Bax (Bcl2-associated X protein) is an apoptosis-inducing protein that participates in cell death during normal development and in various diseases. Bax resides in an inactive state in the cytosol of many cells. In response to death stimuli, Bax protein undergoes conformational changes that expose membrane-targeting domains, resulting in its translocation to mitochondrial membranes, where Bax inserts and causes release of cytochrome c and other apoptogenic proteins. It is unknown what controls conversion of Bax from the inactive to active conformation. Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria. Conversely, reducing HN expression by small interfering RNAs sensitizes cells to Bax and increases Bax translocation to membranes. HN peptides also block Bax association with isolated mitochondria, and suppress cytochrome c release in vitro. Notably, the mitochondrial genome contains an identical open reading frame, and the mitochondrial version of HN can also bind and suppress Bax. We speculate therefore that HN arose from mitochondria and transferred to the nuclear genome, providing a mechanism for protecting these organelles from Bax.
The hydrolysis of ATP by a group of RNA-dependent ATPases (DEAD/H proteins) is required for spliceosome assembly, but not for the subsequent transesterification reactions. Little is known about the function of these ATPases in relation to the RNA conformational changes that occur in formation of active structures, in which U2/U6 small nuclear RNA (snRNA) interactions are essential for splicing to take place. Using a synthetic lethal genetic screen, we have isolated four yeast splicing factors involved in U2/U6 snRNA interactions (D.X. et al., manuscript in preparation). The RNA-dependent ATPase activity associated with one such factor, the Slt22 protein, is stimulated preferentially by annealed U2/U6 snRNAs. Both mutant slt22-1 and U2 snRNA cause a reduction in stimulation. The slt22-1 mutation blocks splicing at or before the first step, resulting in the accumulation of an unusual complex which lacks U5 snRNA. Our results indicate that the U2/U6 snRNA interactions facilitated by Slt22 are also involved in the interaction of U5 snRNA with the spliceosome.
Bax is a proapoptotic member of the Bcl-2 family of proteins which localizes to and uses mitochondria as its major site of action. Bax normally resides in the cytoplasm and translocates to mitochondria in response to apoptotic stimuli, and it promotes apoptosis in two ways: (i) by disrupting mitochondrial membrane barrier function by formation of ion-permeable pores in mitochondrial membranes and (ii) by binding to antiapoptotic Bcl-2 family proteins via its BH3 domain and inhibiting their functions. A hairpin pair of amphipathic ␣-helices (␣5-␣6) in Bax has been predicted to participate in membrane insertion and pore formation by Bax. We mutagenized several charged residues in the ␣5-␣6 domain of Bax, changing them to alanine. These substitution mutants of Bax constitutively localized to mitochondria and displayed a gain-of-function phenotype when expressed in mammalian cells. Furthermore, substitution of 8 out of 10 charged residues in the ␣5-␣6 domain of Bax resulted in a loss of cytotoxicity in yeast but a gain-of-function phenotype in mammalian cells. The enhanced function of this Bax mutant was correlated with increased binding to Bcl-X L , through a BH3-independent mechanism. These observations reveal new functions for the ␣5-␣6 hairpin loop of Bax: (i) regulation of mitochondrial targeting and (ii) modulation of binding to antiapoptotic Bcl-2 proteins.Members of the Bcl-2 family are major regulators of apoptosis and include both pro-and antiapoptotic proteins. Bax is a proapoptotic Bcl-2 family member which participates in the induction of apoptosis in response to a variety of apoptotic signals (4,15,27,31). Furthermore, overexpression of Bax induces apoptosis in many cells (31,50). A number of biochemical functions have been defined for Bax, some of which correlate with its proapoptotic activity, including (i) heterodimerization with the proapoptotic Bcl-2 proteins (9, 48, 49), (ii) homodimerization (8,19,51), (iii) release of cytochrome c from mitochondria (14), and (iv) disruption of the potential across the inner mitochondrial membrane (32, 47). Recently, it has been shown that Bax functionally interacts with components of the mitochondrial inner membrane, the adenine nucleotide transporter (ANT) (22), and the mitochondrial F 0 F 1 ATPase H ϩ pump (24), as well as the outer membrane voltage-dependent anion channel (VDAC) (40).The three-dimensional structures of the Bcl-2 family members Bcl-X L and Bid have been determined, revealing striking resemblance to the pore-forming domains of certain bacterial toxins (2, 25, 35). Moreover, Bcl-2 and Bax can be readily modeled on the same X-ray crystallographic coordinates (36), suggesting that they also possess similar protein folds. This structural homology correlates with the ability of at least four members of the Bcl-2 family, Bcl-X L , Bcl-2, Bid, and Bax, to form ion-conducting pores in synthetic lipid membranes in vitro (1,26,(37)(38)(39). A hairpin pair of ␣-helices within the pore-forming domains of bacterial toxins that share structural similarity t...
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