Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy

Abstract

Autophagy maintains cellular health and homeostasis during stress by delivering cytosolic material captured by autophagosomes to lysosomes for degradation. Autophagosome formation is complex: initiated by the recruitment of autophagy (Atg) proteins to the formation site, it is sustained by activation of Atg proteins to allow growth and closure of the autophagosome. How Atg proteins are translocated to the forming autophagosome is not fully understood. Transport of the ATG8 family member GABARAP from the centrosome occurs during starvation-induced autophagosome biogenesis, but how centrosomal proteins regulate GABARAP localization is unknown. We show that the centriolar satellite protein PCM1 regulates the recruitment of GABARAP to the pericentriolar material. In addition to residing on the pericentriolar material, GABARAP marks a subtype of PCM1-positive centriolar satellites. GABARAP, but not another ATG8 family member LC3B, binds directly to PCM1 through a canonical LIR motif. Loss of PCM1 results in destabilization of GABARAP, but not LC3B, through proteasomal degradation. GABARAP instability is mediated through the centriolar satellite E3 ligase Mib1, which interacts with GABARAP through its substrate-binding region and promotes K48-linked ubiquitination of GABARAP. Ubiquitination of GABARAP occurs in the N terminus, a domain associated with ATG8-family-specific functions during autophagosome formation, on residues absent in the LC3 family. Furthermore, PCM1-GABARAP-positive centriolar satellites colocalize with forming autophagosomes. PCM1 enhances GABARAP/WIPI2/p62-positive autophagosome formation and flux but has no significant effect on LC3B-positive autophagosome formation. These data suggest a mechanism for how centriolar satellites can specifically regulate an ATG8 ortholog, the centrosomal GABARAP reservoir, and centrosome-autophagosome crosstalk.

Keywords: ATG8; GABARAP; LIR; Mib1; PCM1; autophagosome; autophagy; centriolar satellites; centrosome; ubiquitination.

Graphical abstract

Figure 1

PCM1 Directly Binds GABARAP through a LIR Motif (A) Anti-GABARAP immunoprecipitation from HEK293A cells and immunoblot. Beads + Ab, anti-GABARAP antibody with protein G beads; beads + Lys, HEK293A lysate with protein G beads. (B) HEK293A cells in full medium (FM) or EBSS (ES) for 2 hr prior to lysis, followed by treatment as in (A). (C) HEK293A cells expressing indicated constructs in FM, ES, or EBSS + BAFA (EB) for 2 hr prior to lysis and GFP-TRAP. GFP-GABA, GFP-GABARAP; WT, wild-type. (D) Statistical analysis of (C); one-way ANOVA. NS, non-significant. (E) GFP-TRAP of HEK293A cells expressing the indicated GFP-ATG8 constructs and immunoblot. (F) HEK293A cells expressing the indicated GFP-tagged constructs incubated with GST or GST-GABARAP beads and immunoblotted. 3xAla, LIR mutant. (G) Statistical analysis of (F); unpaired Student’s t test; mean ± SEM; n = 3. ^∗∗∗∗p ≤ 0.0001. (H) 24-mer array of PCM1 peptides covering the LIR motif incubated with GST-GABARAP and immunoblot. Each amino acid position was substituted for every other amino acid.

Figure 2

PCM1 and GABARAP Colocalize at the Centrosome and Peripheral CSs (A) HEK293A cells starved for 2 hr in EBSS, fixed and labeled with the mouse anti-PCM1, rabbit anti-GABARAP, and goat anti-gamma tubulin antibodies. The scale bars represent (top) 5 μm and (bottom) 2 μm. Arrowheads and insets show PCM1-GABARAP colocalization. ^∗GABARAP-PCM1 at the PCM. Nuc, nucleus. (B) HEK293A cells starved for 2 hr in EBSS, fixed and labeled with the indicated antibodies. Pearson’s correlation coefficient (PCC) was quantified between non-centrosomal GABARAP-PCM1 or GABARAP-p62 puncta, with and without PCM1 siRNA. Subcellular regions are quantified from two independent experiments. (C) HEK293A cells expressing myc-GABARAP, starved for 2 hr in EBSS, and then fixed and labeled with rabbit anti-PCM1, mouse anti-myc, and goat anti-SSX2IP antibodies; scale bar, 2 μm. (D) HEK293 Flp-In T-Rex cells stably expressing inducible GFP-GABARAP, starved for 2 hr in EBSS, were fixed and labeled with rabbit anti-pericentrin antibodies. The scale bar represents 2 μm. In (C) and (D), arrowheads show GABARAP colocalization with CS markers. See also Figures S1 and S2.

Figure 3

CSs Are Found at Sites of Autophagosome Formation (A) HEK293A cells stably expressing GFP-WIPI2b starved for 2 hr in EBSS, fixed and labeled with mouse anti-PCM1 and rabbit anti-GABARAP antibodies. The scale bars represent 5 μm. Arrows indicate triple colocalization; arrowhead indicates GABARAP-PCM1 double colocalization. (B) HEK293A cells, or MEF cells stably expressing GFP-DFCP1, starved for 2 hr in EBSS, fixed and labeled with the indicated antibodies. The scale bars represent 5 μm. Arrows indicate colocalization or points of contact (GFP-DFCP1) between PCM1 and autophagy markers. Hoechst DNA staining is shown in blue in the merge. In all panels, rabbit anti-PCM1 antibody was used; mouse anti-WIPI2 or LC3B and guinea pig anti-p62 antibodies were used in the remaining panels. (C) HEK293 cells stably expressing GFP-DFCP1 starved for 2 hr in EBSS followed by immunoisolation with control immunoglobulin G (IgG) or anti-GFP and immunoblot analysis with the indicated antibodies. See also Figure S2.

Figure 4

PCM1 Controls GABARAP Localization at the PCM, CS, and GABARAP Autophagosome Formation (A) HEK293A cells were treated with RISC free (RF) or PCM1 siRNA for 72 hr, fixed, and labeled with the mouse anti-PCM1, rabbit anti-GABARAP, and goat anti-gamma tubulin antibodies. The scale bar represents 20 μm. (B) Quantification of (A). Signal intensities at the pericentriolar material (γ-tubulin positive structures) were quantified and normalized to RF. Each measurement represents one centrosome. Statistical analysis using Mann-Whitney test; mean ± SEM; data from three independent experiments. ^∗∗∗∗p ≤ 0.0001. (C) HEK293A cells expressing GFP-PCM1 WT or 3xAla LIR mutant starved for 2 hr in EBSS, fixed, and labeled with rabbit anti-GABARAP and mouse anti-WIPI2 antibodies. The scale bars represent 2 μm. Arrowheads, triple colocalization; arrows, GABARAP-WIPI2 colocalization. ^∗PCM1 and GABARAP at the PCM. (D) HEK293A cells expressing GFP-PCM1 were treated with 50 μM nocodazole for 5 hr in total and starved for 2 hr in EBSS prior to fixation and labeling with rabbit anti-GABARAP and mouse anti-GM130 antibodies. Arrowheads, GFP-PCM1-GABARAP colocalization; arrows, GM130-GABARAP colocalization. ^∗GABARAP at PCM. The scale bar represents 2 μm. (E) HEK293A cells were treated with RF or PCM1 siRNA for 72 hr and then incubated in ES or in ES with bafilomycin A1 (EB) for 2 hr, fixed, and labeled with the indicated antibodies before confocal microscopy and quantification of intracellular puncta. Statistical analysis using unpaired Student’s t test; mean ± SEM; ^∗p ≤ 0.05. Number of independent experiments: WIPI2, three; GABARAP, five; p62, three; and LC3, two. (F) HEK293A cells were treated with RF or PCM1 siRNA for 72 hr and then incubated in EBSS for 2 hr and fixed before confocal microscopy and quantification of intracellular GABARAP-p62 double-positive puncta. Mean ± SEM two independent experiments. (G) HEK293A cells expressing the indicated constructs were incubated in ES or in EB for 2 hr, fixed, and labeled with the indicated antibodies before confocal microscopy and quantification of intracellular puncta. Statistical analysis using unpaired Student’s t test; mean ± SEM; ^∗∗p ≤ 0.01. Three independent experiments. See also Figures S3 and S4.

Figure 5

PCM1 Specifically Regulates GABARAP Protein Levels through Its LIR Motif (A) HEK293A cells treated with RF or PCM1 siRNA incubated in full medium (FM) or EBSS with or without BAFA for 2 hr. (B) Quantifications from (A). For LC3-II/actin, n = 3. For p62/actin, n = 5. For NBR1/actin, n = 5. For PCM1/actin, n = 5. Mean ± SEM. One-way ANOVA; ^∗p ≤ 0.05. (C and D) HEK293A cells treated with RF or PCM1 siRNA incubated in FM or EBSS with or without BAFA1 for 2 hr. In (C), GABARAP-I and GABARAP-II are resolved, whereas in (D) only GABARAP-I is resolved in the western blot. (E) Quantifications from (C) and (D). For GABARAP-II/actin, n = 5. For GABARAP-I/actin, n = 3. Mean ± SEM. One-way ANOVA; ^∗p ≤ 0.05. (F) HEK293A cells expressing the indicated constructs were subjected to immunoblot. (G) Quantification of (F). For p62/actin, n = 4. For GABARAP-I/actin, n = 5. Mean ± SEM; unpaired Student’s t test; ^∗∗p ≤ 0.01. (H) HEK293A cells treated with RF or PCM1 siRNA (72 hr total) and transfected with the indicated siPCM1-resistant constructs (last 24 hr) and immunoblotted. (I) Quantifications from (H). For RF+GFP, siPCM1+GFP, and siPCM1+GFP-PCM1 WT, six experiments are shown. For siPCM1+GFP-PCM1 3xAla, three experiments are shown. Mean ± SEM; unpaired Student’s t test; ^∗∗p ≤ 0.01.

Figure 6

PCM1 Regulates GABARAP Proteasomal Degradation (A) Control or PCM1 knockout RPE-1 cells were subjected to cycloheximide (CHX) treatment for the indicated number of hours prior to immunoblotting. (B) Quantification of (A). Mean ± SEM; n = 3; unpaired Student’s t test; ^∗p ≤ 0.05. (C) HEK293A cells treated with RF or PCM1 siRNA were incubated in DMSO, cycloheximide (CHX), MG132 (MG), and/or bafilomycin A1 (BAFA1) for 8 hr. (D) Quantification of (C). Mean ± SEM; n = 3; one-way ANOVA; ^∗p ≤ 0.05.

Figure 7

Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ^∗p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ^∗, ^∗∗, ^∗∗∗, mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ^∗, ^∗∗, ^∗∗∗, mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ^∗∗. Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ^∗∗ and ^∗∗∗, respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 (^∗) between ATG8 orthologs. See also Figure S5.