Extended Data Fig. 5: Interface variants disrupt interaction of TMEM230 with endosomal P4 lipid flippases ATP11B and ATP8A1/2.

a, Individual and overlay AF-M and AlphaLink2 predictions for TMEM230 and ATP11B. AF-M: TMEM230 (light blue), ATP11B (cyan), cross-link (red line and arrowhead). AlphaLink2: TMEM230 (dark blue), ATP11B (teal), cross-link (wheat line and arrowhead). b, Overlay of yeast DNF1-LEM3 structure (PDB:7DRX) in the EP2 conformation with AF-M prediction for ATP11B-TMEM30A-TMEM230. c, Co-precipitation of Flag-ATP11B and TMEM30A-V5 with HA-TMEM230. The indicated plasmids were transfected into HEK293 cells and α-HA immunoprecipitates or input samples were immunoblotted for the indicated proteins. Black dots indicate proteins expressed in each sample. d, Sequence validation of TMEM230^-/- and TMEM230^X121W clones in H9^{AAVS1-NGN2;Flag-EEA1} cells (H9-Flag-EEA1), showing the location of the sgRNA used (green) and base pairs deleted to create an out of frame mutation and point mutation, respectively. e, Immunoblot of total cell lysates from the indicated H9-Flag-EEA1 cell lines probed with α-TMEM230. The X121W mutation adds a six-residue extension (WHPPHS), which can be detected as a band with slightly higher molecular weight. Stain-free gel was used to indicate equal loading of extracts. f, Volcano plots (log₂FC relative to TMEM230^-/- cells) of TMEM230 immunoprecipitations in H9-TMEM230^-/- iNeurons with or without lentiviral expression of WT and interface variant HA-TMEM230 proteins. g, Mass spectrometry (MS) TMT reporter signal for ATP11B and TMEM30A in the indicated TMEM230 variant immunoprecipitation from iNeurons. Dots indicate individual biological replicates (n = 2, except n = 3 for Control given the limitation of the maximum number of TMT channels). h, Immunoblots of total cell extracts from TMEM230^-/- iNeurons transduced with lentiviruses expressing the indicated variants of HA-TMEM230 protein. Stain-free gel was used as loading control. i, AF-M prediction for a TMEM230-ATP8A1-TMEM30A complex (Y29, R78, and C-terminal D120-D121, purple space fill). The location of a cross-link between ATP8A1 and TMEM30A is indicated by the red line and arrowhead. ipTM = 0.74 for ATP8A1-TMEM230 prediction. j, Volcano plot for Endo-IP proteomic analysis from H9-Flag-EEA1 iNeurons (21 days) (n = 3 biologically independent replicates). Proteins annotated as endosomal (green), lysosomal (blue), or plasma membrane (PM, orange) are indicated. k, Immunofluorescence microscopy showing the colocalization of Flag-EEA1 (green) with RAB5 (magenta) in iNeurons from H9-Flag-EEA1 cells. l, Violin plot showing the fold-change enrichment (log₂) of proteins from individual organelle compartments (color-coded as panel j) in Endo-IP samples from H9-Flag-EEA1 iNeurons (day 21). m, Immunoblots of Endo-IP or input samples (PNS) from H9-Flag-EEA1 iNeurons and untagged H9 control (21 days). Blots were probed with the indicated antibodies.