No abstract

In mammalian cells, the Bcl-2 and Bcl-x(L) proteins suppress programmed cell death whereas the topographically similar Bax protein accelerates the apoptotic process. Recently published data suggest that expression of the human Bax-a gene is lethal for the yeast Saccharomyces cerevisiae and that this toxicity can be overcome by co-expressing Bcl-2 or Bcl-x(L). Our findings corroborate these results. However, we find that although Bax induction invariably stops cell growth under all circumstances, it does not lead to death in 'petite' cells. Petites cannot respire because they lack functional mitochondria. It seems that in 'grande' cells, which do possess normal mitochondrial DNA, nutritional limitation is critical for increased mortality. Surprisingly, murine Bcl-2 lacking the membrane anchor of human Bcl-2 has no effect on grande cells, but can efficiently rescue petites in rich medium. It has been suggested that the C-terminal membrane anchor of human Bcl-2 may have a crucial role in rescuing apoptosis in mammalian cells. When murine Bcl-2 is fused to the membrane anchor of yeast mitochondrial Mas70 protein, the Bcl-2 variant mBci-2-mma rescues not only petites but also grandes, just like human Bcl-x(L). The rescuing ability of Bcl-x(L), which contains its own membrane anchor, surpasses that of mBcl-2-mma. Our results indicate that the process involving Bax-induced growth inhibition followed by possible lethality, and the rescuing effect of Bcl-2 or Bci-x(L) is linked to yeast mitochondrial function. We propose a model which is consistent with these observations.

No abstract

We have isolated a pigment cell-specific cDNA clone from a B16 mouse melanoma cDNA library by differential hybridization. The mRNA of isolated cDNA is highly expressed in B16 melanoma cells and in black mouse (C57BL/6) skin, but is not detectable in mouse neuroblastoma cells nor in K1735 mouse amelanotic melanoma cells. The protein sequence deduced from the nucleotide sequence of the cloned cDNA shows significant similarity to the entire region of Neurospora tyrosinase. To know the identity of cDNA, we transfected K1735 amelanotic melanoma and COS-7 cells with the cDNA carried in a simian virus 40 vector (pKCRH2). We confirmed that the isolated cDNA encodes mouse tyrosinase by immunofluorescence staining of transfected cells using two different anti-T4-tyrosinase monoclonal antibodies. Tyrosinase is composed of 513 amino acids with a molecular weight of 57,872 excluding a hydrophobic signal peptide of 24 amino acids.

Recent studies that attempt to explore the action of pro-and anti-apoptotic proteins of the bcl2 family demonstrate the crucial role of relocalization of cytochrome c from the mitochondrial intermembrane space to the cytosol. This early event of apoptosis can be mimicked in the yeast Saccharomyces cerevisiae following expression of bax. In mammalian mitochondria, the mechanism of relocalization is thought to involve the opening of the so-called permeability transition pore. We show in this paper: (a) that bax-induced release of cytochrome c in yeast does not involve any permeability transition of the inner mitochondrial membrane but involves a general alteration of the permeability of the outer mitochondrial membrane to macromolecules. This suggests that a permeability transition of the inner mitochondrial membrane is not an event required for the relocalization of cytochrome c in yeast. (b) The outer-membrane voltage-dependent anion channel (VDAC), a putative component of the permeability transition pore, is not involved in bax-induced release of cytochrome c or in the prevention of this release by bcl-x L . (c) Bax devoid of its C-terminal putative hydrophobic a-helix is as efficient as full-length bax to allow the relocalization of cytochrome c, demonstrating this segment of the protein is not required for membrane-targeting. (d) We finally observe that the action of bax on the outer mitochondrial membrane requires the presence of ATP both in vitro and in vivo, and it is shown that ATP directly increases the amount of bax inserted to mitochondria.Keywords: apoptosis, bax, cytochrome c, mitochondria, yeast.There is an ongoing effort to understand the mechanism by which proteins of the bcl-2 family act to induce or prevent programmed cell death ([1] for a review). Recently, it was observed that cytochrome c, a protein located in the mitochondrial intermembrane space, can induce apoptotic phenomena in isolated nuclei [2] just like in intact cells [3]. A number of investigators have lately inquired if cytochrome c is really involved in some way in the action of pro-apoptotic proteins, such as bax, and anti-apoptotic proteins, such as bcl-2 or bcl-x L . It has been described by two groups that, in the course of apoptosis, cytochrome c is actually released from the intermembrane space to the cytosol where it participates in the activation of caspase-3, a step critical for the final apoptotic event [4,5]. Moreover, both these groups have demonstrated that bcl-2 can prevent cytochrome c release, thus preventing caspase-9 activation and the later steps of apoptosis.It is still unclear how cytochrome c is released. Several groups have shown that bax, bcl-x L and bcl-2 can create ionic channels in liposomes [6±8]. This feature was originally suspected from the 3-D structure of one of the members of the family, bcl-x L [9]. It has also been shown that bax, like bcl-2, localizes to the mitochondria [10]. Therefore, one could propose that the proapoptotic bax protein induces a massive entry of ions which is followed by a s...

No abstract

A new system for bax-expression in yeast has been devised to investigate bax's effect under fermentative and respiro-fermentative conditions. This has allowed us to show unambiguously that the ability of bax to kill yeast is higher under respiratory conditions than under purely fermentative conditions. The extent of killing under respiro-fermentative conditions (nonrepressive sugars) is intermediate. It has been proposed that the two proteins adenine nucleotides carrier (ANC) and cytochrome c play a crucial role in bax-induced cell death. We have investigated the effects of deletion of the genes encoding the two proteins on the toxicity induced by bax, using this new system. The absence of ANC did not modify bax-induced lethality in any way. Moreover, the absence of cytochrome c also did not prevent bax-induced death. Only the kinetics of lethality were altered. All these effects are prevented by co-expression of bcl-x L .z 1999 Federation of European Biochemical Societies.

It has been suggested