Eukaryotic cells, such as those of mammals and plants, have developed distinct structures within their cytoplasm called organelles. Organelles have specific roles within the cell from protein recycling to energy production.
One of the most important of these is the mitochondria. Mitochondria, along with plastids (mainly chloroplasts), are thought to have arisen by the engulfment of a prokaryotic cell by a more evolved eukaryotic-like cell. With the increasing size of the eukaryotic cell and the lower surface area to volume ratio this brings, the capacity of the membrane to carry out all of the important funtions required for cell survival became challenging. Incorporating another organism into the cytoplasm and utilising its energy production circumvents the need for all reactions to be carried out within the membrane enclosing the cell.
Mitochondria are the power houses of the cell, producing energy in the form of ATP for the many reactions that are happening continuously. This energy is produced from the electrochemical gradient formed from the proton pumps present within the inner membrane of the mitochondria. This causes the intermembrane space to become positively charged and the matrix space to become negatively charged. Like all the organelles within the cell, mitochondria have to transport most of their proteins through the outer membrane and into the various compartments present, although mitochondria do carry some DNA themselves for certain proteins. As the mitochondria are present within the cell they do not use the endoplasmic reticulum/Golgi pathway to import proteins. Instead, they have an array of complex machinery that utilise both ATP and the electrochemical gradient to import fully translated proteins across both of their membranes.
The theory that mitochondria were once free-living prokaryotic cells is further augmented by the fact that they are enclosed by two membranes. The outer membrane is derived from the larger, more developed cell. The inner membrane is derived from the original membrane that enclosed the prokaryotic cell. This leads to the formation of two soluble compartments, the intermembrane space and the matrix space along with the two membranes. The inner membrane is highly specialised and contains many invaginations or folds called cristae. This increases the surface area of the inner membrane, maximising ATP generation.
Unlike proteins found within the endoplasmic reticulum and Golgi aparatus (where translation is coupled to translocation into the endoplasmic reticulum) proteins destined for the mitochondria are translated on free ribosomes found within the cytoplasm. Once translated, the protein chain is bound by cytosolic chaperones, including those of the heat-shock protein (Hsp)70 family, to prevent proteins folding into their native structure and aggregating.
Found within the amino acid sequence are specific signals that direct the protein to each compartment within the mitochondria. Proteins destined for the matrix space are usually characterised as having an amphipathic alpha-helix at their N-terminus (Figure 2A). This usually has a hydrophobic side and a basic side, which are recognised by membrane proteins. Once the protein is in the matrix space, the signal sequence is cleaved by the matrix-processing protease (MPP), leaving the polypeptide chain to be refolded (Figure 2B (A)).
Proteins required in the inner membrane and intermembrane space have several routes through the mitochondria to end up in the correct place. These proteins also usually have an N-terminal helix and are directed to the matrix. However, directly following the matrix-targeting helix is another signal sequence that directs the protein to either area. In one such case the helix is cleaved and a stop transfer signal is reached, causing translocation to halt and movement of the protein into the inner membrane. This can then be cleaved and the protein released into the inter membrane space (Figure 2B(B)). Proteins can be entirely translocated into the matrix space and the helix cleaved, exposing a hydrophobic sequence which directs the protein bach through the membrane and into the intermembrane space (Figure 2B (C)).
Proteins destined for the outer membrane can either be partitioned into the membrane during transloction, when specific hydrophobic signals are reached (Figure 2B (D)), or they can be tranlslocated into the intermembrane space entirely. When in the intermembrane space, chaperones bind to internal hydrophobic signal sequences to prevent aggregation and direct them to another membrane complex. This inserts them into the outer membrane and aids in their correct folding (Figure 2B (E)).
Mitochondrial proteins derived from nuclear genes are all transported into the mitochondria from the cytosol by a multi-subunit protein called TOM (Translocase of the Outer Mitochondrial Membrane). TOM is made up of TOM20, TOM70, TOM22, TOM6, TOM40, TOM5 and TOM7.
TOM20 and TOM 70 are the initial receptors. TOM22 contains a hydrophobic groove which recognises the hydrophobic side of the amphipathic alpha helix. TOM70 mainly recognises proteins with many internal hydrophobic regions such as transmembrane proteins. These proteins are initially recognised by TOM70 before being transferred to TOM20/22.
TOM22 has multiple roles within the TOM complex. It's main role is as a docking point for TOM20 and TOM70 and is the receptor for charged pre-sequences. It also has roles in organising the general import pore and is the binding site for pre-sequences on the trans-side of the membrane.
TOM5 is responsible for passing on the unfolded proteins from TOM22 to TOM40, the main channel, and is also the receptor for some intermembrane space proteins. TOM40 as mentioned above, forms the channel through which proteins are translocated.
The other two subunits provide minor roles, with TOM6 being the organiser of TOM22 and TOM40 and TOM7 important in dissociating the translocase.
All these subunits assemble and work together in order to import all mitochondrial proteins.
After proteins have been translocated through the TOM complex they can be recognised by two membrane complexes, TIM23 and TIM22 (Translocase of the Inner Mitochondrial Membrane). TIM 23 recognises most of the proteins whilst TIM22 is the receptor which inserts proteins required for the transport of metabolites across the inner mitochondrial membrane and will be discussed later.
TIM23 is also a multi-subunit protein made up of TIM23, TIM17, TIM50, TIM21, and a number of other subunits, found on the matrix side involved in refolding the protein, which make up the PAM complex, the latter of which will not be described in detail here.
TIM23 and TIM17 are essential for cell viability and form a cation selective voltage gated channel which responds to peptide signals present on the protein. TIM17 also regulates the opening of TIM23. TIM50 binds to the protein as it emerges from the TOM complex and also closes the TIM23 channel in the absence of protein. TIM21 links both the TOM and TIM complexes through interaction with TOM22's intermembrane space domain. TIM21 also has a role in dissociating TIM23 from the presequence translocase-associated motor (PAM) complex and binding TIM23's interacting partners.
The PAM complex is assembled on the matrix side of the inner membrane and is involved in binding to the protein as it emerges in the matrix space. The main role of the PAM complex is to aid in refolding the protein by bringing mitochondrial Hsp70 to the protein through binding to TIM44 and then to stimulate the ATPase activity of mtHsp70 through PAM18/16/17.
Whilst the TOM complex is responsible for all protein import from the cytoplasm and TIM23 is responsibe for the majority of protien import into the matrix and inner membrane, certain membrane proteins within the mitochondria require additional complexes to help them fold and orientate properly within the membrane.
Beta-barrel proteins are highly abundant in the outer membrane and are involved mainly in the transport of ions and other such molecules into the mitochondria intermembrane space. These proteins, of which TOM40 is a member, require another complex to help them fold in the membrane, the sorting and assembly machinery (SAM) complex. The beta-barrel proteins are first translocated into the intermembrane space by the TOM complex where they are bound by tiny Tims, a specific chaperone in the intermembrane space, to prevent aggregation. The proteins are then directed to the SAM complex, which inserts the proteins from the mitochondrial inter-membrane space side and assists in their correct folding and positioning within the outer membrane.
Within the inner membrane there are a further two complexes in addition to TIM23 that are required for the integration of certain membrane proteins. The first is TIM22, this complex is needed for the insertion of members of the metabolite carrier family of proteins, involved in transporting molecules such as ADP, ATP and phosphate in and out of the mitochondria. TIM 22 is made up of the subunits TIM22, which forms the voltage-gated channel and has homology to TIM23 and TIM17; TIM54, which protrudes into the intermembrane space and binds the pre-protein; TIM18, which may regulate the assembly of the complex and TIM12, which links the TIM9/10 complex and TIM22. The protein is first translocated through the TOM complex and is bound by the TIM9/10 complex, which is found within the intermembrane space, this then directs the pre-protein to the TIM22 complex and it is translocated through and inserted into the inner membrane.
The final complex is the OXA complex, this complex is only involved in inserting a select few proteins into the innner membrane. Its main job rather, is to insert membrane proteins that are a product of the mitochondrial genome, possibly reflecting the mechanism of the prokaryotic cell from where it was derived.
Alberts et al Molecular Biology of the Cell p.713-719. 4th Edition (2002) Garland Science
Neupert, W. (1997). Protein import into mitochondria. Annual Review of Biochemistry 66: 863-917.
Neupert, W. and J. M. Herrmann (2007). Translocation of proteins into mitochondria. Annual Review of Biochemistry. 76: 723-749.
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