Molecular Biology of the Cell - Volume 2 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1993

Intracellular sorting of macromolecules and maintenance of cellular compartments
Protein transport into mitochondria and chloroplasts

As we already know well (Chapter 7), Cell/35.html">Mitochondria and METABOLISM/14.html">Chloroplasts are double-membrane-bounded Organelles specialized in ATP synthesis via Electron Transport and Oxidative Phosphorylation in mitochondria, and photosynthetic phosphorylation in chloroplasts. Although both organelles possess their own DNA and Protein Synthesis machinery, the majority of their Proteins are encoded by nuclear DNA and imported from the Cytosol. Moreover, each imported protein must reach a specific subcompartment where it Functions. Mitochondria contain four subcompartments: the mitochondrial matrix, the inner membrane, the intermembrane space, and the outer membrane facing the cytosol (Fig. 8-26, A). Chloroplasts additionally contain the thylakoid membrane and the thylakoid space (Fig. 8-26, B). Each of these subcompartments contains a distinct set of proteins. The growth of mitochondria and chloroplasts relies on the import of cytoplasmic proteins, which involves the sequential, selective translocation of proteins across one, two, or (in chloroplasts) even three membranes.

Those relatively few proteins encoded by the organelles' own genomes are located primarily in the inner membrane of mitochondria and the thylakoid membrane of chloroplasts. The Polypeptides encoded by these organellar genomes typically form subunits of Structure/178.html">Protein Complexes, whose other components are encoded by nuclear genes and imported from the cytosol. The assembly of such hybrid protein aggregates requires a balanced synthesis of both types of subunit; how the protein synthesis on Different types of Ribosomes separated by two membranes is coordinated remains an enigma.

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8.4.1. Mitochondrial signal Peptides are amphipathic Amino acid sequences [21]

The Study of mitochondrial biogenesis has greatly benefited from The Use of Yeast as an experimental model. Hybrid genes encoding "chimeric" proteins (generated using Recombinant DNA technology) can be efficiently introduced into yeast Cells. Considerably more is known about protein translocation mechanisms in mitochondria than in chloroplasts. Most likely, these mechanisms are identical, although chloroplasts contain an additional, innermost membrane compartment—the thylakoid.

Proteins imported into the mitochondrial matrix typically arrive from the cytosol within one to two minutes after their release from polyribosomes. These proteins almost invariably bear an N-terminal signal peptide ranging from 20 to 80 amino acid residues in length. Upon protein entry into the mitochondrion, the signal peptide is rapidly cleaved by a specific matrix protease (signal peptidase) and is subsequently presumed to be degraded into Amino Acids within the matrix. The signal peptide can be remarkably simple. Molecular genetic experiments in which the signal sequence was progressively truncated demonstrated that as few as 12 Amino acids are required to ensure the import of a mitochondrial protein. These 12 residues can be attached to any non-mitochondrial protein, directing it to the mitochondrial matrix. Physical analyses of complete signal peptides confirm that they can form amphipathic α-Helical structures (Fig. 8-27) in which all positively charged residues are aligned on one side of the helix, while uncharged hydrophobic residues face the opposite side.

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Fig. 8-27. Signal peptide for mitochondrial protein import. Cytochrome c oxidase is a large, multi-subunit protein complex located in The inner mitochondrial membrane, where it functions as the terminal enzyme in the Electron Transport Chain. A. The first 12 amino acids of the precursor to subunit IV of this enzyme serve as the signal peptide for importing this subunit into the mitochondrion. B. When viewed from the top, with the signal peptide folded along its entire length into an α-Helix with 3.6 residues per turn, it is evident that the positively charged residues (highlighted in color) are clustered on one side of the helix, whereas the uncharged residues (boxed) are gathered on the opposite side. Mitochondrial signal peptide sequences are almost universally capable of forming such an amphipathic helix. It is believed that helices of this type play a crucial role in protein translocation across mitochondrial membranes.

It is thought that the mitochondrial outer membrane contains receptor proteins that bind mitochondrial signal peptides and thereby facilitate the translocation process; however, these putative receptors have not yet been fully characterized.

8.4.2. Protein translocation into the mitochondrial matrix depends on both the Electrochemical Potential across the inner membrane and ATP Hydrolysis [22]

Virtually all knowledge regarding the Molecular Mechanism of protein import into mitochondria has been derived from the analysis of cell-free transport systems. The experimental approach is as follows. First, mitochondria are isolated from homogenized cells by differential centrifugation and then incubated with radioactively labeled proteins destined for these organelles (mitochondrial precursor proteins). The purified precursor proteins are incorporated into these mitochondria very rapidly and efficiently.

All forms of directed movement and transport require energy. In most cases, this energy is utilized in the form of ATP. However, protein import into mitochondria additionally requires an electrochemical gradient across the inner mitochondrial membrane. This gradient is generated during electron transport as Protons are pumped from the matrix into the intermembrane space (see Section 7.1.7). The outer mitochondrial membrane is freely permeable to ions and therefore maintains no gradient. The electrochemical gradient across the inner membrane serves as a rechargeable battery that drives the bulk of cellular ATP synthesis. In addition, the energy of this gradient is expended to drive the import of proteins bearing positively charged mitochondrial signal peptides. If ionophores that dissipate the mitochondrial Membrane Potential are added (see Section 7.2.10), this import is blocked. How exactly the electrochemical gradient promotes protein translocation remains an unanswered question.

8.4.3. Mitochondrial proteins enter the matrix via contact sites bridging both membranes [23]

Does a protein crossing the mitochondrial matrix cross the two membranes one after another, or does it penetrate both membranes simultaneously? To answer this question, a cell-free system can be cooled to the Temperature of melting ice, thereby arresting proteins at an intermediate stage of transport. It turned out that their N-termini are located within the matrix under these conditions (they can be cleaved by a matrix protease), while the remainder of the molecules resides outside the mitochondrion (as it is accessible to exogenously added Proteolytic Enzymes). This result demonstrates that the precursor protein passes through both mitochondrial membranes simultaneously upon entry into the matrix. Electron microscopists have observed numerous contact sites where the outer and inner mitochondrial membranes are apposed, suggesting that these are precisely the regions through which proteins are transferred into the matrix. Recently, these contact sites have been biochemically identified (by their binding to precursor proteins partially translocated into the mitochondrion) and purified.

Fig. 8-28. During protein translocation into the matrix, the proteins transiently bridge the inner and outer mitochondrial membranes. When isolated mitochondria are incubated with a precursor protein at 5°C, the precursor is translocated only partially. Within the matrix, the N-terminal signal peptide is cleaved off; most of the polypeptide chain remains outside the mitochondrion (and is accessible to proteolytic enzymes). Upon warming to 37°C, translocation goes to completion. The initial insertion of the protein into the mitochondrial membrane at 5°C requires a membrane potential across the inner membrane. Subsequent translocation can proceed in the absence of this membrane potential, but requires the presence of ATP on the cytosolic side of the inner membrane. It is believed that ATP hydrolysis is required during the unfolding of the polypeptide chain to allow the protein to pass through the membrane.

If chilled mitochondria containing partially translocated intermediates are rewarmed, translocation is rapidly completed (Fig. 8-28), even if the membrane potential across the inner membrane is dissipated. Apparently, the membrane potential is required only for the initial stage of protein translocation across the membrane, which occurs even at low temperatures. Subsequent events, however, require ATP. These facts indicate that normally translocation proceeds in two steps: 1) an electrically driven penetration of the signal peptide and associated sequences through both mitochondrial membranes, and 2) the translocation of the remainder of the chain into the mitochondrial matrix, which requires ATP hydrolysis and physiological temperatures (Fig. 8-29).

8.4.4. Proteins unfold as they enter the mitochondrial matrix [24]

In all likelihood, precursor proteins unfold before crossing the two mitochondrial membranes at a contact site. It is difficult to conceive how a folded Water-soluble protein could "force its way" through two (or even one) lipid bilayers while retaining its native three-dimensional conformation. Likewise, it is impossible to imagine that a pore could accommodate Globular proteins that vary widely in size and shape. Were it to do so, it would become permeable to protons, thereby collapsing the electrochemical gradient across the inner membrane. Meanwhile, in their unfolded state, all proteins share a similar conformation and can be translocated via a common mechanism. However, because proteins in a folded state possess lower Free energy than in an unfolded state (which is why polypeptides spontaneously fold), unfolding a protein molecule requires an input of energy. It is postulated that this energy is supplied by ATP hydrolysis.

To test whether precursor proteins unfold as they cross the mitochondrial membrane, a hybrid Gene was constructed encoding a "chimeric" protein. In this engineered protein, a mitochondrial signal peptide was fused to the N-terminus of a cytosolic enzyme, Dihydrofolate Reductase (DHFR). Such a hybrid protein retained nearly unimpaired enzymatic activity, meaning that DHFR was in its native three-dimensional conformation. Upon mixing with a mitochondrial preparation, this protein entered the matrix. However, if it was pretreated with methotrexate, which binds tightly to the Active Site of the enzyme and prevents the unfolding of its molecule, translocation was sharply inhibited. Genetic experiments in yeast confirm that certain GENES OF THE hsp70 family are required for the ATP-dependent protein-unfolding reaction. If these genes are inactivated, both mitochondrial precursor proteins and proteins destined for the ER fail to cross their respective membranes and instead accumulate in the cytosol.

Fig. 8-29. Import of proteins into mitochondria. The N-terminal signal peptide of a precursor protein is recognized by a receptor thought to be located in the outer membrane. The protein is translocated across both mitochondrial membranes at specialized contact sites. Initiation of this process requires an electrochemical gradient across the inner membrane. In the matrix, the signal peptide is cleaved off by a specific protease, yielding the mature protein.

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8.4.5. Two signals are required for The transport of proteins into the mitochondrial intermembrane space [25]

The transport of certain precursor proteins into the mitochondrial intermembrane space begins with their translocation into the matrix (Fig. 8-29). However, immediately following the N-terminal signal peptide that initiates this translocation lies a highly hydrophobic Amino Acid Sequence. As soon as the signal peptide is cleaved by the matrix protease, this hydrophobic sequence in turn begins to act as a signal peptide for the re-insertion of the given protein back into the inner membrane. This translocation probably occurs via a mechanism similar to that of protein insertion into the ER membrane. A analogous pathway is used for the insertion into the inner mitochondrial membrane of proteins encoded by the Mitochondrial Genome (Fig. 8-30, A).

After proteins destined for the intermembrane space are integrated into the inner membrane, they are cleaved by a protease within the intermembrane space (Fig. 8-30B). Many of these mature soluble proteins ultimately attach to the "outer" surface of the inner membrane, where they form subunits of complexes that also contain transmembrane proteins.

The transport of proteins from the cytosol into the mitochondrial inner membrane also requires a hydrophobic signal peptide. While this transport likely follows the pathway shown in Fig. 8-30A, this has not been directly proven. Experimentally, such a two-step pathway is difficult to distinguish from an alternative pathway in which transport at the contact site is halted upon reaching the hydrophobic signal, leaving the protein embedded within the inner membrane bilayer.

Fig. 8-30. Importing proteins from the cytosol into the mitochondrial intermembrane space or inner membrane requires multiple signals. The protein is first translocated into the matrix space, as shown in Fig. 8-29. However, Cleavage of the signal peptide used for the initial transfer exposes an adjacent hydrophobic signal peptide at the new N-terminus. This signal directs the insertion of the protein into the inner membrane in a manner analogous to how proteins encoded by the mitochondrial genome are integrated (A). This mechanism is presumably similar to the one that bacterial ancestors of mitochondria used to insert proteins into The Plasma Membrane, and is also believed to resemble The Mechanism of protein insertion into the ER. Transport into the intermembrane space requires a Third Stage, in which a protease with its active site facing the transmembrane space cleaves the protein from its transmembrane signal peptide residing in the inner membrane (B). The pathway depicted in (A) may also be utilized for transferring proteins from the cytosol to the inner membrane, which likewise requires a hydrophobic peptide.

8.4.6. The translocation of proteins from the cytosol to the outer mitochondrial membrane also requires their unfolding [26]

The mitochondrial outer membrane features an unusual structure (resembling the outer membrane of Gram-negative Bacteria), whose lipid bilayer contains large amounts of pore-forming porin proteins. Consequently, the outer membrane is freely permeable to inorganic ions, metabolites, and protein molecules smaller than 10 kDa. For larger proteins, however, the outer membrane acts as a barrier, thereby helping to prevent intermembrane space proteins from leaking back into the cytosol.

The incorporation into the outer membrane of proteins encoded by the main nuclear genome—such as porin—occurs via an ATP-dependent mechanism, but does not require specialized cleavable signal peptides. A membrane potential is also not required. Very little is known about how this incorporation takes place. At least one outer membrane protein possesses a normal matrix-targeting signal, followed by a sequence that somehow halts transfer at the outer membrane.

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8.4.7. Directing proteins into the thylakoid membrane of chloroplasts requires two signal peptides [27]

Protein transport into chloroplasts resembles mitochondrial transport in many respects: both processes occur post-translationally, both require energy, and both utilize hydrophilic N-terminal signal peptides that are subsequently cleaved. However, there is at least one important difference: in mitochondria, this transport is further driven by the electrochemical gradient across the inner membrane. In chloroplasts, where the electrochemical gradient is located across the thylakoid membrane rather than the inner membrane (see Chapter 7), ATP hydrolysis appears to serve as the sole energy source for Transport Across the outer double-membrane envelope.

Signal peptides for protein import into chloroplasts resemble the previously described peptides for mitochondrial import. However, plant cells contain both mitochondria and chloroplasts, meaning that proteins must "choose" between them. For example, in plant cells, a bacterial enzyme genetically fused to the N-terminal sequence of a mitochondrial protein is targeted to the mitochondria. The same protein fused to the N-terminal sequence of a chloroplast protein ends up in the chloroplasts.

Fig. 8-31. Transferring proteins into the thylakoid lumen (thylakoid space) of chloroplasts requires two signal peptides and occurs in two stages. The precursor polypeptide contains an N-terminal chloroplast-targeting signal peptide, immediately followed by a thylakoid signal peptide. The chloroplast signal peptide directs protein translocation into the stroma via contact sites in the membrane (see Fig. 7-73); the mechanism of this transfer is similar to that of import into the mitochondrial matrix (Fig. 8-29). The signal peptide is then cleaved, revealing the thylakoid signal peptide, which drives translocation across the thylakoid membrane. This process shares many features with protein translocation into the ER.

Chloroplasts contain yet another membrane-bounded compartment, the thylakoid. Many chloroplast proteins, including subunit Proteins of the photosynthetic apparatus and ATP synthase, are imported into the thylakoid membrane from the cytosol. Like certain precursor mitochondrial proteins, these proteins are delivered to their destination in two steps. First, they cross the double-membrane envelope into the chloroplast matrix (known as the stroma), and are subsequently transferred into the thylakoid membrane (or across it into the thylakoid space). In addition to the N-terminal chloroplast signal peptide, precursors of these proteins possess a hydrophobic thylakoid signal peptide. After the protein enters the stroma via the N-terminal signal peptide, this peptide is removed by a stromal protease (analogous to the mitochondrial matrix protease). This cleavage exposes the thylakoid signal peptide, which then initiates transport across the thylakoid membrane (Fig. 8-31). As in mitochondria, this Second Stage serves to integrate chloroplast-encoded proteins into the thylakoid membrane; the translocator protein required for this process is likely derived from the bacterial ancestor of chloroplasts.

Summary

Most proteins enter mitochondria and chloroplasts from the cytosol in a similar manner. This mechanism has been most thoroughly studied in mitochondria, particularly in yeast. A protein is translocated into the mitochondrial matrix through zones of apposition (contact sites) between the outer and inner membranes. This transfer requires ATP hydrolysis as well as an electrochemical gradient across the inner membrane. The transported protein unfolds as it crosses the mitochondrial membranes. Only proteins containing a specific signal peptide are translocated into mitochondria or chloroplasts. This signal peptide is typically located at the N-terminus of the protein molecule and is cleaved off after import into the organelle. During the second stage of transport, the protein may be further transferred to the inner membrane. For this, it must possess an additional hydrophobic signal peptide, which is exposed after the removal of the first signal. In the case of chloroplasts, a second signal peptide is similarly required to transport proteins from the stroma into the thylakoid.