Exposure of any of two proapoptotic domains of presenilin 1-associated protein/mitochondrial carrier homolog 1 on the surface of mitochondria is sufficient for induction of apoptosis in a Bax/Bak-independent manner
Introduction
Apoptosis is a type of programmed cell death required for the correct development of multicellular organisms and for the elimination of unnecessary or damaged cells (Danial and Korsmeyer, 2004). Two major apoptotic pathways have been described. In the extrinsic pathway, receptors on the cell surface are activated by binding of their ligands (Schmitz et al., 2000), resulting in activation of caspase 8, which then activates caspase 3, responsible for the activation of other caspases and cleavage of different cellular proteins. In the intrinsic pathway, the outer mitochondrial membrane is permeabilized or broken, releasing intermembrane-space proteins that can activate the apoptotic process in a caspase-dependent or -independent manner (Newmeyer and Ferguson-Miller, 2003). Among these proteins, cytochrome c (cyt c) is required for activation of caspase 9, which then activates caspase 3, where both apoptotic pathways meet. In some cells, caspase 8 can also cleave a cytosolic protein, Bid, and truncated Bid, tBid, can then translocate to mitochondria, activating the mitochondrial pathway (Li et al., 1998).
The mechanisms leading to release of proapoptotic proteins from the intermembrane space are not yet fully understood. On one side, several members of the Bcl-2 family act on the outer membrane of mitochondria to induce or inhibit the formation of pores (Cory and Adams, 2002), with proapoptotic members like Bax and Bak apparently being able to form pores large enough to let cyt c and other proteins pass through (Martinou and Green, 2001), although this is still a matter of debate. On the other side, the mitochondrial inner membrane can loose its membrane potential, which can lead to swelling that ruptures the outer membrane (Skulachev, 1996). Mitochondrial depolarization and release of intermembrane-space proteins can be independent from each other (Martinez-Caballero et al., 2005).
Mitochondrial depolarization takes place through opening of the permeability transition pore complex (PTPC), the protein composition of which is not unequivocally known (for review, see Grimm and Brdiczka, 2007). The inner membrane adenine nucleotide translocator (ANT), the outer membrane voltage-dependent anion channel (VDAC) and matrix cyclophilin D have been reported to be part of this pore, although some of these components have been shown not to be necessary for permeability transition (Forte and Bernardi, 2005), as it has been recently reported for VDAC (Baines et al., 2007). Some proapoptotic and antiapoptotic members of the Bcl-2 family interact with components of the PTPC (Halestrap et al., 2002). On the other hand, a new outer membrane channel that functions in apoptosis has been recently described, the mitochondrial apoptosis-induced channel (MAC) (Martinez-Caballero et al., 2005), the composition of which is not yet known, although Bax and Bak appear to be two of its components. For a recent review on mitochondrial membrane permeabilization, see Kroemer et al. (2007).
PSAP/Mtch1 was identified as a presenilin 1-associated protein (PSAP) (Xu et al., 1999) and later reported to be a proapoptotic mitochondrial protein (Xu et al., 2002). It is also known as mitochondrial carrier homolog 1 (Mtch1) due its homology with this protein family. A PSAP/Mtch1 homolog, Mtch2, also known as Met-induced mitochondrial protein (Mimp), has been reported to be a target of tBid in mitochondria, being part of a protein complex where Bax is also present (Grinberg et al., 2005). Furthermore, Mimp/Mtch2 has been reported to participate in Met-hepatocyte growth factor/scatter factor (Met-HGF/SF) signal transduction (Yerushalmi et al., 2002; Leibowitz-Amit et al., 2006). Very recently, it has been described that Mimp/Mtch2 is also able to induce apoptosis upon overexpression and that it interacts with VDAC1 (Alcala et al., 2007). The mechanisms by which these two proteins induce apoptosis are so far unknown.
We reported recently that two independent regions have to be both removed from PSAP/Mtch1 to eliminate its proapoptotic activity (Lamarca et al., 2007). In order to find out if these regions were able to induce apoptosis without the rest of the protein, and therefore gain insight into its mechanism of action, we targeted each of both regions to the surface of mitochondria by fusion to the outer membrane-targeting transmembrane domain of Bcl-xL. We report here that independent exposure of any of these regions on the cytosolic side of the outer membrane is sufficient for induction of apoptosis, which involves mitochondrial depolarization. Furthermore, PSAP/Mtch1 is able to induce apoptosis in the absence of proapoptotic proteins Bax and Bak. We discuss the possible mode of action of this protein based on our results.
Section snippets
General reagents
All reagents were of molecular biology grade. Restriction enzymes were from Roche, Stratagene and Invitrogen. Accuprime Pfx DNA polymerase used for PCR cloning and custom-made primers were from Invitrogen.
Construction of expression vectors
Some of the expression vectors used in this work have been previously described (Lamarca et al., 2007). The new vectors described here were constructed using similar approaches to the ones described previously. See Fig. 1 for a scheme of the different constructs. Amino acid numbering relates
Insertion of the amino-terminal region of PSAP/Mtch1 into the outer mitochondrial membrane is sufficient to induce apoptosis
We reported recently (Lamarca et al., 2007) that the amino-terminal region of PSAP/Mtch1 is required for induction of apoptosis upon overexpression in cultured cells, and a transmembrane domain, TM1, is required for insertion into the outer mitochondrial membrane. We have now investigated if a fragment of PSAP/Mtch1 containing part of the amino-terminal region of the protein, including TM1, (PSAP65–277, Fig. 1) was able to induce apoptosis. PARP cleavage induced by overexpression of this
Discussion
Two new players in the apoptosis-related events that take place in mitochondrial membranes have been reported in recent years, namely (1) presenilin-1-associated protein (PSAP), first identified as a presenilin 1 interactor (Xu et al., 1999) and also known as Mtch1 due to its homology with mitochondrial inner membrane carriers, and (2) Mtch2, first reported as Mimp, a protein induced by the activation of the Met receptor tyrosine kinase by its ligand hepatocyte growth factor/scatter factor
Acknowledgments
We thank María Royo and María José Martínez for excellent technical assistance and help, respectively, with the confocal microscope, and the I+CS for access to the microscope. We thank all BIFI members for support, especially Dr. Javier Sancho and Dr. José Luis Alonso. We thank Cristina Muñoz Pinedo for the gift of wild-type and DKO MEFs. We thank Dr. Javier Naval for access to the cytometer and for critical discussions, people in his lab for their help, and Julián Pardo for critical
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Present address: National Institute of Neurological Disorders and Stroke, Receptor Biology Unit, Building 35, Room 2C-905, 35 Convent Drive, MSC 3704, Bethesda, MD 20892-3704, USA.