AP-4 mediates export of ATG9A from the trans-Golgi network to promote autophagosome formation

Abstract

AP-4 is a member of the heterotetrameric adaptor protein (AP) complex family involved in protein sorting in the endomembrane system of eukaryotic cells. Interest in AP-4 has recently risen with the discovery that mutations in any of its four subunits cause a form of hereditary spastic paraplegia (HSP) with intellectual disability. The critical sorting events mediated by AP-4 and the pathogenesis of AP-4 deficiency, however, remain poorly understood. Here we report the identification of ATG9A, the only multispanning membrane component of the core autophagy machinery, as a specific AP-4 cargo. AP-4 promotes signal-mediated export of ATG9A from the trans-Golgi network to the peripheral cytoplasm, contributing to lipidation of the autophagy protein LC3B and maturation of preautophagosomal structures. These findings implicate AP-4 as a regulator of autophagy and altered autophagy as a possible defect in AP-4-deficient HSP.

Keywords: AP-4; ATG9A; adaptor protein complexes; autophagy; protein sorting.

Fig. 1.

Specific interaction of ATG9A with AP-4. (A) Schematic representation of the AP-4 complex depicting its subunits, structural domains, and recognition of YXXØE signals by the μ4 subunit. (B) Schematic representation of ATG9A showing the cytosolic, transmembrane, and luminal segments, an N-linked glycosylation site (CHO), and the sequence of the N-terminal (NH₂) cytosolic tail (amino acids 1–66 in human ATG9A). A consensus YXXØE motif (YQRLE) and a variant dileucine motif (EEDLLV) are highlighted in red. (C) Y2H analysis of the interaction of (i) the ATG9A N-terminal tail (ATG9A-N) fused to the Gal4 DNA binding domain (BD), with (ii) μ-subunits of different AP complexes fused to the Gal4 transcriptional activation domain (AD). p53 and T antigen (T-Ag) were used as controls. Growth in the absence of histidine (−His) and presence of 3-amino-1,2,4-triazole (AT) is indicative of interactions. Growth in the presence of histidine (+His) is a control for viability and loading of yeast double transformants. Images shown are composites of plates from the same experiment. (D) Y2H analysis of the interaction of: (i) ATG9A N-terminal tail constructs with different alanine substitutions fused to Gal4 BD, with (ii) μ4 fused to Gal4 AD, as described for C. (E) H4 cells stably expressing TSF-tagged AP-4 ε used in the original affinity purification and mass spectrometry analyses (11) were transiently transfected with plasmids encoding GFP or ATG9A-GFP. Detergent extracts were subjected to immunoprecipitation with antibody to GFP, and both lysates (2.8% of input) and immunoprecipitates were analyzed by immunoblotting with antibodies to AP-4 β4 (Top) and GFP (Bottom). The positions of molecular mass markers are indicated on the Left, and those of the target proteins on the Right. The asterisk indicates aggregated ATG9A, a common occurrence for multispanning membrane proteins. (F) Coimmunoprecipitation of AP-4 β4 with ATG9A bearing alanine substitutions in the N-terminal cytosolic tail was analyzed as described in E (lysate samples correspond to 1.4% of input). The complete list of proteins identified by mass spectrometry from the AP-4 ε tandem affinity purification is shown in Dataset S1.

Fig. 2.

Colocalization of ATG9A and AP-4 at the TGN. (A) Immunofluorescence microscopy of endogenous ATG9A and AP-4 ε in WT, ATG9A KO, and AP-4 ε KO cells. Arrows point to TGN structures containing both ATG9A and AP-4 ε. The Bottom Right image is an enlargement of the box in the Top Right image. (Scale bars, 10 μm.) (B–D) Immunofluorescence microscopy colocalization of endogenous ATG9A and TGN46 in WT HeLa cells (B), AP-4 ε and TGN46 in WT HeLa cells (C), and AP-4 ε and TGN46 in ATG9A KO HeLa cells (D). Images in the Right column are enlargements of the boxed areas. (Scale bars, 10 μm; enlarged images in B–D, 1 μm.)

Fig. 3.

AP-4-ε KO causes accumulation of ATG9A at the TGN. (A–C) Immunofluorescence microscopy of endogenous ATG9A and TGN46 or GM130 in WT or AP-4-ε KO HeLa (A), HAP1 (B), or MEF (C) cells. (Scale bars, 10 μm.) The fluorescence intensity for ATG9A (green lines), TGN46 or GM130 (red lines), and DAPI (blue lines) as a function of distance from the center of the cell, measured using the Radial Profile plugin of ImageJ, is shown on the Right. Values are the mean ± SEM of fluorescence intensity relative to the total from 15 cells in each group. Wider fields of cells are shown in Fig. S1. Airyscan microscopy of the colocalization of ATG9A with TGN46 or GM130 is shown in Fig. S2.

Fig. 4.

Specificity of ATG9A redistribution by AP-4 ε KO. (A) Rescue of endogenous ATG9A dispersal by expression of HA-tagged AP-4 ε into AP-4 ε KO MEFs, as analyzed by immunofluorescence microscopy. The asterisk indicates a transfected cell. (B) Quantification of the distribution of ATG9A in rescued (transfected) (red line) vs. nonrescued (untransfected) (green line) cells such as those in A. Values are the mean ± SEM fluorescence intensity relative to the total from 14 cells in each group. (C) Immunofluorescence microscopy of endogenous AP-4 and ATG9A in HeLa cells treated with control siRNA or siRNA targeting the μ4 subunit of AP-4. (D) Immunofluorescence microscopy of endogenous giantin, TfR, CI-MPR, and AP-1 γ1 in WT and AP-4 ε KO HeLa cells. (E and F) Immunofluorescence microscopy of endogenous ATG9A and TGN46 in tepsin KO (E) and AP-5 ζ KO (F) HAP1 cells. Images on the Right are enlargements of the boxed areas. (Scale bars, 10 μm.)

Fig. 5.

Increased levels of ATG9A in AP-4 ε KO cells. (A) Immunoblot analysis of AP-4 ε, ATG9A, and α-tubulin (loading control) in WT, AP-4 ε KO, and ATG9A KO HeLa, HAP1 and MEF cells. (B) Quantification of ATG9A relative to α-tubulin levels in HeLa cells from experiments such as that in A. Values are the mean ± SEM from six independent experiments. **P < 0.01. The level of ATG9A in AP-4 KO HeLa cells relative to WT was 2.5 ± 0.2 (mean ± SEM; n = 6). (C) WT and AP-4 ε KO HeLa cells were incubated for different times at 37 °C with 100 nM bafilomycin A1 and analyzed by SDS/PAGE and immunoblotting for ATG9A, LC3B, and α-tubulin. (D) WT and AP-4 ε KO HeLa cells were incubated for different times at 37 °C with 0.35 mM cycloheximide (CHX) and analyzed by SDS/PAGE and immunoblotting for ATG9A, LC3B, and α-tubulin. In A, C, and D, the positions of molecular mass markers (in kilodaltons) are indicated Left of the immunoblots.

Fig. 6.

Increased levels of LC3B and decreased ratio of LC3B-II to LC3B-I in AP-4 ε KO cells. (A) Immunoblot analysis of LC3B, SQSTM1, and α-tubulin in WT, AP-4 ε KO, and ATG9A KO HeLa cells. (B and C) Quantification of total LC3B (relative to the α-tubulin control) (B) and LC3B-II/LC3B-I ratio under basal conditions in WT and AP-4 ε KO HeLa cells (C). Values are the mean ± SEM from seven assays carried out with samples from six independent experiments (total LC3B) or six assays carried out with samples from five independent experiments (LC3B-II/LC3B-I ratios). ***P < 10⁻⁴; *P < 0.05. The total level of LC3B in AP-4 ε KO relative to WT HeLa cells was 3.2 ± 0.4 (mean ± SEM; n = 7). (D and E) Effect of amino acid and serum starvation on the conversion of endogenous LC3-I to LC3-II in WT and AP-4 ε KO HeLa cells (D) and MEFs (E). Cells were incubated in amino acid- and serum-free DMEM for the indicated times at 37 °C and analyzed by SDS/PAGE and immunoblotting for LC3 and α-tubulin (loading control). The ratio of LC3B-II to LC3B-I was calculated and plotted as a function of time. In A, D, and E, the positions of molecular mass markers (in kilodaltons) are indicated Left of the immunoblots.

Fig. 7.

Abnormal LC3B structures in starved AP-4 ε KO cells. (A and B) Immunofluorescence microscopy for endogenous ATG9A (A) or LC3B (B) in WT and AP-4 ε KO MEFs incubated for 0 or 60 min in amino acid- and serum-free DMEM. Images in the Right column are magnifications of the boxed areas. (Scale bars, 10 μm; enlarged images at Right, 1 μm.) The arrow indicates the irregular shape of an LC3B structure. (C) Quantification of the number and size of LC3 puncta from experiments such as that in B using ImageJ with Analyze Particles. Values are the mean ± SEM from 20 cells per sample. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 1 × 10⁻⁵.