Abstract
The ARF tumor suppressor controls a well-described p53/Mdm2-dependent oncogenic stress checkpoint. In addition, ARF has recently been shown to localize to mitochondria, and to induce autophagy; however, this has never before been demonstrated for endogenous ARF, and the molecular basis for this activity of ARF has not been elucidated. Using an unbiased mass spectrometry-based approach, we show that mitochondrial ARF interacts with the Bcl2 family member Bcl-xl, which normally protects cells from autophagy by inhibiting the Beclin-1/Vps34 complex, which is essential for autophagy. We find that increased expression of ARF decreases Beclin-1/Bcl-xl complexes in cells, thereby providing a basis for ARF-induced autophagy. Our data also indicate that silencing p53 leads to high levels of ARF and increased autophagy, thereby providing a possible basis for the finding by others that p53 inhibits autophagy. The combined data support the premise that ARF induces autophagy in a p53-independent manner in part by virtue of its interaction with Bcl-xl.
Highlights
Autophagy is an evolutionarily conserved homeostatic process whereby cytosolic components are targeted for removal or turnover in membrane-bound compartments
We report the identification of Bcl-xl as a mitochondrial ARF-binding protein, and show that ARF-mediated autophagy is enhanced in cells with Bcl-xl silenced
We induced ARF in these cells using doxycycline, and mitochondria were purified using our previously published protocols [15]; the purity of these mitochondria was verified by the enrichment for the mitochondrial proteins BAK, cytochrome c, and GRP75, relative to cytosol or whole cell lysate (Fig. 1A)
Summary
Autophagy is an evolutionarily conserved homeostatic process whereby cytosolic components are targeted for removal or turnover in membrane-bound compartments We report the identification of Bcl-xl as a mitochondrial ARF-binding protein, and show that ARF-mediated autophagy is enhanced in cells with Bcl-xl silenced. Western analysis indicated that a significant fraction of LC3 accumulated in the faster mobility form (LC3 II) following ARF induction, but not in doxycycline-treated parental cells (Fig. 2A).
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