Fig 2: Enrichment of organellar markers in ATG9 and RAB26 vesicles. (A) box plots showing the enrichments of organelle-specific proteins detected in the ATG9 and RAB26 proteome (see Figure S1 for the respective 2D scatter plots). SV, synaptic vesicles (blue), EL, endosomes and lysosomes (orange), PM, plasma membranes (gray), GA, Golgi apparatus (yellow), ER, endoplasmic reticulum (light blue), Mito, mitochondria (green), Px, peroxisomes (dark blue). The Y-axis shows the average log2-fold enrichment (log2 iBAQ ATG9/Input + log2 iBAQ ATG9/control IgG) divided by 2 of the individual proteins of each group. Bottom: graphs representing the fraction (in %) of the proteins listed in the respective organelle database (see text for details) that were recovered in the respective immuno-isolates (the numbers on top of the bars represent the total number of proteins in the organelle proteomes). (B) distribution of selected proteins between vesicles immuno-isolated for SYP and ATG9, respectively. Immuno-isolated fractions were analyzed by immunoblotting for select proteins, confirming MS-based quantification of proteins enriched in ATG9-containing vesicles. These include TMEM9B, M6PR, LC3B, SNAP23, VTI1A, VAMP7, ATG2A and PIP4K2A found in ATG9-containing vesicles compared to Synpatophysin vesicles. See Figure 1 for details. (C) scatter plot showing the proteins enriched > 2-fold in both dimensions, corresponding to a cutoff of log2-transformed iBAQ (ATG9/Control) and (ATG9/Input) >1, for both ATG9 (red, upper graph) and RAB26 (violet, lower graph). (D) Venn diagram of the > 2-fold enriched proteins in the ATG9 (red) and RAB26 (violet) proteome showing that overlap is limited. (E) as in (C) but limited to the recovered organelle-specific proteins, respectively.

Fig 3: PI5P4Ks support cholesterol homeostasis through lysosome positioning and mTOR activation.(A) Free cholesterol staining of Trp53−/− MEFs with filipin. Cells were either non-treated (NT=10% FBS) or treated with an NPC1 inhibitor for 16 hrs (U18666A) to induce cholesterol accumulation. WT=Pip4k2a+/+ Pip4k2b+/+, abDKO=Pip4k2a−/− pip4k2b−/−, aWT=functional PI5P4Ka rescue, aKD=PI5P4Ka kinase dead. Scale bar=50uM. Shown are mean+SD. ANOVA followed by Dunnett’s multiple comparison correction, compared to WT. p=*0.05, **0.01, ***0.001, ****0.0001. (B) Filipin and lysotracker overlap in untreated Trp53−/− MEFs. Scale bar=50uM. Shown is mean+SD. Student’s t-test. p=*0.032 (C) LBPA staining in untreated Trp53−/− MEFs. Shown is mean+SD obtained from CellProfiler analysis. ANOVA followed by Dunnett’s multiple comparison correction, compared to WT. p=*0.05, ***0.001. (D) Lysosome positioning analysis of Trp53−/− MEFs using LAMP1 as a lysosomal marker. Images were acquired with confocal microscopy at 63x magnification (oil) and representative of 5 cross-sections of 0.5uM Z-stacks with maximum intensity projection. Scale bar=10uM. (E) Immunoblot of Trp53−/− MEFs for PI5P4Ks and mTORC1 target S6 in a cholesterol depletion and repletion assay. Sterol depletion was performed using methyl-β-cyclodextrin (MCD, 1% w/v) for 1.5 hours, followed by refeeding for 1.5 hours with 150 μM cholesterol. (F) Immunofluorescence of endogenous mTOR and LAMP2 colocalization in Trp53−/− MEFs. Cholesterol depletion with MCD for 1.5 hr (-Chol) compared to depletion + readdition of cholesterol for 1.5 hr each (+Chol). Images were acquired with confocal microscopy at 63x magnification (oil). Scale bar=10uM. Shown is the mean+SD of the calculated overlap. Unpaired t-test followed by Holm-Sidak correction. P=**0.0037.

Fig 4: PIP4K2A binds specifically with RAD51 3′UTR, and RNA binding activity is independent of kinase activity. (A). PIP4K2A and labeled 3′UTR of RAD51 were incubated at 16°C in 1x GSB for 1 h in the presence or absence of 10-fold excess of unlabeled self- or non-specific competitor. The reaction was then resolved on a 4% polyacrylamide gel for 8 h at 4°C, and the gels were scanned in a LI-COR scanner. The shifted bands were quantified using Image J software, and the mean (± SEM) relative band intensity was plotted (right panel), n = 3; unpaired t-test, one tailed, **** = p < 0.0001, ** = p < 0.01, and ns = p > 0.1. (B). RNA EMSA was performed with RAD51-3ÚTR and varying amounts of PIP4K2A, the band intensity was quantified using Image J software, and the mean (± SEM) ratio of bound to unbound fraction was plotted for each PIP4K2A concentration (n = 3). The value of Kd was obtained by extrapolating 50% bound fraction on the X-axis (PIP4K2A conc.) (C). The predicted secondary structure of RAD51 3′UTR RNA indicating the formation of a stem loop structure by MFold algorithm. The RAD51-short sequence is boxed inside the arrow shape. (D). PIP4K2A and labeled RAD51-short were incubated at 16°C in 1x GSB for 1 h in the presence or absence of 10-fold excess of unlabeled self- or non-specific competitor. The reactions were then resolved on a 1% agarose gel at 4°C, and the gels were scanned in a LI-COR scanner. The shifted bands were quantified using ImageJ software, and the mean (± SEM) relative band intensity was plotted (right panel) (n = 3); unpaired t-test, one tailed, *** = p < 0.001, ** = p < 0.01, and ns = p > 0.1). (E). RNA EMSA was performed with RAD51-short RNA and varying amounts of PIP4K2A, the band intensity was quantified, and the mean (± SEM) ratio of bound to unbound fraction was plotted for each PIP4K2A concentration. The value of Kd was obtained by extrapolating 50% of bound fraction on the X-axis (n = 3). (F). PIP4K2AG131L Y138F, the kinase-dead mutant, and labeled RAD51-short were incubated at 16°C in 1x GSB for 1 h in the presence or absence of 10-fold excess of unlabeled self- or non-specific competitor. The reactions were then resolved on a 1% agarose gel at 4°C, and the gels were scanned in a LI-COR scanner. (G). RNA EMSA was performed with RAD51-short RNA and varying amounts of PIP4K2AG131L Y138F, the band intensity was quantified, and the mean (±SEM) ratio of bound to unbound fraction was plotted for each PIP4K2A G131L Y138F concentration (n = 3). The value of Kd was obtained by extrapolating 50% bound fraction on the X axis.

Fig 5: Validation of Tools to Eliminate All Three PIP4K Isoforms Reveals Paradoxical Increase in PI(4,5)P2(A) Western blots showing the efficiency of knockdown of PIP4K isoforms in HeLa cells. Cell line notation and their descriptions are listed in Table S2.(B) Western blots showing 293T clones with CRISPR-mediated knockout of PIP4K2A, PIP4K2B, and PIP4K2C, with cell line descriptions in Table S2. Parental cell line is in the first lane, and the CRISPR clonal cell line with intact PIP4K is designed as WT. A faint non-specific band that runs slightly faster than PIP4Ks is just visible.(C and D) Quantification by HPLC of PI(5)P, which increases in cells with the loss of PIP4K, shown in HeLa TKD cells (C) as well as 293T TKO cells (D).(E) Knock down of PIP4K enhances insulin signaling in HeLa cells, as shown by western blot. Quantification by Li-Cor indicated the respective bands below.(F) Immunofluorescence detection of lysosomal marker Lamp1 showing increased lysosomal accumulation in 293T TKO versus control cells. Representative images shown.(G and H) PI(4,5)P2 increases in cells with loss of PIP4K, shown in HeLa TKD cells (G) and 293T TKO cells (H), measured by HPLC.(I and J) PI(4)P decreases in cells with loss of PIP4K, shown in HeLa TKD cells (I) and 293T TKO cells (J), measured by HPLC.(K) 293T TKO cells have higher levels of PI(3,4,5)P3, as measured by HPLC.Significance calculated using ANOVA with Holm-Sidak multiple comparisons to control cell line with intact PIP4K. **p < 0.01, ***p < 0.001, and ****p < 0.0001. Data are represented as means ± SEMs. n = 3.