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GE and Science Prize

Endonuclease G Regulates Cellular Fate

Sabrina Büttner

Though the apoptotic mode of cell death holds widespread biological significance, being involved in development, differentiation, tissue homeostasis, and a variety of pathological conditions, yeast altruism in the form of apoptosis is a quite young field of study. The phenomenon of yeast cells undergoing apoptosis has long been contentious, on the one hand due to the assumed lack of conserved yeast homologs of the basic molecular machinery executing apoptosis in higher eukaryotes, and on the other hand due to the general belief that yeast as a unicellular organism would seem to benefit most from self-preservation.

However, within the past decade a plethora of studies dealing with yeast apoptosis repealed both assumptions. Yeast has not only been shown to die exhibiting typical hallmarks of apoptosis, but has been demonstrated to commit suicide for good reasons. The death of old, infertile, virus-infected, or otherwise damaged cells during various physiological processes such as mating, sporulation, colony development, and aging ensures the survival of the clonal population and thus introduces the concept of an altruistic aging and death program (1). In addition, the crucial components of the self-destructive enzymatic machinery responsible for apoptotic cell death have been discovered in yeast, and various conserved mitochondrial, proteasomal, and histone-regulated pathways have been delineated (2, 3).

In organisms ranging from yeast to mammals, fragmentation of nuclear DNA constitutes a typical apoptotic event eventually leading to cell death. This complex process involves and is executed by various nucleases, harboring distinct activities and specificities, which may or may not act cooperatively to promote stepwise chromosomal fragmentation (4). One of the major human apoptotic nucleases is the mitochondrial endonuclease G (EndoG), whose translocation to the nucleus coincides with large-scale DNA fragmentation (5). Though EndoG is an intensively studied molecule with suggested implications in various cancer forms as well as degenerative diseases, considerable concerns have been raised against EndoG as a causative player in apoptotic cell death. The nuclear localization of EndoG during several diseases was argued to be merely correlative, indicating that EndoG was "just at the wrong place in the wrong time" (6). In addition, contradictory data were obtained applying different EndoG knockout mice, ranging from early embryonic lethality to no apparent phenotype at all (7-9).

Identifying and characterizing yeast EndoG, we could dissect vital and lethal functions of this mitochondrial nuclease and elucidate the so far nebulous molecular mechanisms of EndoG-mediated cell killing (10). The nuclear occurrence of yeast EndoG not only coincided with but was essential for apoptotic DNA fragmentation. Mutation of the catalytically active site completely abolished apoptosis, whereas exclusion from mitochondria strongly enhanced death mediated by yeast EndoG. The question arose of how EndoG might escape from mitochondria, shuttle into the nucleus, and eventually bind to DNA for fragmentation, either autonomously or dependent on cofactors necessary for binding or cleavage. A biochemical approach to identify interactors of yeast EndoG provided further insights into the mechanisms of EndoG translocation and cell killing. Among identified interacting proteins of yeast EndoG were several capable of either completely inhibiting or reducing cell death upon EndoG expression. Interestingly, yeast homologs of components of the mitochondrial permeability transition pore were found to interact with and to be indispensable for apoptotic death via EndoG. Furthermore, nuclear import was identified as an essential step during this death process, as the deletion of one specific karyopherin (KAP123) was able to abolish death upon EndoG expression. Within the nucleus, specific phosphorylation of histone H2B at serine 10 and attachment of EndoG to H2B was found to be required for its deadly nucleolytic action (10).

Referring to the quite controversial results obtained in mammalian cell culture regarding caspase dependency or caspase independency of EndoG, we could exclude an impact of the yeast caspase YCA1 on EndoG-mediated cell death in any tested apoptotic scenario. Thus, we picture a caspase-independent apoptotic pathway for cell death execution via EndoG that successively involves mitochondrial pore opening, nuclear import, and chromatin association (see the figure). As EndoG has been implicated to play a role in several degenerative disorders, the identified rate-limiting factors for cell death execution via EndoG could potentially influence the outcome of these pathologies.


A potential pathway for yeast EndoG-mediated apoptosis. Components of the yeast permeability transition pore (VDAC, voltage-dependent anion channel; AAC, ADP/ATP translocator), the karyopherin KAP123, and phosphorylation of histone H2B are successively involved in EndoG-mediated death.

At first glance quite surprisingly, inhibition of apoptosis via deletion of yeast EndoG was only detectable upon increase of mitochondrial respiration. On the contrary, deletion of EndoG along with repressed oxidative phosphorylation drastically enhanced necrotic death. This suggests that EndoG, like other death effectors, exerts vital functions besides its role in apoptosis execution. Several years ago, mammalian EndoG was shown to generate primers necessary for mitochondrial DNA replication (11). In this line, yeast EndoG participates in cell proliferation, as its deletion manifested in a prominent G2 arrest (10). Aside from that, we came across another cellular function of EndoG outside the apoptosis realm. Taking a closer look at the distribution of cells within distinct phases of the cell cycle, hardly any polyploid cells were present in an EndoG-deleted yeast population (compared with 10 to 20% in wild-type cultures). Thus, EndoG seemed to be essential for the maintenance of polyploid cells. Intriguingly, these effects proved to be conserved in and valuable for mammalian cells, since depletion of human EndoG provoked death in tetraploid but not in diploid colon cancer cells (12). In a recent paper, the viability of polyploid cells has been linked to proteins required for homologous recombination and genomic stability (13). As mammalian EndoG was suggested to influence the frequency of recombination events in the herpes simplex virus -- 1a sequence (14), it is tempting to speculate that EndoG directly or indirectly participates in genome maintenance by homologous recombination. Still, the profound mechanisms through which EndoG contributes to the survival of polyploid cells remain elusive. Polyploidization occurs frequently during evolution, metazoan development, and disease, and often constitutes an important step toward tumor development. As polyploid tumor cells are particularly resistant against a variety of DNA-damaging agents used in anticancer chemotherapy, the unique susceptibility to killing by EndoG depletion might bear the potential to selectively eliminate polyploid cancer cells.

References

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  2. T. Eisenberg, S. Büttner, G. Kroemer, F. Madeo, Apoptosis 12, 1011 (2007).
  3. F. Madeo et al., Curr. Opin. Microbiol. 7, 655 (2004).
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  6. P. G. Ekert, D. L. Vaux, Curr. Opin. Cell Biol. 17, 626 (2005).
  7. K. K. David, M. Sasaki, S. W. Yu, T. M. Dawson, V. L. Dawson, Cell Death. Differ. 13, 1147 (2006).
  8. R. A. Irvine et al., Mol. Cell. Biol. 25, 294 (2005).
  9. J. Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 100, 15782 (2003).
  10. S. Büttner et al., Mol. Cell 25, 233 (2007).
  11. J. Cote, A. Ruiz-Carrillo, Science 261, 765 (1993).
  12. S. Büttner et al., Cell Cycle 6, 1072 (2007).
  13. Z. Storchova et al., Nature 443, 541 (2006).
  14. K. J. Huang, B. V. Zemelman, I. R. Lehman, J. Biol. Chem. 277, 21071 (2002).

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Science. ISSN 0036-8075 (print), 1095-9203 (online)