Abstract
In many forms of apoptosis, cytochrome c released from mitochondria induces the oligomerization of Apaf-1 to form a caspase-activating apoptosome complex. Activation of lysates in vitro with dATP and cytochrome c results in the formation of an active caspase-processing approximately 700-kDa apoptosome complex, which predominates in apoptotic cells, and a relatively inactive approximately 1.4-MDa complex. We now demonstrate that assembly of the active complex is suppressed by normal intracellular concentrations of K(+). Using a defined apoptosome reconstitution system with recombinant Apaf-1 and cytochrome c, K(+) also inhibits caspase activation by abrogating Apaf-1 oligomerization and apoptosome assembly. Once assembled, the apoptosome is relatively insensitive to the effects of ionic strength and processes/activates effector caspases. The inhibitory effects of K(+) on apoptosome formation are antagonized in a concentration-dependent manner by cytochrome c. These studies support the hypothesis that the normal intracellular concentrations of K(+) act to safeguard the cell against inappropriate formation of the apoptosome complex, caused by the inadvertent release of small amounts of cytochrome c. Thus, the assembly and activation of the apoptosome complex in the cell requires the rapid and extensive release of cytochrome c to overcome the inhibitory effects of normal intracellular concentrations of K(+).
Highlights
Apoptotic cell death is characterized by morphological and biochemical changes, which are the consequence of the activation of a family of cysteine proteases known as caspases
Since cytochrome c-dependent caspase activation is antagonized by increasing ionic strength [21], it is possible that the regulatory control point is the formation of the apoptosome complex
We show that normal [Kϩ]i strongly inhibits the formation of this apoptosome complex and that high levels of cytochrome c overcome this inhibition
Summary
Apoptotic cell death is characterized by morphological and biochemical changes, which are the consequence of the activation of a family of cysteine proteases known as caspases (for review, see Refs. 1 and 2). In receptor-mediated apoptosis, such as CD95/Fas/APO-1, receptor ligation and trimerization induce the formation of a death-inducing signaling complex, located at the cell membrane, which recruits procaspase-8, via the FADD/MORT1 adaptor molecule [4] This causes a conformational change in procaspase-8, which results in caspase-8 cleavage and activation (4 –7). Initial studies showed that cytochrome c binds to Apaf-1 in a 2:1 stoichiometry with high affinity (Ka ϭ 1011 MϪ1) that is markedly reduced (Ka ϭ 4 ϫ 107 MϪ1) in the presence of normal intracellular Kϩ concentrations [Kϩ]i [17, 18] These findings imply that the ionic conditions in the cell may regulate cytochrome c binding to Apaf-1, thereby modulating apoptosome formation. The smaller ϳ700-kDa complex is the most active caspase-
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