Extended Data Fig. 7: TMEM9/9B are core subunits of endosomal CLCN3/4/5 Cl−-H+ antiporters.
From: EndoMAP.v1 charts the structural landscape of human early endosome complexes
a, Endosomal score and rank of TMEM9/9B and CLCN3/5/7, with higher values corresponding to endosomal proteins. b, Individual and overlay AF-M and AlphaLink2 predictions for CLCN3-TMEM9. AF-M: TMEM9 (dark blue), CLCN3 (cyan), cross-link (red bar and arrowhead). AlphaLink2: TMEM9 (light blue), CLCN3 (teal), cross-link (wheat bar and arrowhead). c,d AF-M predictions for CLCN3-TMEM9B and selected CLCN-TMEM9/9B heterotetramers. The locations of DSSO cross-links are indicated with the red line and arrowhead. The location of variants found in CLCN5 in Dent’s Disease retrieved from UniProt are shown in red (right, panel c). e, Overlay of the CLCN5-TMEM9 heterotetramer prediction with the CLCN7-OSTM1 heterotetramer structure (PDB: 7JM7). f, Example of TMEM9-GFP, mCh-CLCN3, and α-LAMP1 staining in a cell expressing high levels of CLCN3, which promotes the formation of swollen endolysosomes. Line traces show the overlap of the 3 proteins in the limiting membrane of endolysosomes (bottom right panel). g, Co-precipitation of CLCN3/5-Flag and TMEM9/9B-HA. The indicated plasmids were transfected into HEK293 cells and α-HA or α-Flag immunoprecipitations or input samples were immunoblotted with the indicated antibodies. Loading controls as stain-free gels are shown. Black dots indicate proteins expressed in each sample. h,i, Sequence validation of H9 TMEM9-/- cells, showing the location of the sgRNA used (green) to create frameshift mutations in TMEM9 (panel h) and subsequently, used the indicated sgRNA (green) to create frameshift mutations in TMEM9B (panel i). j, (Left) Volcano plot for Endo-IP proteomic analysis from H9-Flag-EEA1 iNeurons (n = 3 biologically independent replicates). (Right) Violin plot showing the fold-change enrichment (log2) of proteins from individual organelle compartments in Endo-IP from H9-Flag-EEA1 iNeurons. Proteins annotated as endosomal (green), lysosomal (blue), or plasma membrane (PM, orange) are indicated.
