Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32

Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.