Response of yeast to the regulated expression of proteins in the Bcl-2 family

Abstract

The mechanisms by which pro-apoptotic members of the Bcl-2 family of proteins promote the release of mitochondrial factors like cytochrome c, subsequently activating the apoptotic cascade, or by which anti-apoptotic family members block this release, are still not understood. When expressed in yeast, Bcl-2 family members act directly upon conserved mitochondrial components that correspond to their apoptotic substrates in mammalian cells. Here we describe a system in which the levels of representative pro- and anti-apoptotic members of the Bcl-2 family can be regulated independently in yeast. Using this system, we have focused on the action of the anti-apoptotic family member Bcl-x(L), and have defined the quantitative relationships that underlie the antagonistic action of this protein on the lethal consequences of expression of the pro-apoptotic family member Bax. This system has also allowed us to demonstrate biochemically that Bcl-x(L) has two actions at the level of the mitochondrion. Bcl-x(L) is able to inhibit the stable integration of Bax into mitochondrial membranes, as well as hinder the action of Bax that does become stably integrated into these membranes. Taken together, our results suggest that both the functional and biochemical actions of Bcl-x(L) may be based on the ability of this molecule to disrupt the interaction of Bax with a resident mitochondrial target that is required for Bax action. Finally, we confirm that VDAC (voltage-dependent anion channel) is not required for the functional responses observed following the expression of either pro- or anti-apoptotic members of the Bcl-2 family.

Studies of the Interaction of Substituted Mutants of BAX with Yeast Mitochondria Reveal That the C-terminal Hydrophobic α-Helix Is a Second ART Sequence and Plays a Role in the Interaction with Anti-apoptotic BCL-xL

“…As noted before, from the proposed structure of soluble BAX (10), a part of ␣9 is localized in close proximity to the BH3 domain, which is widely considered as the domain responsible for the dimerization of BCL-2 family members. It is noteworthy that, from a study in yeast (39), BCL-x L may have at least two inhibitory effects in the process of BAX activation: it would prevent the addressing/ insertion step, and also prevent the acquisition of the final conformation able to drive the release of cytochrome c. From experiments realized with a hemagglutinin-tagged BAX in yeast, Polcic and Forte (39) suggested that BCL-x L was acting both on the "insertion" step and on the "activation" step. Similarly, the cytochrome c release activity of a c-Myc-tagged BAX is prevented by BCL-x L (24).…”

Mitochondrial carriers and pores: Key regulators of the mitochondrial apoptotic program?

“…This theory is supported by data showing no influence of BAX on the properties of VDAC channels under a variety of conditions [30], and an inability to detect a direct interaction between BAX and VDAC in several experimental systems [30,31]. The holders of this theory also question the results obtained with the expression of BAX in the VDAC-disrupted yeast strain, as it appears that although release of Cyt c was blocked, there was no influence on overall cell death, induced by similar BAX levels in both disrupted and wild type cells [32]. Furthermore, it was reported that removal of growth factors leads to adenine nucleotide exchange deficiency, most likely accounted by the closure of VDAC.…”

Cardiolipin and phosphatidylglycerol are not required for the in vivo action of Bcl-2 family proteins

“…Earlier studies had demonstrated that the level of Bax expression under these conditions is proportional to the concentration of doxycycline present in the media; higher concentrations lead to higher levels of Bax expression. 25 Accordingly, Dcrd1, Dpgs1 and wild-type cells were transformed with plasmid-containing sequences encoding an Nterminally, HA-tagged Bax protein downstream of the Tetinducible promoter in pCM252 in strain CML282, allowing us to regulate expression of Bax following addition of doxycycline to the media. 25 When grown on media containing 1 mg/ml doxycycline, all three strains express similar levels of Bax (Figure 1b).…”

“…In wild-type cells, coexpression of Bcl-X L is able to protect cells completely from the toxic effects of Bax expression. 16,18,20,25 Accordingly wild-type, Dcrd1 and Dpgs1 strains containing Bax expression plasmids were cotransformed with plasmids that mediate the expression of N-terminally, HAtagged Bcl-X L in response to the addition of doxycycline to the media; similar levels of Bcl-X L are expressed in response to doxycycline addition in each strain (Figure 1b). Strain viability was again tested by the serial dilution of each strain onto the plates with varying concentrations of doxycycline (Figure 1d).…”

“…These studies led to a model in which the action of Bax and Bcl-X L on the mitochondrial membranes involves the interaction of Bax and Bcl-X L with the hypothetical target in the outer mitochondrial membrane. 25 In this model, Bax and Bcl-X L compete for a common target; interaction of Bax with this target leads to outer membrane permeabilization and cell death, while Bcl-X L , which interacts with the target preferentially, prevents the target from the interaction with Bax. Clearly, in this model the availability of the target would determine the sensitivity of the cell to both Bax-mediated killing and Bcl-X L -mediated rescue.…”