Figure 10.

Knockout of reference isoforms of BDCPs leads to missorting of plasma membrane proteins. (A) The plasma membrane proteome of wild-type HeLa cells and two clones of HeLa cells lacking the reference isoforms of ALFY, LRBA, NBEA, NBEAL1, and NBEAL2 (KO1 and KO2) were isolated and analyzed by mass spectrometry. The Venn diagram and heatmap depict proteins that were significantly up- or downregulated in knock-out clones compared with wild-type cells. (B) Western blot of the indicated proteins from the cell surface proteome and total cell lysates of wild type Hela cells and HeLa cells lacking reference isoforms of ALFY, LRBA, NBEA, NBEAL1, and NBEAL2 (KO1). (C) Knockout of single BDCPs differentially affects the level of proteins identified as up or down-regulated on the plasma membrane of 5KO cells. Western blot of the indicated proteins from total cell lysates of wild-type HeLa and HeLa cells lacking reference isoforms of either ALFY, LRBA, NBEA, NBEAL1, or NBEAL2. (D) Quantification of data in C from three independent experiments. Values shown are mean ± SD. (E) Binding of ALFY to cytosolic tails of selected transmembrane proteins found to be up- or down-regulated in BDCPs 5KO cells. GST-tagged cytosolic tails of CTLA4, EPHA7, EFNB2, ROBO2, FGFR4, FLRT2, JAG1, TGFBR3, or SDK1 were immobilized on glutathione-sepharose beads and incubated with HeLa 3xFlag-EGFP-ALFY cell lysate. Bound proteins were detected by staining with an anti-flag antibody, while GST-tagged proteins were detected by staining with an anti-GST antibody. (F) Quantification of data in C from three independent experiments. Values shown are mean ± SD. (G) Schematic model of BDCPs function as transmembrane cargo sorting adaptors on trans-Golgi network and tubular sorting endosomes/tubular endosomal network. Source data are available for this figure: SourceData F10.