The recruitment of STX17 to autophagosomes is dependent on its positively charged C-terminal region.

(A) Schematic representation of the structures of STX17 and its C-terminal variants. The positively (orange) and negatively (blue) charged residues are shown. Alanine substitutions are shown in green. TMH, transmembrane helix; CTR, C-terminal region.

(B) Schematic representation of the localization of ATG5, LC3B, and STX17 during autophagosome formation and maturation.

(C–E) Mouse embryonic fibroblasts (MEFs) stably expressing mRuby3-LC3B and GFP–STX17TM (containing the two transmembrane helices and the C-terminal region) or its mutants were cultured in starvation medium for 1 h. Bars indicate 10 μm (main images) and 1 μm (insets). Quantification of GFP–STX17TM intensity of mRuby3– LC3B-positive ring-like structures (n > 30) are shown in the graphs. Solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), and whiskers indicate the 5th to 95th percentiles. Differences were statistically analyzed by Welch’s t-test (C) or one-way ANOVA followed by Dunnett’s multiple comparison test (D and E).

Autophagosomes become negatively charged during maturation.

(A) GFP–STX17TM translated in vitro was incubated with rhodamine-labeled liposomes containing the indicated concentrations of phospholipids: 70% phosphatidylcholine (PC), 20% phosphatidylethanolamine (PE), and 10% of either PE, phosphatidylserine (PS), phosphatidylinositol 3-phosphate (PI3P), or phosphatidylinositol 4-phosphate (PI4P). Bars indicate 1 μm. GFP intensities of liposomes are quantified and shown as in Figure 1C (n > 30).

(B) Amino acid sequences of GFP-tagged membrane surface charge probes. The positively charged residues are shown in orange. The farnesylation motif is indicated in green.

(C and D) Mouse embryonic fibroblasts (MEFs) stably expressing one of the GFP-tagged charge probes and mRuby3-STX17TM (C) or mRuby3–LC3B (D) were cultured in starvation medium for 1 h. GFP intensities of mRuby3–STX17TM-positive (C) or mRuby3–LC3B-positive (D) ring-like structures were quantified (n > 70).

(E and F) Time-lapse analysis of MEFs stably expressing the GFP-tagged 1K8Q (E) or 5K4Q (F) charge probes and mRuby3–STX17TM or mRuby3–LC3B cultured in starvation medium. Autophagosomes are indicated by arrows. Bars indicate 1 μm.

(G) Summary of electrostatic dynamics of autophagosome formation.

Solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), and whiskers indicate the 5th to 95th percentiles. Differences were statistically analyzed by one-way ANOVA followed by Sidak’s multiple comparison test (A, C, and D).

Phosphatidylinositol 4-phosphate (PI4P) is enriched in the autophagosomal membrane during maturation.

(A) Mouse embryonic fibroblasts (MEFs) stably expressing GFP–CERT(PHD) and mRuby3–STX17TM or mRuby3–ATG5 were cultured in starvation medium for 1 h. Bars indicate 10 μm (main images) and 1 μm (insets). GFP intensities of mRuby3-positive structures (n > 60) were quantified. Solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), and whiskers indicate the 5th to 95th percentiles. Differences were statistically analyzed by Welch’s t-test.

(B–D) Time-lapse analysis of MEFs stably expressing GFP–CERT(PHD) and mRuby3– ATG5 (B), WIPI2B–mRuby3 (C), or mRuby3–STX17TM and HaloTag7–LC3B (visualized with SaraFluor 650T HaloTag ligand) (D) cultured in starvation medium.

Autophagosomes are indicated by arrows. Bars indicate 1 μm.

STX17 recruitment to autophagosomes depends on phosphatidylinositol 4-phosphate (PI4P) in vitro.

(A) Schematic representation of the in vitro autophagosome recruitment assay. Isolated autophagosomes were mixed with mGFP–STX17TM and either recombinant Sac1-phosphatase domain (Sac1PD) or its phosphatase-dead mutant (C392S).

(B) Isolation of mature autophagosomes prior to their fusion with lysosomes. Homogenates of STX17 knockout HeLa cells stably expressing mRuby3–LC3B cultured in starvation medium at 1 h were separated by the OptiPrep membrane flotation method. The autophagosome-containing fraction (#1: LC3-positive and LAMP1-negative) was collected. The positions of mRuby3–LC3B (black arrowhead) and endogenous LC3B (white arrowhead) are indicated.

(C) Purification of recombinant yeast Sac1 (phosphatase domain, PD) and its phosphatase-dead (C392S) mutant and mGFP–STX17TM from High Five cells.

(D) In vitro autophagosome association assay. Isolated autophagosomes were mixed with recombinant Sac1 (WT or C392S) for 30 min and then with mGFP–STX17TM for another 30 min. Representative images are shown. Bars indicate 2.5 μm. STX17-positivity rates were determined across three independent experiments (two of the three experiments were performed in a blind manner, and 80 autophagosomes were counted in each experiment). Solid horizontal lines indicate means. Differences were statistically analyzed by one-way ANOVA followed by Tukey’s test.

Molecular dynamics simulations of phosphatidylinositol 4-phosphate (PI4P-dependent STX17TM insertion into membranes.

(A and C) An example of a time series of simulated results of STX17TM insertion into a membrane consisting of 70% phosphatidylcholine (PC), 20% phosphatidylethanolamine (PE), and 10% PI4P (POPI14) (A) or 70% PC and 30% PE (C). STX17TM is shown in blue. Phosphorus in POPC, POPE, and PI4P are indicated by yellow, cyan, and red, respectively. Short-tailed lipids are represented as green sticks. The time evolution series of (A) is shown in Video 1.

(B and D) Time evolution of the z-coordinate of the center of mass (Zcm) of the transmembrane helices of STX17TM in the case of membranes with (B) and without (D) PI4P. Five independent simulation results are represented by solid lines of different colors. The gray dashed lines indicate the locations of the lipid heads. The time evolution series of (B) is shown in Video 2.

(E) Model of the PI4P-driven electrostatic maturation of the autophagosome and STX17 recruitment.

Recruitment of STX17 depends on the abundance of cationic amino acids in the C-terminal region but not on its specific amino acid sequence.

(A) Multiple sequence alignment of STX17 proteins from Homo sapiens (Hs), Mus musculus, Danio rerio, Ciona intestinalis, Drosophila melanogaster (Dm), and Caenorhabditis elegans (Ce). Identical residues between more than two species are indicated with gray boxes. Domains of STX17 are indicated with different colors: green, the Habc domain; magenta, the SNARE domain; blue, transmembrane helix (TMH); orange, a linker between the TMDs; and purple, the C-terminal region.

(B) Mouse embryonic fibroblasts (MEFs) stably expressing mRuby3–LC3B and either GFP-tagged HsSTX17TM, DsSTX17TM, or CeSTX17TM were cultured in starvation medium for 1 h. GFP–STX17TM intensities of mRuby3–LC3B-positive ring-like structures were quantified (n > 30). Solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), and whiskers indicate the 5th to 95th percentiles.

(C) MEFs stably expressing one of the GFP-tagged alanine replacement mutants (shown in Figure 1A) and mRuby3–LC3B were cultured in starvation medium for 1 h. Quantification results are shown in Figure 1D. Bars indicate 10 μm (main images) and 1 μm (insets).

(D) MEFs stably expressing one of the GFP-tagged charge replacement mutants (shown in Figure 1E) and mRuby3–LC3B were cultured in starvation medium for 1 h. Quantification results are shown in Figure 1E. Bars indicate 10 μm (main images) and 1 μm (insets).

Autophagosomes become negatively charged during maturation.

(A) Mouse embryonic fibroblasts stably expressing one of the GFP-tagged charge probes and mRuby3–LC3B were cultured in starvation medium for 1 h. Bars indicate 10 μm (main images) and 1 μm (insets).

(B) Time-lapse analysis of MEFs stably expressing GFP–3K6Q and mRuby3– STX17TM or mRuby3–LC3B cultured in starvation medium. Autophagosomes are indicated by arrows. Bars indicate 1 μm.

(C) Time-lapse analysis of MEFs stably expressing one of the GFP-tagged charge probes and mRuby3–ATG5 cultured in starvation medium. Autophagosomes are indicated by arrows. Bars indicate 1 μm.

Localization of phospholipids in mature autophagosomes.

(A) Mouse embryonic fibroblasts (MEFs) stably expressing the indicated GFP-tagged phospholipid probe and mRuby3–STX17TM were cultured in starvation medium for 1 h. The following phospholipid probes were used: phosphatidic acid (PA), Spo20(PABD); PS, Evectin-2; diacylglycerol (DAG), PKD C1ab; PI3P, 2×FYVE; PI4P, CERT(PHD); PI5P, ING2(PlantHD); PI(3,4)P2, TAPP1(PHD); PI(4,5)P2, PLCd1(PHD); PI(3,5)P2, TRPML1(PHD); and PIP3, Btk(PHD). Bars indicate 10 μm (main images) and 1 μm (insets).

(B) MEFs stably expressing GFP–CERT(PHD) or TRPML1(PHD) were cultured in starvation medium containing LysoTracker Deep Red for 1 h. Bars indicate 10 μm (main images) and 1 μm (insets).

(C) MEFs stably expressing GFP–PI4KB or GFP–PI4K2A were cultured in starvation medium for 1 h. Bars, 10 μm (main images) and 1 μm (insets).

Phosphatidylinositol 4-phosphate (PI4P) is enriched in mature autophagosomes before fusion with lysosomes.

(A and B) Mouse embryonic fibroblasts (MEFs) stably expressing the indicated GFP-tagged indicated PI4P probe, FAPP(PHD), OSBP(PHD) or P4M-SidMx2, and mRuby3– STX17TM or mRuby3–ATG5 were cultured in starvation medium for 1 h. Bars indicate 10 μm (main images) and 1 μm (insets) (A). GFP intensities of mRuby3-positive structures (n > 50) were quantified (B). Solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), and whiskers indicate the 5th to 95th percentiles. Differences were statistically analyzed by Welch’s t-test.

(C) Time-lapse analysis of MEFs stably expressing GFP–CERT(PHD) cultured in starvation medium containing LysoTracker Deep Red. Bars indicate 1 μm.

(D) U2OS cells stably expressing GFP–CERT(PHD) and mRuby3–LC3B were transfected with siSTX17 and siYKT6. After 3 days, cells were cultured in starvation medium for 1 h, and immunostained with anti-LAMP1 antibodies. Bars indicate 10 μm (main images) and 1 μm (insets).