Inner membrane protease

Fig-i Mitochondrial protein import machinery as defined in S. cerevisiae. TOM translo-case of the outer mitochondrial membrane SAM sorting and assembly machinery TIM translocase of the inner mitochondrial membrane MIA mitochondrial IMS import and assembly machine PAM presequence translocase associated motor IMP inner membrane protease MPP mitochondrial processing peptidase. The numbers on the individual Tom, Sam, Tim or Pam components represent their approximate molecular masses in kDa. See text for mechanistic details. Adopted from Dolezal et al. 2006... 

Schleyer M, Schmidt B, Neupert W (1982) Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria. Eur J Biochem 125 109-116 Schneider A, Behrens M, Scherer P, Pratje E, Michaelis G, Schatz G (1991) Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast. EMBO J 10 247-254... 

Pathway A, the major one for delivery to the intermembrane space, is similar to pathway A for delivery to the inner membrane (see Figure 16-29). The major difference is that the internal targeting sequence in proteins such as cytochrome b2 destined for the intermembrane space is recognized by an inner-membrane protease, which cleaves the protein on the... 

The biogenesis of a gram-negative bacterial envelope requires subsequent sorting mechanisms for its component proteins after their translocation across the inner membrane (Duong et al., 1997 Danese and Silhavy, 1998). In addition, some proteins such as proteases and toxins are secreted through the outer membrane to the extracellular space. Three major secretion pathways have been characterized (Salmond and Reeves, 1993). 

It is now well estahlished that activation of the caspase cascade is an indispensable and sufficient process in the execution phase of apoptosis (Nunez et al, 1998). As for mitochondria-mediated apoptosis, cytochrome c released from the mitochondrial inner membrane is well known to play an important role in the activation of caspase 9, one of the upstream proteases in the cascade (Zou et al, 1997). For activation of caspase 9, cytochrome c or apoptotic protease activating factor 2 (Apaf 2) induces the formation of the complex between Apaf 1 and caspase 9. The resultant activated caspase 9 then activates caspase 3, which in turn leads to the genomic DNA fragmentation and apoptotic cell death. 

The pill protein is expressed as a precursor having an amino-terminal signal peptide necessary for addressing the protein through the periplasm of E. coli. The signal peptide is removed by a specific protease after secretion, and the pill ends up anchored in the bacterial inner membrane. Its assembly in the phage particle is concomitant with phage extrusion. 

Cytochrome c (Cyt c), the peripheral protein loosely associated with the inner membrane of mitochondria, is one of the most well-known factors involved in apoptosis (Green 2005). In healthy cells, Cyt c functions as an electron shuttle in the respiratory chain and its activity is necessary for life. Cyt c is released by the mitochondria as the consequence of elevated permeability of the outer membrane in responses to proapoptotic stimuli (Li et al. 1997). In the cytosol, Cyt c binds to the apoptosis-protease activating factor 1 (Apaf-1), which then recruits caspase-9 to form the apoptosome (Li et al. 1997). Caspase-9 in turn cleaves and activates executioner caspase-3, resulting in apoptotic cell death as described above. The whole process requires energy and relatively intact cell machinery. 

The pharmacological evidence compiled for y-secretase is indicative of the activity of an aspartic protease requiring at least one additional cofactor. The location of the active site within the membrane makes y-secretase quite unique. Currently, there is only one precedent for a similar, tricky enzyme, signal peptidase, which shares several features and most of the problems associated with inner-membrane location [25]. It will not, unfortunately, be easy to isolate and purify the membrane-stabilized protease while retaining its activity. It has, therefore, so far escaped crystallization and X-ray structure determination. Mutation analysis of the two conserved aspartic acids of all presenilins supports their key role in y-secretase... 

Fig. 12.5. Biogenesis and assembly of cytochrome 6-c, complex in the inner mitochondrial membrane. Cytochrome fc-Cj complex contains at least five different subunits COREI (corl), COREII (corll), nonheme iron protein (Fe-S), cytochrome c, (cyt Cj), and cytochrome b (cyt b). Cytochrome f> is a mitochondrial gene product and is probably assembled into the inner membrane (IM) via vectorial translation by mitochondrial ribosomes. The other subunits are synthesized on cytoplasmic ribosomes as larger precursors. The precursors, perhaps in association with a cytoplasmic factor , are attached to receptors on the mitochondrial outer membrane (OM). The complex laterally diffuses to the junctions of the outer and inner membranes, and with the help of a hypothetical translocator the precursors are imported across the membrane. Pre-Corl, pre-Corll, and the pre-nonheme iron protein cross the two membranes, whereas cytochrome c, becomes anchored to the outer face of the inner membrane, facing the intermembrane space (IMS). Cytochrome b is assembled inside the inner membrane, and the nonheme iron protein and Corl and Corll are assembled into the matrix side of the inner membrane. The N-terminal extensions are removed by a soluble matrix protease. The N-terminal extension of cytochrome c, is removed in two steps the first is catalyzed by the matrix protease and the second probably by a protease located on the outer face of the inner membrane.

After its synthesis in cytoplasm, cytochrome c, must be translocated into the mitochondrial inner membrane space. (Recall that cytochrome c, is a component of complex III of the ETC.) The targeting of cytochrome q requires two sequences. The first targets the polypeptide to the matrix. After this sequence is removed by a protease, the second sequence targets the molecule to the inner membrane space. The second targeting sequence is then also removed. After folding and binding a heme, the molecule associates with complex HI in the inner membrane. 

Burri L, Strahm Y, Hawkins CJ et al (2005) Mature DIABLO/Smac is produced by the IMP protease complex on the mitochondrial inner membrane. Mol Biol Cell 16(6) 2926-2933... 

Additional work with closed vesicles derived from B. megaterium membranes demonstrates that NBD analogs of PE, PG, and PC can translocate across the membrane with a /i/2 of 30 s at 37°C (S. Hraffnsdottir, 1997). Similar types of experiments conducted with closed vesicles isolated from E. coli inner membrane reveal that NBD phospholipids traverse the bilayer with a of 7 min at 37°C (R. Huijbregts, 1996). This latter process is insensitive to protease and A-ethylmaleimide treatments and does not require ATP. Collectively, the data indicate that transbilayer lipid movement is rapid and does not require metabolic energy in bacterial membranes that harbor the biosynthetic enzymes for phospholipids. The basic characteristics of lipid translocation in the intact cell appear to be retained in isolated membranes. 

Opal, also known as Mgml in yeast, is a mitochondrial member of the dynamin family. Unlike other dynamin family members. Opal has an N-terminal mitochondrial targeting sequence, suggesting that this protein is imported into mitochondria. Here, we describe biochemical techniques, such as mitochondrial isolation, digitonin extraction, a protease protection assay, and carbonate extraction, that were used to determine that mammalian Opal resides in the intermembrane space where it is tightly bound to the inner membrane. In addition, we describe bacterial expression of the Opal GTPase domain, methods for purification, and an in vitro assay for GTP hydrolysis. 

The Rieske protein in mitochondrial bci complexes is assembled when the protein is incorporated into the complex. The Rieske protein is encoded in the nucleus and synthesized in the cytosol with a mitochondrial targeting presequence, which is required to direct the apoprotein to the mitochondrial matrix. The C-terminus is then targeted back to the outside of the inner mitochondrial membrane where the Rieske cluster is assembled. In addition, the presequence is removed and the protein is processed to its mature size after the protein is inserted into the bci complex. In mammals, the presequence is cleaved in a single step by the core proteins 1 and 2, which are related to the general mitochondrial matrix processing protease (MPP) a and (3 subunits the bovine heart presequence is retained as a 8.0 kDa subunit of the complex (42, 107). In Saccharomyces cerevis-iae, processing occurs in two steps Initially, the yeast MPP removes 22 amino acid residues to convert the precursor to the intermediate form, and then the mitochondrial intermediate protease (MIP) removes 8 residues after the intermediate form is in the bci complex (47). Cleavage by MIP is independent of the assembly of the Rieske cluster Conversion of the intermediate to the mature form was observed in a yeast mutant that did not assemble any Rieske cluster (35). However, in most mutants where the assembly of the Rieske cluster is prevented, the amount of Rieske protein is drastically reduced, most likely because of instability (35, 44). 

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