Fig. 4: TMEM9 and TMEM9B are core subunits of endosomal CLCN3, CLCN4 and CLCN5 Cl⁻–H⁺ antiporters.

a, EndoMAP.v1 interactions for CLCN3, CLCN4, CLCN5, TMEM9 and TMEM9B. Diamonds and circular nodes represent endosomal and other proteins, respectively. Solid and dashed edges represent interactions identified by at least one crosslink or only co-fractionation, respectively. b, BN–MS profiling for CLCN3, CLCN4, CLCN5, TMEM9 and TMEM9B. c, AF-M predictions for CLCN3–TMEM9 pair and heterotetramer. The locations of DSSO crosslinks are indicated with the red line and arrowhead. d,e, Co-localization analysis of TMEM9–GFP and mCh–CLCN3 in SUM159 cells by live-cell imaging. Mander’s coefficients of GFP and mCh puncta are shown in e (n = 39 in 3 independent replicates, mean ± s.e.m.), with an example of a cell shown in d. f, Mander’s coefficient analysis of co-localization between TMEM9–GFP, mCh–CLCN3, anti-EEA1 and anti-LAMP1 in fixed SUM59 cells as determined by immunofluorescence. The number of fields of view across three biological replicates is indicated (mean ± s.e.m.) and P values from linear mixed-effects model analysis of variance. g, Example of TMEM9–GFP, mCh–CLCN3 and anti-EEA1 staining in a cell expressing high levels of CLCN3 (left panels), which promotes the formation of swollen endolysosomes. Traces of the white line in the bottom panel show the overlap of the three proteins in the limiting membrane of endosomes (right panel). h, Volcano plot showing the proteomic analysis of anti-HA IPs from TMEM9^−/− iNeurons with or without lentiviral expression of TMEM9–HA (n = 4 biologically independent replicates). i, Schematic of experimental design for proteomic analysis of early endosomes and PNS in 21-day iNeurons derived from WT cells, TMEM9^−/− cells and two different clones of TMEM9^−/−TMEM9B^−/− (DKO) cells in biological triplicate (Supplementary Table 5). j, Volcano plot showing the proteomic analysis of Endo-IPs from TMEM9^−/−TMEM9B^−/− (DKO clone 2) versus WT iNeurons (day 21; n = 3 biologically independent replicates). CTSF, cathepsin F. k, TMT reporter signal intensity for CLCN3, CLCN5, TMEM9 and TMEM9B in Endo-IPs from iNeurons with the indicated genotypes (n = 3 biologically independent replicates). DKO1, TMEM9^−/−TMEM9B^−/− (clone 1); DKO2, TMEM9^−/−TMEM9B^−/− (clone 2). Scale bars (d and g), 5µm. Panel i adapted from ref. ⁴⁴, CC BY 4.0; illustration of MS machine from NIAID NIH BioArt Source.

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