TRAPPC13 modulates autophagy and the response to Golgi stress

Abstract

Tether complexes play important roles in endocytic and exocytic trafficking of lipids and proteins. In yeast, the multisubunit transport protein particle (TRAPP) tether regulates endoplasmic reticulum (ER)-to-Golgi and intra-Golgi transport and is also implicated in autophagy. In addition, the TRAPP complex acts as a guanine nucleotide exchange factor (GEF) for Ypt1, which is homologous to human Rab1a and Rab1b. Here, we show that human TRAPPC13 and other TRAPP subunits are critically involved in the survival response to several Golgi-disrupting agents. Loss of TRAPPC13 partially preserves the secretory pathway and viability in response to brefeldin A, in a manner that is dependent on ARF1 and the large GEF GBF1, and concomitant with reduced caspase activation and ER stress marker induction. TRAPPC13 depletion reduces Rab1a and Rab1b activity, impairs autophagy and leads to increased infectivity to the pathogenic bacterium Shigella flexneri in response to brefeldin A. Thus, our results lend support for the existence of a mammalian TRAPPIII complex containing TRAPPC13, which is important for autophagic flux under certain stress conditions.

Keywords: Autophagy; Brefeldin A; Golgi apparatus; Shigella flexneri; TRAPP complex.

Fig. 1.

Loss of TRAPPC13, a TRAPPC-interacting protein, provides resistance against several Golgi-disrupting agents. (A) TRAPPC13 co-immunoprecipitates TRAPPC3, TRAPPC4, TRAPPC12 and itself. Control and TRAPPC subunits were transiently co-overexpressed in HEK293T cells before Flag IP and western blotting with the indicated antibodies. (B) Viability of several cancer cell lines infected with lentiviral control or TRAPPC13 shRNAs in response to Golgi stress inducers. Relative viability was calculated by dividing fluorescence values (arbitrary units, CTB assay) of BFA-treated cells by their corresponding counterparts under vehicle-treated conditions. The means of TRAPPC13 knockdown and control cells are shown; data are representative of at least three independent experiments. Average controls (shAvg CTRL): the averaged mean survival ratio of one to three control shRNA-infected cell lines (LUC shRNA, RFP shRNA, GFP shRNA); relative viability is shown as mean±s.e.m. Treatment duration, drug concentrations: HeLa: 3 days, 12.5 ng/ml BFA treatment, 2 µM GCA; A549: 3 days 20 ng/ml BFA treatment, 2 µM GCA, 2 µM Mon, 20 µM tyrphostin (AG1478); HT29, BCPAP: 3 days 20 ng/ml BFA treatment, 2 µM GCA; six wells were measured for each genotype and condition. *P<0.05, **P<0.01, ***P<0.001 (two-tailed Student's t-test). Western blot analysis confirmed TRAPPC13 knockdown in the entire cell line panel shown below the viability bar graphs. Quantification of TRAPPC13 levels normalized to GAPDH or p84 are shown; a.u., arbitrary units. (C) Viability of A549 cell lines infected with control or two different hairpins targeting the indicated TRAPPC subunits in response to BFA. Relative viability was calculated as in B. The mean viability values of TRAPPC subunit knockdowns and control cells are shown and are representative of at least three independent experiments. shAvg CTRL: the averaged mean survival ratio of two control shRNA-infected cell lines (LUC shRNA, GFP shRNA); survival ratio is shown as mean±s.e.m. *P<0.05, **P<0.01. Knockdown of TRAPPC3, TRAPPC8, TRAPPC11 and TRAPPC12 transcript levels in A549 cells was confirmed by Q real-time PCR (right graph).

Fig. 2.

TRAPPC13 depletion reduces apoptosis and ER stress while partially preserving the secretory pathway in response to BFA. (A) Stable shRFP or shTRAPPC13 HeLa knockdown cells were left untreated or treated with 7.5 or 12.5 ng/ml BFA or vehicle control (0.1% ethanol) in the presence or absence of 20 µM QVD (caspase inhibitor) or 10 µM Nec-1 for 48 h. Viability was determined using the CTB assay (upper graph) and quantification of LDH release (lower graph). Graphs show the mean±s.d. of six replicates from two independent experiments. (B) Stable shRFP or shTRAPPC13 HeLa knockdown cells were cultured for the indicated times in the presence of 12.5 ng/ml BFA or vehicle control (0.1% ethanol). Indicated proteins were resolved on SDS-PAGE gel and detected by immunoblotting. Vehicle control cells (labelled as C) were incubated for 24 h. *N.S., a nonspecific band detected by the anti-caspase-3 antibody. Representative blots from three independent experiments are shown. (C) IF micrographs of control (shLUC) and two stable TRAPPC13 knockdown A549 cell lines reveal that TRAPPC13 depletion leads to a less disrupted Golgi compared to the control cell line when treated with BFA, as assessed by staining for the Golgi markers GM130 and GBF1. Cells were treated with 20 ng/ml BFA for 24 h. Images are representative of three independent experiments. Scale bars: 10 µm. Quantification of Golgi dispersal was calculated using Knime software. a.u., arbitrary units. ***P<0.001, **P<0.01 (one-way ANOVA, Dunnett post test). (D) HeLa cells transfected with Gaussia luciferase were seeded in six-well plates and treated with 40 nM BFA for the indicated duration. Luciferase activity in the medium was assayed at the indicated time points after treatment with BFA. Data are expressed as a percentage of the maximum luciferase signal after 8 h, obtained from the untreated sample of each pair per genotype. Results represent the mean±s.e.m from three independent experiments. **P<0.01, ***P<0.001 (two-way ANOVA with Bonferroni post test for multiple groups).

Fig. 3.

ARF1 and GBF1 contribute to BFA resistance in TRAPPC13 knockdown cells. (A) Stable shLUC-transduced control, shARF4 (positive control) or TRAPPC13 knockdown A549 cells were analyzed for total ARF-GTP levels in the absence or presence of 20 ng/ml BFA (24 h treatment) using a GST-VHS-GAT pulldown assay. In this assay, increased ARF binding to VHS-GAT, a truncated GGA3 form and ARF-substrate that only interacts with GTP-bound ARFs, serves as an indicator of ARF activity. Pan-ARF indicates the use of an antibody (1D9), which recognizes all five mammalian ARF isoforms, whereas (1A9/5) specifically detects ARF1 (see also Materials and Methods). A representative quantification from two independent experiments is shown; a.u., arbitrary units. (B,C) Reducing (B) ARF1 or (C) GBF1 function in A549 TRAPPC13 knockdown cells using several independent shRNAs reverses BFA resistance observed in TRAPPC13 single knockdown cells. Cells were treated (B) for 3 days with 6 or 8 ng/ml BFA or (C) for 2 days with 1.5, 3, 12.5 ng/ml BFA; at least five wells per condition were measured. The survival ratio was determined by performing a CTB assay. Data represent at least three independent experiments. **P<0.01, *P<0.05. Western blots of co-depleted cells are shown to confirm knockdown of the indicated proteins.

Fig. 4.

TRAPPC13 regulates Rab1-GTP levels, and BFA resistance of TRAPPC13 knockdown cells depends on Rab1. (A) Rab1-effector pulldown assay. Stable shTRAPPC13 or shLUC knockdown A549 cells were stably transduced with Flag-tagged γTubulin or Golgin-84. Cells were treated with 20 ng/ml BFA or left untreated for 24 h. After treatment, Flag IP was performed followed by western blotting with the indicated antibodies. A representative blot of two independent experiments is shown; the bars in the right graph represent Rab1a-IP levels normalized to p84-input levels; a.u., arbitrary units. (B) A549 cells were stably transduced with lentiviral control or Rab1a hairpins. Cell viability was assessed after 3 days of treatment with either 20 or 35 ng/ml BFA or 2 µM GCA using CTB. Graphs display mean±s.e.m. of three independent experiments, each with six wells. A western blot of Rab1a-depleted A549 cells is shown below the survival graph to confirm knockdown. (C) Left panel: A549 cells stably transduced with Flag-tagged γTubulin as a control, Rab1b, Q67L mutant (GTP-bound) or the N121I mutant (GDP-bound) were treated for 3 days as indicated after which cell viability was measured as in B. Overexpression of dominant-negative N121I made cells highly resistant to different Golgi disrupting agents. Cells expressing the dominant-negative Rab1b form are also significantly more protected from Golgi stress than Rab1(wt) or dominant-active (Q67L) overexpressors. Drug concentrations: 12.5, 20, 35 ng/ml BFA, 2 µM GCA, 20 µM AG1478. Results are presented as the mean±s.e.m of three independent experiments (GCA two independent experiments). Right panel: western blot of overexpressed wild-type Flag-Rab1b and Rab1b mutants. Cell lysates were obtained and processed by SDS-PAGE and immunoblotted with anti-Flag epitope antibody. (D) Left panel: reducing Rab1a levels in A549 TRAPPC13 knockdown cells using multiple independent shRNAs induces increased BFA resistance relative to the respective single knockdowns. Cells were treated for 3 days with various BFA concentrations. Six wells were measured per condition and knockdown combination. Graphs display the mean±s.e.m of at least three independent experiments. Right panel: western blot analysis of co-depleted A549 cells to confirm Rab1a knockdown in stable TRAPPC13 knockdown cells. (E) Left panel: viability of A549 cells stably overexpressing wild-type Flag-tagged Rab1b or Rab1b mutants in a TRAPPC13 knockdown background. Cells were treated for 3 days with the indicated BFA concentrations, and six wells were measured per condition and knockdown combination. The graph displays a representative experiment of three, with the mean±s.d. provided. In comparison to TRAPPC13 knockdown cells expressing a control protein (Flag-γTubulin), the same knockdown cells overexpressing Flag-Rab1b(N121I) have a higher survival rate in response to 20 ng/ml BFA. Right panel: western blots of genotypes shown in the survival graph on the left. *P<0.05, **P<0.01, ***P<0.001 (two-tailed Student's t-test).

Fig. 5.

Loss of TRAPPC13 leads to impaired autophagic flux in response to BFA treatment. (A) Stable shTRAPPC13 or shRFP knockdown HeLa cells were treated with 12.5 ng/ml BFA for the indicated times in the presence of 20 nM bafilomycin for the last 3 h of incubation. Untreated control cells were incubated in DMEM for 6 h in the presence (labelled as B) or absence (labelled as C) of bafilomycin A1 (Baf) for the last 3 h of incubation. Protein lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Quantification of relative LC3 II levels was performed as described in the Materials and Methods. Samples shown in the upper two western blot panels were additionally run and evaluated on the same gel as displayed in the lower western blot panel to confirm reduced LC3-II levels in TRAPPC13 knockdown cells compared with control shRFP cells. Data are representative of three independent experiments. (B) HeLa cells stably expressing GFP-LC3 and transduced with shRFP or shTRAPPC13 hairpins were incubated with DMEM in the absence or presence of 20 ng/ml BFA for 24 h with or without Baf for the last 3 h of incubation. The expression of GFP-LC3 was examined by confocal microscopy. LC3 dots in cells were measured as described in the Materials and Methods. Representative quantification results from two independent experiments are shown. **P<0.01 (two-tailed Student's t-test). (C) HeLa cells stably expressing ATG16L1-Flag and transduced with shRFP or shTRAPPC13 hairpins were treated with BFA for 24 h or left untreated. ATG16L1-Flag accumulation was assessed by IF using confocal microscopy. Representative quantification results from two independent experiments are shown. ** P<0.01 (two-tailed Student's t-test). (D) TRAPPC13 interacts with ATG9. The indicated proteins were transiently expressed in HEK293T cells before Flag IP and blotting for endogenous ATG9.

Fig. 6.

Loss of TRAPPC13 increases S. flexneri propagation upon BFA exposure. (A) Increased growth of S. flexneri upon BFA treatment following TRAPPC13 or TRAPPC11 knockdown. Cells were infected with S. flexneri and treated with gentamicin. The cells were then lysed after 24 h growth in medium with or without 20 ng/ml BFA, and dilution plating was used to count the number of CFUs present. Upper panel: TRAPPC13 and TRAPPC11-depleted cells show higher numbers of CFUs compared to control shRNA-infected cells. Data are mean±s.e.m. from three independent experiments performed in triplicate. **P<0.01, *P<0.05. Lower panel: survival of S. flexneri-infected HeLa cells with or without 20 ng/ml BFA for 24 h was determined by a CellTiter-Glo (CTG) assay. Results are representative of three independent experiments; RLU, relative luminescence units. (B) Stable shRFP or shTRAPPC13 HeLa cells were infected with S. flexneri and left untreated or treated with 20 ng/ml BFA for 24 h. Protein lysates were resolved by SDS-PAGE and samples probed with the indicated antibodies. Relative LC3-II levels were calculated. Results are representative of two independent experiments performed in triplicate.