Robert Eskes

The University of Texas Southwestern Medical Center

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Jean‐Claude Martinou

University of Geneva

Bruno Antonsson

Merck Serono (Switzerland)

Sylvie Montessuit

University of Geneva

Solange Desagher

Université de Montpellier

Philip S. Perlman

Howard Hughes Medical Institute

Alan M. Lambowitz

The University of Texas at Austin

Sandra Lauper

Anthony C. Nichols

London Health Sciences Centre

Astrid Osen‐Sand

ETH Zurich

John C. Kennell

Saint Louis University

Cooperative Institutions

The University of Texas Southwestern Medical Center

University of Lausanne

Howard Hughes Medical Institute

The Ohio State University

Instituto Biofisika

Harvard University

Centre National de la Recherche Scientifique

ETH Zurich

University of Vienna

Vienna Biocenter

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Multiple Homing Pathways Used by Yeast Mitochondrial Group II Introns

Molecular and Cellular Biology (2000)

Robert Eskes Lu Liu Hongwen Ma Michael Y. Chao Lorna M. Dickson

The yeast mitochondrial DNA group II introns aI1 and aI2 are retroelements that insert site specifically into intronless alleles by a process called homing. Here, we used patterns of flanking marker coconversion in crosses with wild-type and mutant aI2 introns to distinguish three coexisting homing pathways: two that were reverse transcriptase (RT) dependent (retrohoming) and one that was RT independent. All three pathways are initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, with the sense strand cleaved by partial or complete reverse splicing, and the antisense strand cleaved by the intron-encoded protein. The major retrohoming pathway in standard crosses leads to insertion of the intron with unidirectional coconversion of upstream exon sequences. This pattern of coconversion suggests that the major retrohoming pathway is initiated by target DNA-primed reverse transcription of the reverse-spliced intron RNA and completed by double-strand break repair (DSBR) recombination with the donor allele. The RT-independent pathway leads to insertion of the intron with bidirectional coconversion and presumably occurs by a conventional DSBR recombination mechanism initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, as for group I intron homing. Finally, some mutant DNA target sites shift up to 43% of retrohoming to another pathway not previously detected for aI2 in which there is no coconversion of flanking exon sequences. This new pathway presumably involves synthesis of a full-length cDNA copy of the inserted intron RNA, with completion by a repair process independent of homologous recombination, as found for the Lactococcus lactis Ll.LtrB intron. Our results show that group II intron mobility can occur by multiple pathways, the ratios of which depend on the characteristics of both the intron and the DNA target site. This remarkable flexibility enables group II introns to use different recombination and repair enzymes in different host cells.

Group II intron

Homing endonuclease

10.1128/mcb.20.22.8432-8446.2000

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Citations (77)

Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria

Biochemical Journal (2000)

Bruno Antonsson Sylvie Montessuit Sandra Lauper Robert Eskes Jean‐Claude Martinou

Bax is a Bcl-2-family protein with pro-apoptotic activity that can form channels in lipid membranes. The protein has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. Recombinant human Bax isolated in the presence of detergent was found to be present as an oligomer with an apparent molecular mass of approx. 160000 Da on gel filtration. When Bax was isolated in the absence of detergent the purified protein was monomeric with an apparent molecular mass of 22000 Da. Bax oligomers formed channels in liposomes and triggered cytochrome c release from isolated mitochondria, whereas monomeric Bax was inactive in both respects. Incubation of the monomeric Bax with 2% octyl glucoside induced formation of oligomers that displayed channel-forming activity in liposomes and triggered cytochrome c release from mitochondria. Triton X-100, Nonidet P-40 and n-dedecyl maltoside also activated monomeric Bax, whereas CHAPS had no activating effect. In cytosolic extracts from mouse liver, Bax migrated at a molecular mass of 24000 Da on gel filtration, whereas after incubation of the cytosol with 2% octyl glucoside Bax migrated at approximately 140000 Da. These results show that oligomeric Bax possesses channel-forming activity whereas monomeric Bax has no such activity.

Molecular mass

Oligomer

Bcl-2-associated X protein

10.1042/bj3450271

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Citations (620)

Bid-induced Conformational Change of Bax Is Responsible for Mitochondrial Cytochrome c Release during Apoptosis

The Journal of Cell Biology (1999)

Solange Desagher Astrid Osen‐Sand Anthony C. Nichols Robert Eskes Sylvie Montessuit

Here we report that in staurosporine-induced apoptosis of HeLa cells, Bid, a BH3 domain containing protein, translocates from the cytosol to mitochondria. This event is associated with a change in conformation of Bax which leads to the unmasking of its NH2-terminal domain and is accompanied by the release of cytochrome c from mitochondria. A similar finding is reported for cerebellar granule cells undergoing apoptosis induced by serum and potassium deprivation. The Bax-conformational change is prevented by Bcl-2 and Bcl-xL but not by caspase inhibitors. Using isolated mitochondria and various BH3 mutants of Bid, we demonstrate that direct binding of Bid to Bax is a prerequisite for Bax structural change and cytochrome c release. Bcl-xL can inhibit the effect of Bid by interacting directly with Bax. Moreover, using mitochondria from Bax-deficient tumor cell lines, we show that Bid- induced release of cytochrome c is negligible when Bid is added alone, but dramatically increased when Bid and Bax are added together. Taken together, our results suggest that, during certain types of apoptosis, Bid translocates to mitochondria and binds to Bax, leading to a change in conformation of Bax and to cytochrome c release from mitochondria.

Mitochondrial apoptosis-induced channel

Staurosporine

Apoptosome

Bcl-2-associated X protein

Conformational change

10.1083/jcb.144.5.891

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Citations (1,256)

Mobility of Yeast Mitochondrial Group II Introns: Engineering a New Site Specificity and Retrohoming via Full Reverse Splicing

Cell (1997)

Robert Eskes Jian Yang Alan M. Lambowitz Philip S. Perlman

Group II intron

Homing endonuclease

10.1016/s0092-8674(00)81932-7

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Citations (115)

Transposition of group II intron al1 in yeast and invasion of mitochondrial genes at new locations

Nature (1993)

Manfred W. Mueller Martina Allmaier Robert Eskes Rudolf J. Schweyen

Group II intron

Transposition (logic)

10.1038/366174a0

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Citations (100)

Bax-induced Cytochrome C Release from Mitochondria Is Independent of the Permeability Transition Pore but Highly Dependent on Mg2+ Ions

The Journal of Cell Biology (1998)

Robert Eskes Bruno Antonsson Astrid Osen‐Sand Sylvie Montessuit Christoph Richter

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homologue of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). We now report that Bax is sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A-sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg2+ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A, the other Bax dependent, Mg2+ sensitive but cyclosporin insensitive.

Apoptosome

Bcl-2-associated X protein

Mitochondrial apoptosis-induced channel

Voltage-dependent anion channel

Bcl-2 Family

10.1083/jcb.143.1.217

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Citations (648)

The Destabilization of Lipid Membranes Induced by the C-terminal Fragment of Caspase 8-cleaved Bid Is Inhibited by the N-terminal Fragment

Journal of Biological Chemistry (2000)

Grzegorz Kudla Sylvie Montessuit Robert Eskes Catherine Berrier Jean‐Claude Martinou

Bid is a proapoptotic, BH3-domain-only member of the Bcl-2 family. In Fas-induced apoptosis, Bid is activated through cleavage by caspase 8 into a 15.5-kDa C-terminal fragment (t^cBid) and a 6.5 kDa N-terminal fragment (tⁿBid). Following the cleavage, t^cBid translocates to the mitochondria and promotes the release of cytochromec into the cytosol by a mechanism that is not understood. Here we report that recombinant t^cBid can act as a membrane destabilizing agent. t^cBid induces destabilization and breaking of planar lipid bilayers without appearance of ionic channels; its destabilizing activity is comparable with that of Bax and at least 30-fold higher than that of full-length Bid. Consistently, t^cBid, but not full-length Bid, permeabilizes liposomes at physiological pH. The destabilizing effect of t^cBid on liposomes and planar bilayers is independent of the BH3 domain. In contrast, mutations in the BH3 domain impair t^cBid ability to induce cytochrome c release from mitochondria. The permeabilizing effect of t^cBid on planar bilayers, liposomes, and mitochondria can be inhibited by tⁿBid. In conclusion, our results suggest a dual role for Bid: BH3-independent membrane destabilization and BH3-dependent interaction with other proteins. Moreover, the dissociation of Bid after cleavage by caspase 8 represents an additional step at which apoptosis may be regulated.

Cleavage (geology)

C-terminus

10.1074/jbc.m003807200

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Citations (126)

Bid Induces the Oligomerization and Insertion of Bax into the Outer Mitochondrial Membrane

Molecular and Cellular Biology (2000)

Robert Eskes Solange Desagher Bruno Antonsson Jean‐Claude Martinou

AbstractIn many types of apoptosis, the proapoptotic protein Bax undergoes a change in conformation at the level of the mitochondria. This event always precedes the release of mitochondrial cytochrome c, which, in the cytosol, activates caspases through binding to Apaf-1. The mechanisms by which Bax triggers cytochrome c release are unknown. Here we show that following binding to the BH3-domain-only proapoptotic protein Bid, Bax oligomerizes and then integrates in the outer mitochondrial membrane, where it triggers cytochromec release. Bax mitochondrial membrane insertion triggered by Bid may represent a key step in pathways leading to apoptosis. ACKNOWLEDGMENTSWe thank S. Arkinstall and K. Maundrell for critical reading of the manuscript, C. Herbert for artwork, and T. Wells for encouraging support.Part of this work was also supported by grants from the European Community (Biotech grant BIO4CT96 0774 to J.-C. Martinou).

Mitochondrial apoptosis-induced channel

Bcl-2 Family

Bcl-2-associated X protein

10.1128/mcb.20.3.929-935.2000

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Citations (1,151)

The Release of Cytochrome c from Mitochondria during Apoptosis of NGF-deprived Sympathetic Neurons Is a Reversible Event

The Journal of Cell Biology (1999)

Isabelle Martinou Solange Desagher Robert Eskes Bruno Antonsson Élisabeth André

During apoptosis induced by various stimuli, cytochrome c is released from mitochondria into the cytosol where it participates in caspase activation. This process has been proposed to be an irreversible consequence of mitochondrial permeability transition pore opening, which leads to mitochondrial swelling and rupture of the outer mitochondrial membrane. Here we present data demonstrating that NGF-deprived sympathetic neurons protected from apoptosis by caspase inhibitors possess mitochondria which, though depleted of cytochrome c and reduced in size, remained structurally intact as viewed by electron microscopy. After re-exposure of neurons to NGF, mitochondria recovered their normal size and their cytochrome c content, by a process requiring de novo protein synthesis. Altogether, these data suggest that depletion of cytochrome c from mitochondria is a controlled process compatible with function recovery. The ability of sympathetic neurons to recover fully from trophic factor deprivation provided irreversible caspase inhibitors have been present during the insult period, has therapeutical implications for a number of acute neuropathologies.

Apoptosome

Mitochondrial apoptosis-induced channel

10.1083/jcb.144.5.883

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Citations (297)

Mobile Group II Introns of Yeast Mitochondrial DNA Are Novel Site-Specific Retroelements

Molecular and Cellular Biology (1995)

John V. Moran Steven Zimmerly Robert Eskes John C. Kennell Alan M. Lambowitz

Group II introns aI1 and aI2 of the yeast mitochondrial COXI gene are mobile elements that encode an intron-specific reverse transcriptase (RT) activity. We show here that the introns of Saccharomyces cerevisiae ID41-6/161 insert site specifically into intronless alleles. The mobility is accompanied by efficient, but highly asymmetric, coconversion of nearby flanking exon sequences. Analysis of mutants shows that the aI2 protein is required for the mobility of both aI1 and aI2. Efficient mobility is dependent on both the RT activity of the aI2-encoded protein and a separate function, a putative DNA endonuclease, that is associated with the Zn2+ finger-like region of the intron reading frame. Surprisingly, there appear to be two mobility modes: the major one involves cDNAs reverse transcribed from unspliced precursor RNA; the minor one, observed in two mutants lacking detectable RT activity, appears to involve DNA level recombination. A cis-dominant splicing-defective mutant of aI2 continues to synthesize cDNAs containing the introns but is completely defective in both mobility modes, indicating that the splicing or the structure of the intron is required. Our results demonstrate that the yeast group II intron aI2 is a retroelement that uses novel mobility mechanisms.

Group II intron

10.1128/mcb.15.5.2828

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Citations (124)