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.
Sunayama
J
J
Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan.
Ando
Y
Y
Itoh
N
N
Tomiyama
A
A
Sakurada
K
K
Sugiyama
A
A
Kang
D
D
Tashiro
F
F
Gotoh
Y
Y
Kuchino
Y
Y
Kitanaka
C
C
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001254:Astrocytoma / Q000473:pathology; D001665:Binding Sites; D019556:COS Cells; D002352:Carrier Proteins; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012235:Rhodamines; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques
D051017:Apoptosis Regulatory Proteins; C474007:Bax protein (53-86); C087299:C1QBP protein, human; D002352:Carrier Proteins; D005456:Fluorescent Dyes; C484586:HRK protein, human; D024101:Mitochondrial Proteins; D009479:Neuropeptides; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D005982:Glutathione Transferase; D016650:Fluorescein-5-isothiocyanate