Atg18 phosphoregulation controls organellar dynamics by modulating its phosphoinositide-binding activity

Abstract

The PROPPIN family member Atg18 is a phosphoinositide-binding protein that is composed of a seven β-propeller motif and is part of the conserved autophagy machinery. Here, we report that the Atg18 phosphorylation in the loops in the propellar structure of blade 6 and blade 7 decreases its binding affinity to phosphatidylinositol 3,5-bisphosphate in the yeast Pichia pastoris. Dephosphorylation of Atg18 was necessary for its association with the vacuolar membrane and caused septation of the vacuole. Upon or after dissociation from the vacuolar membrane, Atg18 was rephosphorylated, and the vacuoles fused and formed a single rounded structure. Vacuolar dynamics were regulated according to osmotic changes, oxidative stresses, and nutrient conditions inducing micropexophagy via modulation of Atg18 phosphorylation. This study reveals how the phosphoinositide-binding activity of the PROPPIN family protein Atg18 is regulated at the membrane association domain and highlights the importance of such phosphoregulation in coordinated intracellular reorganization.

Figure 1.

Phosphorylation of PpAtg18 modulates PI(3,5)P₂-binding activity. (A) Evaluation of purified GST-PpAtg18 from P. pastoris by SDS-PAGE and Coomassie brilliant blue staining. Lane 1: Purified GST-PpAtg18 was eluted from a GS 4B column using the reduced form of glutathione. Lane 2: Purified GST-PpAtg18 was treated with λ-phosphatase on the GS 4B column. This sample was used for the lipid binding assay. (B) Phosphatase treatment of purified PpAtg18 from P. pastoris. Heat, λ-phosphatase was inactivated at 65°C for 1 h. (C) Phosphatase treatment of cell-free extracts from the strain expressing PpAtg18-5×Flag under the original PpATG18 promoter. The cell lysate was treated with λ-phosphatase at 37°C for 1 h. (D) PIP strip analysis. Membranes were incubated with 0.5 µg/ml protein and detected using the Light Capture II system (ATTO). GST-PpAtg18Wt and GST-Atg18Wt with λ-phosphatase were acquired simultaneously to ensure equal exposure times. (E) PIP array analysis. Membranes were incubated with 1.0 µg/ml protein and detected simultaneously to ensure equal exposure times. (F) Liposome pull-down assay. Purified GST-PpAtg18 (1.0 µg) was incubated with each liposome preparation and centrifuged at 16,000 g for 20 min. The pellets were washed twice, suspended in sample buffer, and analyzed by immunoblotting to detect GST-PpAtg18. No lipid, no liposomes; No PI, liposomes without PIs; PI(3)P, liposome containing PI(3)P; PI(3,5)P₂, liposome containing PI(3,5)P₂. (G) Liposome pull-down with dephosphorylated PpAtg18. Each protein (1.0 µg) was incubated with liposomes containing PI(3,5)P₂ and analyzed by immunoblot to detect GST-PpAtg18. The bands were analyzed by densitometry, and band intensities were normalized to samples not treated with phosphatase. Error bars indicate mean ± SEM. **, P < 0.05.

Figure 2.

PpAtg18 is phosphorylated at two distinct sites, and the loop region in blade 6 is conserved among PROPPIN family members. (A) Sequence alignment of PROPPIN domains between KlHsv2p (K. lactis), PpAtg18 (P. pastoris), and WIPI1 (human). Each blade contains 4 β sheets marked by different colors. The arrowheads indicate phosphorylated regions of PpAtg18. (B) Sequences of phosphorylated regions in the blade 6 and 7 loop structures of PpAtg18. The predicted sites of phosphorylation determined from LC-MS/MS analyses are underlined, and amino acid residues critical for phosphorylation are shown in bold font.

Figure 3.

PpAtg18 is phosphorylated at two distinct sites in vivo. (A) The designated PpAtg18 phosphorylation mutants were grown in SD, collected at the exponential phase, and analyzed (5 µg) by immunoblotting. (B) Analysis of PpAtg18 variants using Phos-tag. The samples are the same as those used in Fig. 3 A. (C) PIP array analysis. Membranes were incubated with 1.0 µg/ml protein and detected simultaneously to ensure equal exposure times. (D) Liposome pull-down assay of PpAtg18 phosphorylation-defective mutants. The samples were subjected to immunoblotting and the bands were analyzed by densitometry. Error bars indicate mean ± SEM. ***, P < 0.1.

Figure 4.

PpAtg18 phosphorylation correlates with the vacuolar dynamics observed under a variety of environmental conditions. (A) PpAtg18-5×Flag from cells grown under a variety of conditions was detected by immunoblotting with the anti-FLAG antibody. Cells were shifted from SD (0 min) to SD medium containing 0.9 M NaCl or 20% glucose, to water, to YNB medium without amino acids (−glucose), to methanol-containing medium, or (as a control) to SD medium for the indicated times (10, 20, 40, or 60 min). (B) Fluorescence microscopy analysis of PpAtg18 localization and vacuolar morphology. Growing conditions were the same as in Fig. 4 A. PpAtg18-tagged YFP was expressed under the original promoter and vacuoles were stained with FM 4-64. (C) Pulse-chase experiment. Cells were shifted from SD medium to either SD + 0.9 M NaCl or SD-glucose, and then shifted again into the opposite medium (see arrows). Analysis was performed by immunoblotting. (D) Oxidative stress induces PpAtg18 phosphorylation. t-BOOH, t-butylhydroperoxide. Analysis was performed by immunoblotting. (E) Morphological analysis and morphometric analysis of vacuoles in cells with or without oxidants. Cells grown on SD were shifted to SD (control) or to SD with 1 mM H₂O₂, 15 mM t-BOOH, or 4 mM diamide. Vacuoles were stained with FM 4-64. Error bars indicate mean ± SEM. *, P < 0.01 (compared with SD [control]). Bars, 2 µm.

Figure 5.

Phosphorylation of PpAtg18 regulates vacuolar membrane dynamics via PI(3,5)P₂ binding. (A) Localization of PpAtg18-YFP in Ppfab1Δ. YFP tagged at the C terminus with PpAtg18 was expressed under its own promoter, and vacuoles were stained with FM 4-64. (B) Localization of PpAtg18-YFP phosphorylation mutants. PpAtg18AA, a phosphorylation-defective mutant, and PpAtg18DD, a phosphorylation mimic mutant, appeared similar to the wild type as observed in Fig. 4 B. Bars, 2 µm. (C) Western blot analysis of PpAtg18Wt-5×Flag in Ppfab1Δ or PpAtg18FTTG-5×Flag in Ppatg18Δ.

Figure 6.

Phosphorylation of PpAtg18 regulates VSM formation in micropexophagy. (A) Intracellular structures observed during micropexophagy and macropexophagy. MIPA, micropexophagy-specific membrane apparatus; VSM, vacuolar sequestering membrane. (B) Fluorescence microscopy analysis during micropexophagy. Cells were shifted from methanol medium to glucose medium for 30–60 min to induce micropexophagy. Vacuoles were stained with FM 4-64, and MIPA was visualized by YFP-tagged PpAtg8 expressed under the PpATG8 promoter. (C) Intracellular localization of PpAtg18. YFP C-terminally tagged with PpAtg18 was expressed in Ppatg18Δ under the PpATG18 promoter. Cells were shifted from methanol medium to ethanol or glucose medium for 30–60 min to induce macropexophagy or micropexophagy, respectively. (D) Immunoblot detection of PpAtg18-5×Flag. PpAtg18-5×Flag was expressed in Ppatg18Δ under the PpATG18 promoter. Cells were shifted from methanol medium to ethanol or glucose medium to induce macropexophagy or micropexophagy, respectively. This blot was incubated with the anti-FLAG antibody. (E) Intracellular localization of PpAtg18 AA and DD during micropexophagy. YFP-tagged PpAtg18 mutants were expressed under the PpATG18 promoter. Cells were shifted from methanol medium to glucose medium for 60 min to induce micropexophagy. Bars, 2 µm. (F) PpPex12 degradation assay to assess micropexophagy and macropexophagy activity in PpAtg18 mutants. Strains were grown in methanol medium and then shifted to glucose or ethanol medium to induce micropexophagy or macropexophagy, respectively. Cell-free extracts were prepared and analyzed by immunoblotting with anti-PpPex12 or anti-Pgk1 antibodies. Optical density measurements showed no significant difference in growth between these mutants.

Figure 7.

Phosphoregulation of PpAtg18 for maintenance of vacuolar shape.