cargoes. (A) Starvation decreases endocytic recycling of GLUT1 in cells expressing SNX27, but not Ser51A. Confocal imaging for GLUT1 (green) and phalloidin (red) immunofluorescence staining of SNX27-KO HeLa cells. Cells were transfected with empty vector, GST-tagged SNX27 WT, or Ser51A for 24 h and starved for 4 h (E+B). Representative images from two independent experiments are shown. Scale bars: 10 µm. Pearson’s correlation coefficients for GLUT1 and phalloidin. Results for individual cells are plotted, along with the mean and SD for each group (GST control, n = 52 cells; GST-SNX27-WT control, n = 70; GST-SNX27-WT E+B, n = 74; GST-SNX27-Ser51A control, n = 44; and GST-SNX27-Ser51A E+B, n = 55); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001. (B) SNX27 Ser51D specifically inhibits the recycling of SEMA4C and GLUT1, as indicated by surface protein biotinylation. SNX27-KO HeLa cells were transfected with GST-tagged SNX27 WT, Ser51A, or Ser51D for 24 h, followed by surface biotinylation and immunoblotting using indicated antibodies. Representative immunoblots show the protein levels at the PM and total. In contrast with GLUT1 and SEMA4C, which are transported by SNX27, CI-MPR, and TRAILR1, integrin β1 and integrin α5 are cargoes of SNX-BARs and SNX17, respectively. HSD, honestly significant difference; ns, not significant.