HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 19/3/464 04-2316fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guscetti, F. Articles by Denko, N. Search for Related Content PubMed PubMed Citation Articles by Guscetti, F. Articles by Denko, N.

Functional characterization of human proapoptotic molecules in yeast S. cerevisiae

Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, USA

²Correspondence: Room 1245 CCSR South, 269 Campus Dr., Stanford University, Stanford CA 94305, USA. E-mail: ndenko{at}stanford.edu

SPECIFIC AIMS

Yeast is sensitive to heterologous expression of the human proapoptotic Bax protein. We used this characteristic to test members of the large family of human proapoptotic proteins for functional differences in their ability to kill yeast. We tested the direct toxicity of these molecules, as well as their ability to interfere with the function of the antiapoptotic Bcl2 protein.

PRINCIPAL FINDINGS

1. Bax causes reproductive death in yeast, and this toxicity is inhibitable by Bcl2
The expression of human Bax in yeast under the control of a galactose-inducible promoter allows for sensitive analysis of how galactose-responsive Bax expression effects growth of wild-type yeast. The inhibition of growth can be determined in liquid culture by the measure of changes in optical density, or it can be determined on solid media by the measure of the decrease in the number of viable colonies. These two measures have complementary value. Comparing these assays of Bax toxicity, we find that galactose-regulated expression of Bax yields an 2.5-fold inhibition of growth in liquid culture, but up to 50-fold inhibition in terms of colony formation on solid media. The toxicity measured by either means can be completely inhibited by the coexpression of Bcl2. These findings are in agreement with the literature.

2. Of various BH3-only molecules, only tBid has direct toxicity to yeast, and this is inhibitable by Bcl2
In mammalian cells, it is thought that to initiate the apoptotic cascades that one "complete" BH1-3 proapoptotic molecule is necessary to initiate mitochondrial breakdown. The activation of these complete killers can be triggered by the accumulation of a BH3-only proapoptotic molecule. To test the toxicity of BH3-only molecules in the absence of complete BH1-3 molecules, we individually tested a series of prototypic BH3-only proteins for toxicity in yeast. Only tBid showed direct killing comparable to Bax in a galactose-inducible expression system. Like Bax toxicity, this toxicity was Bcl2 inhibitable. Neither Bad, Bnip3, Bnip3L, Noxa, nor Puma showed any toxicity when expressed alone.

3. Of various BH3-only molecules, Bad and Puma are able to interfere with Bcl2’s ability to protect against Bax toxicity
Apoptotic signaling is the result of a balance between proapoptotic and antiapoptotic signals. Therefore, one mechanism by which proapoptotic molecules can initiate a death signal is through the blocking of action of one of the various antiapoptotic molecules such as Bcl2. We tested the BH3-only molecules for this activity by coexpressing them along with Bax and Bcl2 (Fig. 1 ). Using a triple galactose-inducible system, we found that Bad and Puma were capable of interfering with Bcl2’s ability to block Bax toxicity. This effect is not a result of the absolute levels of expression because tBid toxicity was inhibitable in the triple expression system. Bnip3, Bnip3L, and Noxa were still not toxic.

View larger version (27K): [in this window] [in a new window]   Figure 1. Coexpression of multiple Bcl-2 family members reveals functional interactions. A) Average colony formation of 3 independent experiments in which yeast were grown for 48 h expressing Bax alone, Bax along with Bcl-2, and a combination of Bax/Bcl-2 with the additional BH3-only protein as indicated. Note that while Bcl-2 can protect against Bax expression, Bad and Puma are capable of antagonizing Bcl-2’s protective effects, while tBid, BNip3, BNip3L, and Noxa cannot. The relative protection by Bcl-2 is even more evident in panel B, where the ratio of colonies is plotted ± Bcl-2 along with the indicated proteins. The nearly 1000-fold protection against the tBid/Bax combination is due to the additive killing of the two molecules, and the protection seen in panel A. Fold protection against other combinations is less dramatic due to reduced killing of the other pairs of proapoptotic proteins.

4. Neither Bnip3 nor Bnip3L are able to directly activate human caspases in yeast
Data in the literature suggests that Bnip3 can form a direct complex with caspase-3, potentially leading to its activation. By expressing human caspases in yeast, we showed that human caspases can be activated and kill yeast, but the coexpression of Bnip3 or Bnip3L along with effector caspase-3 does not lead to enhanced killing. If there is a functional interaction between Bnip3 and caspases in mammalian cells, then additional interacting partners are required (Fig. 2 B).

View larger version (42K): [in this window] [in a new window]   Figure 2. Yeast does not activate endogenous metacaspase in response to expression of toxic Bcl-2 family members. A) Overexpression of the endogenous yeast metacaspase MCA1 with Bax does not result in synergistic killing. Yeast containing inducible constructs was grown for 48 h in galactose media and the survival relative to vector controls of 3 independent experiments was calculated. Note that MCA1 overexpression alone results in modest toxicity, presumably due to autoactivation. B) Autoactivation of overexpressed MCA. Cells containing either MCA1-expressing or empty vector were grown in glucose or galactose media, after which protein was run and blotted for the FLAG epitope tag. Note the spontaneous cleavage of the 59kDA metacaspase to the activated 39 and 12 kDa products. C) Loss of MCA1 does not result in protection from Bax-induced toxicity. Similar toxicity is seen in 3 independent experiments of wild-type (Fig. 1 ) and Mca1–/– strains (YOR197w) that contain pESCBax after growth for 48 h in glucose media (open bars) or galactose media (filled bars).

5. Endogenous yeast metacaspase Mca1 does not accelerate, nor is it required for Bax toxicity
Recent data suggest that environmentally triggered yeast apoptosis is mediated through an evolutionarily conserved yeast "metacaspase." We therefore determined the necessity of this molecule for Bax toxicity. We found that overexpression of MCA1 along with Bax did not lead to super additive killing (Fig. 2A ). Likewise, Bax showed equal toxicity in wild-type yeast and in yeast in which Mca had been deleted (Fig. 2A ). These results suggest that the mechanisms responsible for environmentally initiated apoptosis in yeast probably do not utilize a classical BH3-like molecule.

CONCLUSIONS AND SIGNIFICANCE

Using yeast as a model system to study the biology of human proapoptotic molecules yields information not only about the putative function(s) of these proteins in mammalian cells, but about the newly identified apoptotic death in yeast. Proapoptotic molecules can be biochemically tested for structural motifs and/or physical interactions, but these results show the first genetically defined assay for functional, in vivo interactions.

It is thought that in mammalian cells the apoptotic process is initiated by activation of one or more BH3-only proapototic protein(s), and this signal is then executed through the actions of a complete BH1-3 proapoptotic molecule such as Bax or Bak. Gene knockout studies support this model, and show that Bax/Bak double knockouts are particularly resistant to apoptotic stimuli. However, the complex nature of the BH3-only multigene family (with at least 12 members) makes the extension of these compound knockout studies very difficult. We have taken the converse approach and started in a model organism (yeast Saccharomyces cerevisiae) that does not contain any recognized Bcl2 family members, and, as such, represents a complete knockout. In this true null background we have begun to introduce proapoptotic proteins in various combinations in order to determine the functional consequences of their expression. Using this system, we have identified three functional classes of BH3-only proteins, based on their ability to kill in yeast (Fig. 3 ). These findings are consistent with models in the literature that propose alternative mechanisms for apoptotic activation. One caveat of this cross-species assay system is that the results in one species may not pertain to a similar function in another species. Despite this potential drawback, we can still identify prospective areas for investigation, and then test the validity of our conclusion in more standard model systems.

View larger version (27K): [in this window] [in a new window]   Figure 3. Model showing alternative mechanisms for proapoptotic protein toxicity in yeast. A) Expression of Bax or tBid is sufficient for killing in yeast, presumably through homodimerization at the mitochondria. B) Expression of Bad or Puma is toxic only when it is coexpressed with Bax/Bcl2. Bad or Puma is presumed to disrupt nontoxic Bax/Bcl2 dimers, releasing Bax to form either toxic Bax/Bad heterodimers or toxic Bax/Bax homodimers. Bnip3, Bnip3L, and Noxa are not toxic to yeast, despite dimer formation and mitochondrial targeting.

Recent data have identified an apoptotic-like death in fission yeast. It was not originally thought to be a benefit for a unicellular organism to have such a system, but these studies suggest that it may be advantageous during times of saturated growth when the entire culture is environmentally stressed. The execution of this apoptotic cascade is reported to be through the metacaspase MCA1 that was identified based on its limited structural similarity to the mammalian caspases. Loss of MCA1 reduces environmentally stimulated apoptosis, but it is unclear how this molecule is activated, as yeasts do not contain classical Bcl2 family members. We show that neither overexpression of Mca1 nor its complete loss leads to altered sensitivity of these yeast to Bax. The lack of mechanistic interaction between Bax expression and MCA1 (Fig. 2) suggests a novel means by which MCA1 is activated by environmental stress.

The lack of a Bax/MCA interaction raises questions as to how Bax is actually killing yeast. Several groups have identified mutant yeast that are resistant to killing by Bax, and all have identified different mutations. We have tried to identify Bax-resistant mutants using the recently constructed yeast deletion pool and had only marginal success. The lack of a specific deletion that fully rescues Bax lethality implies one of two possibilities: 1) multiple pathways exist in yeast for Bax to kill so that individual mutations do not confer resistance; or 2) Bax kills through its action on an essential gene, and so the deletion strain is not represented in the pool. We favor the first explanation due to the disparate identification of Bax-resistant yeast in the literature, and to our limited success in identifying mutants that show modest resistance to Bax. The inability to establish a specific molecular target for Bax in yeast, however, does not preclude its usefulness as a model system, as many of the characteristics of Bax killing are recapitulated in yeast. The localization at the mitochondria, the necessity for the BH3 domain, and the inhibition of killing by Bcl2 are all similar in yeast and human cells, and as such, show how valuable information can be obtained in this system.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2316fje;

¹ Current address: Institute of Veterinary Pathology, Vetsuisse Faculty of the University of Zurich, Winterthurerstr. 268, Zurich CH-8057, Switzerland.

This article has been cited by other articles:

				O. Terrones, A. Etxebarria, A. Landajuela, O. Landeta, B. Antonsson, and G. Basanez BIM and tBID Are Not Mechanistically Equivalent When Assisting BAX to Permeabilize Bilayer Membranes J. Biol. Chem., March 21, 2008; 283(12): 7790 - 7803. [Abstract] [Full Text] [PDF]

				L. Bonneau, Y. Ge, G. E. Drury, and P. Gallois What happened to plant caspases? J. Exp. Bot., February 13, 2008; (2008) erm352v1. [Abstract] [Full Text] [PDF]

				I. Kissova, L.-T. Plamondon, L. Brisson, M. Priault, V. Renouf, J. Schaeffer, N. Camougrand, and S. Manon Evaluation of the Roles of Apoptosis, Autophagy, and Mitophagy in the Loss of Plating Efficiency Induced by Bax Expression in Yeast J. Biol. Chem., November 24, 2006; 281(47): 36187 - 36197. [Abstract] [Full Text] [PDF]

				S. Buttner, T. Eisenberg, E. Herker, D. Carmona-Gutierrez, G. Kroemer, and F. Madeo Why yeast cells can undergo apoptosis: death in times of peace, love, and war J. Cell Biol., November 20, 2006; 175(4): 521 - 525. [Abstract] [Full Text] [PDF]

				N.-N. Zhang, D. D. Dudgeon, S. Paliwal, A. Levchenko, E. Grote, and K. W. Cunningham Multiple Signaling Pathways Regulate Yeast Cell Death during the Response to Mating Pheromones Mol. Biol. Cell, August 1, 2006; 17(8): 3409 - 3422. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 19/3/464 04-2316fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guscetti, F. Articles by Denko, N. Search for Related Content PubMed PubMed Citation Articles by Guscetti, F. Articles by Denko, N.