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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 familyinteracting 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.

The protein kinase inhibitor staurosporine is one of the most potent and frequently used proapoptotic stimuli, although its mechanism of action is poorly understood. Here, we show that staurosporine as well as its analog 7-hydroxystaurosporine (UCN-01) not only trigger the classical mitochondrial apoptosis pathway but, moreover, activate an additional novel intrinsic apoptosis pathway. Unlike conventional anticancer drugs, staurosporine and UCN-01 induced apoptosis in a variety of tumor cells overexpressing the apoptosis inhibitors Bcl-2 and Bcl-x(L). Furthermore, activation of this novel intrinsic apoptosis pathway by staurosporine did not rely on Apaf-1 and apoptosome formation, an essential requirement for the mitochondrial pathway. Nevertheless, as demonstrated in caspase-9-deficient murine embryonic fibroblasts, human lymphoma cells, and chicken DT40 cells, staurosporine-induced apoptosis was essentially mediated by caspase-9. Our results therefore suggest that, in addition to the classical cytochrome c/Apaf-1-dependent pathway of caspase-9 activation, staurosporine can induce caspase-9 activation and apoptosis independently of the apoptosome. Since staurosporine derivatives have proven efficacy in clinical trials, activation of this novel pathway might represent a powerful target to induce apoptosis in multidrug-resistant tumor cells.

Modification by the ubiquitin-like protein SUMO affects hundreds of cellular substrate proteins and regulates a wide variety of physiological processes. While the SUMO system appears to predominantly target nuclear proteins and, to a lesser extent, cytosolic proteins, hardly anything is known about the SUMOylation of proteins targeted to membrane-enclosed organelles. Here, we identify a large set of structurally and functionally unrelated mitochondrial proteins as substrates of the SUMO pathway in yeast. We show that SUMO modification of mitochondrial proteins does not rely on mitochondrial targeting and, in fact, is strongly enhanced upon import failure, consistent with the modification occurring in the cytosol. Moreover, SUMOylated forms of mitochondrial proteins particularly accumulate in HSP70- and proteasome-deficient cells, suggesting that SUMOylation participates in cellular protein quality control. We therefore propose that SUMO serves as a mark for nonfunctional mitochondrial proteins, which only sporadically arise in unstressed cells but strongly accumulate upon defective mitochondrial import and impaired proteostasis. Overall, our findings provide support for a role of SUMO in the cytosolic response to aberrant proteins.

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