Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Abstract

Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.

Figure 1.

Generation of Atg13-deficient DT40 cells. (A) Atg13-deficient DT40 B cell lines (atg13^−/−) were generated by targeted disruption of both atg13 alleles. Successful targeting was confirmed by genomic PCR using primers specific for wild-type, hisD- or bsr-targeted alleles (see Fig. S1 for details). Equal amounts of protein from cleared cellular lysates of either wild-type (wt) cells (lane 1), atg13^+/− clones (lanes 2–3) and atg13^−/− clones (lanes 4–7) were analyzed for Atg13 and GAPDH by immunoblotting. The asterisk indicates an unspecific background band. (B) Wild-type and atg13^−/− DT40 cells, retrovirally transfected with cDNA encoding mCitrine-LC3B, were treated with 10 nM bafilomycin A₁ (BafA1) or DMSO (control) for 6 h and directly visualized by confocal laser scanning microscopy (bars: 10 µm). (C) Cleared cellular lysates of cells described in (B) were subjected to anti-Atg13 and anti-LC3 immunoblotting. LC3-II/LC3-I ratios are represented as mean values of three independent experiments ± SEM (D) atg13^−/− cells reconstituted with HA-tagged full-length chicken Atg13 isoform A (lanes 9–12) were incubated in normal growth medium (RPMI) or starvation medium (EBSS) for 1 h in the presence or absence of 10 nM (BafA1). Equal protein amounts from cleared cellular lysates were analyzed for Atg13, GAPDH and LC3 by immunoblotting. As control, wild-type cells (lanes 1–4) and atg13^−/− cells reconstituted with empty vector (lanes 5–8) were analyzed in parallel. LC3-II/GAPDH ratios are represented as mean values of three independent experiments ± SEM.

Figure 2.

Atg13 is essential for autophagosome generation (A) DT40 wild-type and (B) atg13^−/− cells were incubated in normal growth medium (control) or EBSS for 2 h. Cells were fixed and analyzed by transmission electron microscopy. A representative cell from each condition is shown in two different magnifications. Autophagosomes are indicated by black arrow heads in the image with higher magnification, swollen mitochondria are indicated by asterisks (bars: 500 nm). (C) Wild-type and (D) atg13^−/− cells, retrovirally transfected with cDNA encoding mRFP-EGFP-rLC3 were incubated in EBSS for 2 h and analyzed by confocal laser scanning microscopy. The mRFP signal is shown in red and the EGFP signal in green in the merged image. Autolysosomes are indicated by white arrow heads (bars: 10 µm). The percentage of autolysosome containing cells (>200 cells/experiment) is represented as mean ± range of two independent experiments.

Figure 3.

Ulk1 and Ulk2 are dispensable for autophagy induction in DT40 cells. (A) DT40 cells deficient for Ulk1 (ulk1^−/−), Ulk2 (ulk2^−/−) or Ulk1 and Ulk2 (ulk1/2^−/−) were generated by gene targeting and loss of wild-type alleles was confirmed by genomic PCR (for details see Figure S2). The absence of ulk1 and ulk2 transcripts was verified by RT-PCR. (B) Wild-type cells and two independent double deficient ulk1/2^−/− clones (2–6 and 17–16) were incubated in full medium (RPMI) or EBSS in the presence or absence of 10 nM BafA1 for 1 h. Equal amounts of protein from cleared cellular lysates were analyzed for Atg13, GAPDH and LC3 by immunoblotting. LC3-II/GAPDH ratios are represented as mean values of three independent experiments ± SEM (C) ulk1/2^−/− cells (clone 2–6) were incubated in normal growth medium (control) or EBSS for 2 h, cells were fixed and analyzed by transmission electron microscopy. For starvation condition, a representative cell is shown in two different magnifications. Autophagosomes are indicated by black arrow heads in the image with higher magnification (bars: 500 nm). (D) ulk1/2^−/− cells (clone 2–6) retrovirally transfected with cDNA encoding mRFP-EGFP-rLC3 were incubated in EBSS for 2 h and analyzed by confocal laser scanning microscopy. The mRFP signal is shown in red and the EGFP signal in green in the merged image. Autolysosomes are indicated by white arrow heads (bars: 10 µm). The percentage of cells with autolysosomes (> 100 cells/experiment) is represented as mean value ± SD from three independent experiments. n.s. indicates a non-significant difference between wild-type and ulk1/2^−/− cells (Student’s t-test).

Figure 4.

FIP200 binding site in Atg13 is encoded by exon 12. (A) Schematic representation of Atg13 splice variants (named A-G) amplified from DT40 cells. (B) The relative abundance of these splice variants was analyzed by qRT-PCR using splice variant-specific primer combinations (see Supplementary Material and Methods and Fig. S8A). (C) atg13^−/− cells were reconstituted with HA-tagged versions of splice variants A-G and lysates were subjected to anti-HA immunoprecipitation and analyzed for Atg13 and FIP200 by immunoblotting. Asterisk indicates an unspecific background band.

Figure 5.

FIP200 binding site is essential for Atg13 function. (A) atg13^−/− cells were reconstituted with HA-tagged versions of splice variants A-G and incubated in full medium (RPMI) or EBSS for 1 h in the presence or absence of 10 nM BafA1. Equal protein amounts from cleared cellular lysates were analyzed for Atg13, HSP90 and LC3 by immunoblotting. LC3-II/HSP90 ratios from three independent experiments are represented as mean values ± SEM (B) atg13^−/− retrovirally transfected with cDNAs encoding either HA-Atg13 isoform A (full-length) or HA-Atg13 isoform C (?exon12) or the empty vector were stably transfected with pmRFP-EGFP-rLC3. Cells were incubated in EBSS for 2 h and analyzed by confocal laser scanning microscopy. The mRFP signal is displayed in red and the EGFP signal in green in the merged image. The percentage of autolysosome containing cells (>100 cells/experiment) from three independent experiments is represented as mean value ± SD *p < 0.05, Student’s t-test.

Figure 6.

Model of differential Atg13-dependent autophagy induction pathways. Based on previous findings it has been proposed that mTORC1 associates with the Ulk-Atg13-FIP200 complex under nutrient-rich conditions, phosphorylates Ulk1/2 and Atg13 at inhibitory sites and suppresses Ulk1/2 kinase activity. Following starvation or direct mTORC1 inhibition, this negative regulation is released, Ulk1/2 autophosphorylates itself at activating sites and subsequently phosphorylates both Atg13 and FIP200. This in turn leads to autophagy induction (mTORC1-Ulk1/2-axis). In cells that do respond to mTORC1 inhibition by autophagy induction and do depend on Ulk1 and Ulk2, this pathway is most likely favored. However, the incomparable phenotypes of atg13^−/− and ulk1/2^−/− DT40 cells let us assume that Atg13 has a more basal function that is not necessarily regulated by Ulk1 or Ulk2, but necessarily requires FIP200 binding capacity. Thus, Atg13 surprisingly has an additional role, besides its proposed function as an adaptor molecule that bridges Ulk1/2 and its substrate FIP200. Several modes of action are conceivable: (A) Atg13 acts in a kinase-independent way, e.g. by stabilizing or recruiting FIP200, (B) the Atg13-FIP200 complex is regulated in a kinase-independent manner, or (C) Atg13-FIP200 is regulated by other kinases than Ulk1/2.