Mature DIABLO/Smac Is Produced by the IMP Protease Complex on the Mitochondrial Inner Membrane

Abstract

DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX 5 P in Imp1 and NX 5 S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space. INTRODUCTIONProgrammed cell death is a means whereby metazoans can remove unwanted cells, with failure of programmed cell death enabling cancer and autoimmune disease, and inappropriate cell death contributing to neurodegenerative disease. Several of the proteins that regulate cell death, including Bcl-2 family members, signal-transduction receptors, effector proteases and DIABLO/Smac, are specifically enclosed within subcellular structures allowing precise control over the commitment of a cell to die. Understanding where and how these key regulators are localized is crucial in understanding their normal biological function and their role in the pathogenesis of malignant diseases.A family of intracellular proteases called caspases implement programmed cell death (Ekert et al., 1999). The activity of caspases is regulated by a family of inhibitor of apoptosis proteins (IAPs) that bind and neutralize active caspases (Deveraux and Reed, 1999). For example, the inhibitor MIHA/XIAP/hILP/BIRC4 can bind and inhibit processed caspases 3, 7, and 9 (Duckett et al., 1996;Liston et al., 1996;Uren et al., 1996;Deveraux et al., 1997Deveraux et al., , 1998. This damping of caspase activity provides a layer of regulation over the cell death-promoting activities of this family of effector proteases.In mammalian cells, signals for cell death can lead to rupture of the mitochondrial outer membrane, and under these conditions the inhibition of caspases can be antagonized by the mitochondrial proteins DIABLO/Smac Verhagen et al., 2000) and HtrA2/Omi (Suzuki et al., 2001;Martins et al., 2002;Verhagen et al., 2002). Structural studies Liu et al., 2000;Wu et al., 2000) have shown that purified DIABLO is a homodimer , and each DIABLO dimer can bind to the BIR domains of inhibitor proteins via contacts made to the N-terminal residues of DIABLO (Liu et al., 2000;Wu et al., 2000). The avid interaction DIABLO makes with the inhibitor MIHA competes the inhibitor away from active caspase 9, freeing caspase 9 to proteolytically activate downstream caspases .Mouse DIABLO is translated as a 237-residue precursor protein (preDIABLO) with an N-terminal presequence that must be cleaved to generate the mature form with the amino-ter...

The direct route: a simplified pathway for protein import into the mitochondrion of trypanosomes

“…In fungi, animals and plants, two catalytically active subunits, Imp1 and Imp2, provide an ability to process a range of targeting sequences and enable the 'stop-transfer' targeting of proteins into the inner membrane; whereby a substrate protein is targeted to the TIM23 complex, but additional targeting information signals for a 'stop' to translocation and for a lateral 'transfer' from the translocase into the inner membrane [36]. The entire targeting sequence, including the stop-transfer signal, is then processed from the substrate by the IMP complex.…”

“…The entire targeting sequence, including the stop-transfer signal, is then processed from the substrate by the IMP complex. A motif in the catalytic region of the peptidase distinguishes Imp1 and Imp2 [36]; T. brucei has just a single protease subunit (Tb10.70.2630) with the sequence characteristics of Imp2 (Figure 3).…”

Intragenic deletions affecting two alternative transcripts of the IMMP2L gene in patients with Tourette syndrome

“…The structurally important substrate-binding cleft NX 5 Smotif of the long IMMP2L transcript and the highly conserved C-terminal 12 are present in the short isoform.…”

“…7 Mammalian IMMP1L and IMMP2L constitute, like the yeast Imp1 and Imp2, the subunits in the inner membrane peptidase complex, which catalyze the removal of the signal peptides required for the targeting of proteins from the mitochondrial matrix into the intermembrane space across the inner membrane. 12 The two subunits share a conserved core and they are both catalytically active, but process different substrates. 13,14 Cytochrome c1 and mitochondrial glycerol-3-phosphate dehydrogenase 2 are substrates for IMMP2L, and IMMP2L mutations have been shown to impair signal peptide processing of both proteins.…”

Translocation breakpoint at 7q31 associated with tics: further evidence for IMMP2L as a candidate gene for Tourette syndrome

“…The IMP complex generates mature, active proteins in the mitochondrial intermembrane space by proteolytically removing the mitochondrial targeting pre-sequence of nuclear-encoded proteins. 9 Although little is known about the exact function of the protein, of note, other mitochondrial proteins (eg, COXII) have been shown to be involved in neurodegenerative and neuropsychiatric disorders. [10][11][12] Family-based association studies by Díaz-Anzaldú a et al 13 in a French-Canadian GTS sample showed significant association with the region previously described by Petek et al 7 in their case with the inverted duplication of 7q31.…”