Mitochondrial membranes protein insertion

Most of the data available on membrane protein insertion has been generated for members of the mitochondrial carrier family (MCF), particularly for AAC, the model precursor. 

The nuclear-encoded proteins are inserted into both inner and outer mitochondrial membranes, the intermembrane space, and the matrix and there are several different mechanisms involved. As mentioned above there is no apparent requirement for a presequence on proteins which insert specifically into the mitochondrial outer membrane. For proteins destined for the inner mitochondrial membrane, a stop-transfer mechanism is proposed. Thus some information in the peptide must stop the complete transfer of the protein into the mitochondrial matrix, enabling the protein to remain in the inner mitochondrial membrane. For some proteins in the intermembrane space (for example the Rieske iron-sulphur protein associated with the outer face of complex III), a particularly complicated import pathway... 

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). 

The above describes the major pathway of proteins destined for the mitochondrial matrix. However, certain proteins insert into the outer mitochoiidrial membrane facilitated by the TOM complex. Others stop in the intermembrane space, and some insert into the inner membrane. Yet others proceed into the matrix and then return to the inner membrane or intermembrane space. A number of proteins contain two signaling sequences—one to enter the mitochondrial matrix and the other to mediate subsequent relocation (eg, into the inner membrane). Certain mitochondrial proteins do not contain presequences (eg, cytochrome Cy which locates in the inter membrane space), and others contain internal presequences. Overall, proteins employ a variety of mechanisms and routes to attain their final destinations in mitochondria. 

Figure 3. Possible mechanisms of actions of Bcl-2 members. Two prevailing models through which Bcl-2 membas trigger cytochrome c release have been suggested. In both models phospholipids in the bilayer stnicture either individually and/or collectively induce a conformational change in Bcl-2 members, allowing them to insert into the outer mitochondrial membrane. In model 1 proapoptotic proteins destabilize the outer mitochondrial membrane, oligomerize and form channels through which cytochrome c and other proteins of the intermembrane space can escape.BcI-2 proteins such as Bax or tBid act in concert with other proteins of the BcI-2 family to form channels. In model 2 Bcl-2 members such as Bax interact with residoit proteins in the outer membrane (OM) such as the voltage-dependent anion...

Murcha MW et al. (2007) Characterization of the preprotein and amino acid transporter gene family in Arabidopsis. Plant Physiol 143 199-212 Murcha MW, Millar AH, Whelan J (2005a) The N-terminal cleavable extension of plant carrier proteins is responsible for efficient insertion into the inner mitochondrial membrane. J Mol Biol 351 16-25... 

In the absence of a death signal, most of the pro- and anti-apoptotic members are located in separate subcellular compartments. Anti-apoptotic proteins are inserted in intracellular membranes, mainly the mitochondrial membrane, while some proapoptotic members are located in the cytoplasm or cytoskeleton in an inactive form. They are activated and translocated by apoptotic stimuli to their place of action to perform their functions (Gross et al., 1999). 

Allen and Bevan (80) have applied the SMD technique to the study of reversible inhibitors of monoamine oxidase B, and this paper will be used as an example for discussion of the constant velocity SMD pulling method. They used the Gromacs suite of biomolecular simulation programs (18) with the united-atom Gromos 43al force field to parameterize the lipid bilayer, protein, and small-molecule inhibitors. The protein was inserted into their mixed bilayer composed of phosphatidyl choline (POPC) and phosphatidyl ethanolamine (POPE) lipids in a ratio known to be consistent for a mitochondrial membrane. Each inhibitor-bound system studied was preequilibrated in a periodic box of SPC water (20) with the simulations run using the NPT ensemble at 300 K and 1 atm pressure for 20 ns. Full atomic coordinates and velocities were saved in 200-ps increments giving five replicates for each inhibitor-bound system. A dummy atom was attached to an atom (the SMD atom shown in Fig. 7) of the inhibitor nearest to the... 

Rehling P, Model K, Brandner K, Kovermann P, Sickmann A, Meyer HE, Kuhlbrandt W, Wagner R, Truscott KN, Pfanner N (2003) Protein insertion into the mitochondrial inner membrane by a twin-pore translocase. Science 299 1747-1751 Richards TA, Cavalier-Smith T (2005) Myosin domain evolution and the primary divergence of eukaryotes. Nature 436 1113-1118... 

The overall assembly of cytochrome c oxidase on the inner mitochondrial membrane is controlled by a large number of nuclear encoded genes (Tzagoloff and Dieckmann, 1990). Four of these genes, scol, sco2, coxl 1, and coxl 7, encode proteins that appear to be involved in copper incorporation into the catalytic core of the enzyme, though precisely which one(s) is (are) responsible for insertion of copper into the complex remains unclear (Horvath et al., 2000). Scol is anchored in the inner mitochondrial membrane and is essential for the accumulation of Coxl and CoxII subunits as well as the proper assembly of the cytochrome c... 

All three respiratory complexes are typical integral membrane proteins that span the inner mitochondrial membrane. Each consists of several different subunits, the exact number of which is still under debate. The genes of some subunits of cytochrome oxidase and the />c, complex are in mitochondrial DNA (mtDNA). These proteins are synthesised inside the mitochondrion. However, most proteins of these complexes, as well as cytochrome c, are synthesised on cytoplasmic ribosomes and coded by the nuclear genome. This raises intriguing questions of how the latter are imported into the mitochondrion and inserted into the mitochondrial membrane, as well as of how mitochondrial and cytoplasmic transcription and translation are synchronised [3-5]. 

The amino acids of a protein control its location in the cell. Some proteins are water soluble, whereas others are bound to the ceil membrane (plasma membrane), the mitochondrial membrane, and the membranes of the endoplasmic reticulum and nucleus. The association of a protein with a membrane is maintained by a stretch of lipophilic amino acids. Insertion of this stretch into the membrane occurs as the protein is synthesized. Water-soluble proteins are formed on ribosomes that "float" free in the cytoplasm. Membrane-bound proteins are formed on ribosomes that associate with the endoplasmic reticulum (ER). As the amino acids are polymerized in the vicinity of the F,R, a stretch of lipophilic acids becomes inserted into the membrane of the FR. This anchoring of the protein is maintained when it is shuttled from its location in the ER to its desired location in the plasma membrane. 

During acidosis, the cells of the renal tubule can respond by inserting two proteins into the apical region of the plasma membrane. (The apical part is that region that is exposed to the developing urine.) The two proteins are H,K-ATPase and H+-ATPase. H,K-ATPase, and the enzymes that act in concert with it, is better known as a component of the parietal cell where it creates stomach acid. The other proton pump of the renal tubule, which is H" -ATPase, is closely related to FoFiH" -ATPase of the mitochondrial membrane. Hence, anyone who imderstands how protons are pumped out of the mitochondrion and how stomach acid is made will clearly understand how the renal tubule can shuttle protons to the lumen of the renal tubule and into the developing urine. 

FECH (also known as heme synthase) is an iron-sulfur protein located in the inner mitochondrial membrane. This enzyme inserts ferrous iron into protoporphyrin to form heme During this process, two hydrogens are displaced from the ring nitrogens. Other metals in the divalent state will also act as substrate, yielding the corresponding chelate (e.g., incorporation of Zn into protoporphyrin to yield zinc protoporphyrin). In iron-deficient states Zn successfully competes with Fe in developing red cells so that the concentration of zinc protoporphyrin in erythrocytes increases. Furthermore, other dicarboxylic porphyrins will also serve as substrates (e.g., mesoporphyrin). 

The Bcl-2 family of proteins are considered as apoptosis regulating proteins. Members of this family are the Bcl-2 and Bcl-xL which are anti-apoptotic while Bax, Bad, Bid, Bim are pro-apoptotic. Fro-apoptotic and anti-apoptotic Bcl-2 proteins can bind directly to the components of mitochondrial pore, leading to either its opening or closure respectively.16 Figure 1. Alternatively, pro-apoptotic members, such as Bak or Bax, insert into the outer mitochondrial membrane where they oligomerize to form a permeable pore.17 Furthermore, an interaction between the intrinsic and the extrinsic... 

The final pathway for insertion in the inner mitochondrial membrane is followed by multipass proteins that con-... 

Dalbey, R. E., and A. Kuhn. 2000. Evolutionarily related insertion pathways of bacterial, mitochondrial, and thylakoid membrane proteins. Ann. Rev. Cell Devel. Biol. 16 51-87. 

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