Extended Data Fig. 8: zSec14l3 and hSEC14L2 promote lipid transfer and VPS34 kinase activity in vitro.
From: A Golgi-derived vesicle potentiates PtdIns4P to PtdIns3P conversion for endosome fission
a, Cartoon schematic of PI3P/PI4P extraction assays. NBD-PHFAPP protein was mixed with PI3P- or PI4P-containing liposomes with or without DGS-NiNTA in the presence or absence of 3 µM His-zSec14l3 protein. Fluorescence spectra of NBD-PHFAPP were obtained after the addition of proteins at 5 min, 15 min, and 30 min. The percentage of PI3P/PI4P extraction was calculated according to the fluorescence intensity at 540 nm. b, Quantification of PI3P/PI4P extraction by zSec14l3. The lipid extraction assay was performed as in (a) and the amount of extracted PI3P/PI4P was calculated. Data are presented as the mean ± SD from 3 independent experiments. Unpaired, Two-tailed student’s t-test. ***p = 0.0008 and *p < 0.05. c, LC-MS/MS-based PI3P or PI4P transfer assay in vitro. 200 nM hSEC14L2-His protein was incubated for 15 min at 25 °C. d, Quantification of in vitro PI3P or PI4P transfer efficiency by hSEC14L2 protein. The in vitro transfer assay was performed as shown in (c) and acceptor liposomes recovered for LC-MS/MS analysis to quantify acquired PI3P or PI4P content. Data are presented as the mean ± SD of three independent experiments. Unpaired, Two-tailed student’s t-test was used. **p = 0.003 (PI3P) and **p = 0.002 (PI4P). e, Schematic of the liposome-based fusion assay. The change in fluorescence intensity (ΔF = Ft-F0) could reflect the liposome fusion percentage. f, Quantification of fluorescence changes when excited at 460 nm in the liposome-based fusion assay with addition of zSec14l3 or hSEC14L2 protein. Data are presented as the mean ± SD of three independent experiments. Unpaired, Two-tailed student’s t-test was used. ***p = 3.02E-05 and *p = 0.029. g, VPS34 interacts with zSec14l3 and its lipid binding deficient form zSec14l3-M5. h, Quantification of VPS34 catalyzed PI3P content in the presence of zSec14l3 or zSec14l3-M5. SEC14L2 KO COS-7 cells were transfected as indicated and then cells were lysed for immunoprecipitation using anti-EGFP antibody. The immunoprecipitated pellets were used for in vitro kinase assays and lipid extracted for LC-MS/MS. Data are presented as the mean ± SD of three independent experiments. Unpaired, Two-tailed student’s t-test was used. *p = 0.031 (M5), **p = 0.005 (WT).
