Membrane scission driven by the PROPPIN Atg18

Abstract

Sorting, transport, and autophagic degradation of proteins in endosomes and lysosomes, as well as the division of these organelles, depend on scission of membrane-bound tubulo-vesicular carriers. How scission occurs is poorly understood, but family proteins bind these membranes. Here, we show that the yeast PROPPIN Atg18 carries membrane scission activity. Purified Atg18 drives tubulation and scission of giant unilamellar vesicles. Upon membrane contact, Atg18 folds its unstructured CD loop into an amphipathic α-helix that inserts into the bilayer. This allows the protein to engage its two lipid binding sites for PI3P and PI(3,5)P₂ PI(3,5)P₂ induces Atg18 oligomerization, which should concentrate lipid-inserted α-helices in the outer membrane leaflet and drive membrane tubulation and scission. The scission activity of Atg18 is compatible with its known roles in endo-lysosomal protein trafficking, autophagosome biogenesis, and vacuole fission. Key features required for membrane tubulation and scission by Atg18 are shared by other PROPPINs, suggesting that membrane scission may be a generic function of this protein family.

Keywords: autophagy; endosomes; lysosomes; membrane fission; membrane traffic.

Figure 1. Redundancy of PROPPINs in vacuole fission

1. Cells (wt, ∆atg18 or ∆atg18/∆atg21/∆hsv2 (ΔΔΔ), with or without a plasmid expressing wt or a mutant form of Atg18) were grown in YPD; vacuoles were stained with FM4‐64 and imaged by confocal microscopy. At least 10 confocal sections spaced at 300 nm were assembled into maximum projections. Images show cells before (0 min) and 15, 30, and 60 min after induction of vacuolar fragmentation by addition of 0.4 M NaCl. Red: FM4‐64. Gray: DIC. Scale bar 5 μm.

2. The number of vacuoles per cell was quantified for the experiment in (A); 150 cells from three independent experiments were counted for each condition. Values represent the mean, error bars the SEM.

Figure 2. Localization of Atg18‐GFP during salt‐induced vacuole fission

atg18Δ cells expressing plasmids carrying Atg18‐GFP or Atg18^FGGG‐GFP, a mutant in the lipid binding sites, were stained with FM4‐64.

1. Confocal z‐stacks were taken before and 2, 6, and 10 min after addition of 0.4 M NaCl. Stacks were processed into maximum projections using ImageJ. The cell outlines are marked with dotted lines. Scale bar: 5 μm.

2. Same experiment as in (A), but in a fab1Δ or vps34Δ background. Scale bar: 5 μm.

3. The cells were categorized as indicated, and the phenotypes of 150 cells were quantified.

4. Localization of Atg18‐GFP 5 min after salt shock. Experiment as in (A). Scale bar: 1 μm.

5. Line scan analysis of fluorescence intensities along the white lines shown in (D). Red: FM4‐64. Green: Atg18‐GFP. Scale bar: 1 μm.

Figure 3. Amphipathic α‐helix formation

1. The CD loop sequences of PROPPINs from a wide variety of organisms. Potential amphipathic helices were predicted using the online tool Heliquest and are plotted in red. Amino acids removed in Atg18^DLoop (aa 328–384) are printed in bold. In the loop of Pichia pastoris Atg18, bold print mark phosphorylation sites relevant for its membrane insertion (Tamura et al, 2013). Dr: Danio rerio, Dd: Dictyostelium discoideum.

2. Helical wheel projections of the red sequences from (A). A schematic representation of the 7‐bladed beta‐propeller PROPPIN structure is also shown, illustrating the localization of the putative amphipathic α‐helix (blue) of the CD loop between the two lipid binding sites for PI3P/PI(3,5)P₂. Red arrows mark phosphorylation sites in the loop of Pichia pastoris Atg18, which are relevant for its membrane insertion (Tamura et al, 2013). Color code for residues: yellow, hydrophobic; purple, serine and threonine; blue, basic; red, acidic; pink, asparagine and glutamine; gray, alanine and glycine; green, proline; light blue, histidine.

Figure 4. Lipid‐triggered folding of the amphipathic helix of CD loop peptides and its requirement for vacuole scission

1. A

   CD loop folding in hydrophobic buffer. Peptides were synthesized that correspond to the predicted amphipathic helix from Saccharomyces cerevisiae Atg18 (TRLAREPYVDASRKTMGRMIRYSSQ) or to a sequence with reduced tendency to form amphipathic helices, generated by a swap of two amino acid pairs (indicated by green and red arrows; SLoop). Helical wheel projections of the sequences are shown.

2. B, C

   Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro‐2‐propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30% PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for (B) the wild‐type peptide, and (C) the SLoop peptide.

3. D

   Vacuole morphology of ΔΔΔ cells expressing Atg18^Sloop‐GFP, imaged before and after osmotic shock as in Fig 1A. Scale bar: 5 μm.

4. E

   Number of vacuoles per cell in (D), assayed as in Fig 1B. n = 3.

5. F

   Vacuole morphology of ΔΔΔ cells expressing Atg18^DLoop‐GFP, imaged as in (D).

6. G

   Number of vacuoles per cell in (E) assayed as in Fig 1B. n = 3.

Figure 5. Binding of Atg18^Sloop and Atg18^Dloop to small unilamellar vesicles

1. A–D

   Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C) and centrifuged, and the supernatants (S) and pellets (P) analyzed by SDS–PAGE and Coomassie staining. Binding is shown for (A) Atg18^wt; (B) Atg18^FGGG; (C) Atg18^DLoop; (D) Atg18^SLoop.

2. E

   The bands from (A–D) were quantified from three independent experiments. The mean with SEM is shown.

Figure 6. Different functional aspects of Atg18 displayed in vacuole fission and autophagy

1. Autophagy measured with the pho8∆60 assay in atg18Δ cells complemented with wt, FGGG, SLoop, or DLoop alleles of ATG18. Error bars represent SEM of 3 independent experiments.

2. Same assay as in (A), but in atg18Δ atg21Δ cells complemented with wt or SLoop versions of ATG18 and ATG21. Atg21^SLoop contains K426F and F429K substitutions. Helical wheel projections for both Atg21 helices are given below. The arrows indicate the magnitude of the hydrophobic moment. Error bars represent SEM of 3 independent experiments.

3. Vacuole fragmentation in atg2Δ cells, assayed 15 min after a moderate hypertonic shock as in Fig 1. Scale bar: 5 μm.

4. Number of vacuoles per cell in (C), assayed as in Fig 1B. n = 3.

Figure 7. Time lapse analysis of the effects of Atg18 on giant unilamellar vesicles

1. A

   Time lapse analysis of Atg18 added to liposomes. 10 μl of 10 μM purified recombinant Atg18^wt was added to 100 μl buffer containing GUVs with 5% PI3P and 1% PI(3,5)P₂. The sample was imaged on a spinning disk confocal microscope. Most of the tubules underwent scission into small vesicles (see Movie EV6). Scale bar 5 μm.

2. B, C

   Areas in boxes 1 and 2 of (A) are shown at higher magnification in (B and C). Scale bars: 2.5 μm.

3. D

   Quantification of fission activity. For each movie, the whole frame was divided into squares of 10 per 10 μm and the number of small vesicles per 100 μm² counted. The mean of three independent experiments is shown with SEM.

Figure 8. Effect of Atg18 mutants on GUVs of different lipid composition

1. 1 μM Atg18 was added to wells containing sedimented GUVs with 10% PI3P or PI(3,5)P₂ while imaging a single plane by spinning disk microscopy. Membrane tension exerted by Atg18 binding suffices to induce liposome collapse, which allows to better judge the mobility of the tubular or clustered membranes that remain. Black arrow points to a PI(3,5)P₂ induced tubule collapsing to the GUV surface. Images are extracted from [Link], [Link], [Link], [Link]. Scale bar 5 μm.

2. Atg18 was covalently labeled with the green fluorescent dye Alexa488 TFP (ThermoFisher) and used in experiments as in (A), with rhodamine‐phosphatidylethanolamine‐labeled (Rh‐PE) GUVs containing the indicated phosphoinositides. Scale bar 5 μm.

3. Effect of 1 μM of purified recombinant Atg18 variants on GUVs containing 10 mol% of the indicated phosphoinositides. Vesicles are shown after 1–3 min of incubation. Scale bar: 7 μm. Arrow and arrowhead point to the structures quantified in (D).

4. Quantification of the experiments in (C). The percentage of GUVs showing an enrichment of Atg18 flexible tabulated (arrowhead) or stiff aggregated structures (arrow) was determined. Shown are the mean and SEM from three independent experiments.

5. Quantification of the effect of different Atg18 version on the formation of tubules in (C). Shown is the mean from three independent experiments with SD.

Figure 9. Oligomerization of Atg18

1. Cross‐linking of Atg18. Purified recombinant Atg18^wt or Atg18^FGGG (1.5 μM) was incubated with liposomes containing 5% PI3P, PI(3,5)P₂, PI(4,5)P₂, or 15% PS as a control for negative charge. After addition of a cleavable cross‐linker or buffer only, liposomes were pelleted, dissolved in sample buffer, incubated in the presence or absence of 100 mM dithiothreitol (DTT) for 5 min at room temperature, and analyzed by SDS–PAGE and Coomassie staining. Cross‐link products migrate at the upper limit of the separating gel.

2. Cross‐linking experiment with recombinant Atg18^wt as in (A), performed in the presence of increasing concentrations of liposomes containing 5% PI(3,5)P₂.