Extended Data Fig. 5: PI4K2A-mediated PtdIns4P signaling recruits ORP9, ORP10, and ORP11 to damaged lysosomes.

a, Antibody verification by western blot using CRISPR knockout pools. Asterisk indicates a nonspecific band. b, ORP10 is mainly found in the P20 fraction and absent from the S20 fraction. P20, pellet after 20,000 xg centrifugation. S20, supernatant after 20,000 xg centrifugation. Asterisk indicates nonspecific bands. c, LMP induces new puncta of EGFP-ORP9/11 on damaged lysosomes. EGFP-ORP9 and -ORP11 are recruited to puncta outside of the Golgi complex upon lysosomal damage by LLOME (top). LMP-induced EGFP-ORP9 puncta colocalize with LAMP1 and IST1 (bottom). d, Endogenous ORP9 and stably expressed EGFP-ORP11 completely colocalize when forming puncta in response to lysosomal damage by LLOME. e, EGFP-ORP11 colocalizes with both PI4K2A and LAMP1 upon lysosomal damage. f, ORP9, but not EGFP-ORP3, forms puncta upon lysosomal damage. g, EGFP-ORP5 is not recruited to damaged lysosomes. Note that the ORP5 punctate structures observed here are not random diffuse signals; they are morphologically distinct from the diffuse signal of ORP3 in (f). When the confocal images were captured at different planes of the cell, the ORP5 structures appear dramatically different³¹. If the focus plane is away from the bottom of the cell, ORP5 appears as punctate structures on the PM with only diffuse signal in the cytosol. However, when the plane is near the bottom of the cell, as is the case here, ORP5 appears as extensive puncta throughout the cells corresponding to ER-plasma membrane contact sites at the bottom. h, i, Knockdown of PI4K2A by RNA interference (RNAi) blocks the recruitment of EGFP-ORP9 (h) and EGFP-ORP11 (i) to damaged lysosomes. U2OS cells stably expressing EGFP-ORP9 or EGFP-ORP11 were transfected with indicated siRNAs. After 72 h, cells were treated or not with 1 mM LLOME for 10 min and fixed for co-staining of endogenous LAMP1 and IST1. See Supplementary Table 4 for siRNA sequences. j–l, The kinase activity of PI4K2A is required for the recruitment of EGFP-ORP9/10/11 to damaged lysosomes. EGFP-ORP9 (j), EGFP-ORP10 (k), or EGFP-ORP11 (l) were stably expressed in wild type (WT) and PI4K2A knockout (PI4K2A-KO) cells, as well as in PI4K2A-KO cells re-expressing WT (KO + WT) or kinase dead PI4K2A (KO + KDAA). Cells were treated or not with 1 mM LLOME for 10 min and fixed for immunostaining of endogenous IST1. Note, in the absence of PI4K2A activity, all cells lost the recruitment of ORP9/10/11 to damaged lysosomes, as exemplified by the quantification of endogenous ORP9/11 in Fig. 2c. m, LMP-induced puncta of endogenous ORP9 and ORP11 are dependent on PI4K2A and its kinase activity. Indicated cell lines were treated with LLOME for 10 min and fixed for co-staining of endogenous ORP9 and ORP11. See Fig. 2c for quantification. n, o, ORP9 binding to PtdIns4P through its PH ___domain and to VAPA/B through its FFAT motif are both necessary for its efficient recruitment/accumulation on damaged lysosomes. n, schematic illustration of ORP9 mutants. o, indicated ORP9 mutants no longer accumulate on damaged lysosomes. Note, despite the presence of intact PH ___domain for PtdIns4P binding, the ORP9-FYAA mutant is no longer efficiently recruited to damaged lysosomes, suggesting that PtdIns4P-binding alone is insufficient for stable ORP9 association with lysosomes. p, The LMP-induced lysosomal recruitment of ORP9/10/11 requires PtdIns4P-binding through their PH ___domain. RE indicates the point mutation of a conserved arginine residue required for PtdIns4P-binding to the PH ___domain of all the three ORPs. U2OS cells stably expressing mCherry-ORP9, ORP10, or ORP11 or their RE mutants were treated with LLOME for 10 min and then fixed for immunostaining of endogenous LAMP1. Note, while all RE mutants lost lysosomal targeting downstream of LMP, ORP9/10-RE but not ORP11-RE can still be localized to the Golgi complex in resting conditions, indicating different mechanisms for their basal Golgi targeting. See Supplementary Results and Supplementary Table 3 for more details regarding the Golgi and lysosomal targeting of the ORPs. DAPI stains the nuclei. Bar, 10 μm. Uncropped western blot images are provided in Supplementary Fig. 1.