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Proteins sequestered within mitochondria are resistant to trypsin

Protein taken up into mitochondria; uptake-targeting sequence removed and degraded

Trypsin

Proteins sequestered within mitochondria are resistant to trypsin

Uptake-targeting sequence and mitochondrial protein degraded

Uptake-targeting sequence and mitochondrial protein degraded

M EXPERIMENTAL FIGURE 16-25 The post-translational uptake of precursor proteins into mitochondria can be assayed in a cell-free system. The imported protein must contain an appropriate uptake-targeting sequence. Uptake also requires ATP and a cytosolic extract containing chaperone proteins that maintain the precursor proteins in an unfolded conformation. Protein uptake occurs only with energized (respiring) mitochondria, which have a proton electrochemical gradient (proton-motive force) across the inner membrane. This assay has been used to study targeting sequences and other features of the translocation process.

and then consider how some proteins subsequently are targeted to other compartments of the mitochondrion.

After synthesis in the cytosol, the soluble precursors of mitochondrial proteins (including hydrophobic integral membrane proteins) interact directly with the mitochondrial membrane. In general, only unfolded proteins can be imported into the mitochondrion. Chaperone proteins such as cytosolic Hsc70 keep nascent and newly made proteins in an unfolded state, so that they can be taken up by mitochondria. Import of an unfolded mitochondrial precursor is initiated by the binding of a mitochondrial targeting sequence to an import receptor in the outer mitochondrial membrane. These receptors were first identified by experiments in which antibodies to specific proteins of the outer mitochon-drial membrane were shown to inhibit protein import into

M FIGURE 16-26 Protein import into the mitochondrial matrix. Precursor proteins synthesized on cytosolic ribosomes are maintained in an unfolded or partially folded state by bound chaperones, such as Hsc70 (step Ml). After a precursor protein binds to an import receptor near a site of contact with the inner membrane (step |2), it is transferred into the general import pore (step |3|). The translocating protein then moves through this channel and an adjacent channel in the inner membrane (steps |4|, |5|). Note that translocation occurs at rare "contact sites" at which the inner and outer membranes appear to touch. Binding of the translocating protein by the matrix chaperone Hsc70 and subsequent ATP hydrolysis by Hsc70 helps drive import into the matrix. Once the uptake-targeting sequence is removed by a matrix protease and Hsc70 is released from the newly imported protein (step |6|), it folds into the mature, active conformation within the matrix (step |7|). Folding of some proteins depends on matrix chaperonins. See the text for discussion. [See G. Schatz, 1996, J. Biol. Chem. 271:31763, and N. Pfanner et al., 1997, Ann. Rev. Cell Devel. Biol. 13:25.]

isolated mitochondria. Subsequent genetic experiments, in which the genes for specific mitochondrial outer-membrane proteins were mutated, showed that specific receptor proteins were responsible for the import of different classes of mitochondrial proteins. For example, N-terminal matrixtargeting sequences are recognized by Tom20 and Tom22. (Proteins in the outer mitochondrial membrane involved in targeting and import are designated Tom proteins for translocon of the outer membrane.)

The import receptors subsequently transfer the precursor proteins to an import channel in the outer membrane. This channel, composed mainly of the Tom40 protein, is known as the general import pore because all known mito-chondrial precursor proteins gain access to the interior compartments of the mitochondrion through this channel. When purified and incorporated into liposomes, Tom40 forms a transmembrane channel with a pore wide enough to accommodate an unfolded polypeptide chain. The general import pore forms a largely passive channel through the outer mi-tochondrial membrane, and the driving force for unidirectional transport into mitochondria comes from within the mitochondrion. In the case of precursors destined for the mitochondrial matrix, transfer through the outer membrane occurs simultaneously with transfer through an innermembrane channel composed of the Tim23 and Tim17 proteins. (Tim stands for iranslocon of the inner membrane.) Translocation into the matrix thus occurs at "contact sites" where the outer and inner membranes are in close proximity.

Soon after the N-terminal matrix-targeting sequence of a protein enters the mitochondrial matrix, it is removed by a protease that resides within the matrix. The emerging protein also is bound by matrix Hsc70, a chaperone that is localized to the translocation channels in the inner mitochondrial membrane by interacting with Tim44. This interaction stimulates ATP hydrolysis by matrix Hsc70, and together these two proteins are thought to power translocation of proteins into the matrix.

Some imported proteins can fold into their final, active conformation without further assistance. Final folding of many matrix proteins, however, requires a chaperonin. As discussed in Chapter 3, chaperonin proteins actively facilitate protein folding in a process that depends on ATP. For instance, yeast mutants defective in Hsc60, a chaperonin in the mitochondrial matrix, can import matrix proteins and cleave their uptake-targeting sequence normally, but the imported polypeptides fail to fold and assemble into the native tertiary and quaternary structures.

Studies with Chimeric Proteins Demonstrate Important Features of Mitochondrial Import

Dramatic evidence for the ability of mitochondrial matrixtargeting sequences to direct import was obtained with chimeric proteins produced by recombinant DNA techniques. For example, the matrix-targeting sequence of alcohol dehydrogenase can be fused to the N-terminus of dihydrofolate reductase (DHFR), which normally resides in the cytosol. In the presence of chaperones, which prevent the C-terminal DHFR segment from folding in the cytosol, cellfree translocation assays show that the chimeric protein is transported into the matrix (Figure 16-27a). The inhibitor methotrexate, which binds tightly to the active site of DHFR and greatly stabilizes its folded conformation, renders the chimeric protein resistant to unfolding by cytosolic chaper-ones. When translocation assays are performed in the presence of methotrexate, the chimeric protein does not completely enter the matrix. This finding demonstrates that a precursor must be unfolded in order to traverse the import pores in the mitochondrial membranes.

Additional studies revealed that if a sufficiently long spacer sequence separates the N-terminal matrix-targeting se quence and DHFR portion of the chimeric protein, then a stable translocation intermediate forms in the presence of methotrexate (Figure 16-27b). In order for such a stable translocation intermediate to form, the spacer sequence must be long enough to span both membranes; a spacer of 50 amino acids extended to its maximum possible length is adequate to do so. If the chimera contains a shorter spacer—say, 35 amino acids—no stable translocation intermediate is obtained because the spacer cannot span both membranes. These observations provide further evidence that translocated proteins can span both inner and outer mitochondrial membranes and traverse these membranes in an unfolded state.

Microscopic studies of stable translocation intermediates show that they accumulate at sites where the inner and outer mitochondrial membranes are close together, evidence that precursor proteins enter only at such sites (Figure 16-27c). The distance from the cytosolic face of the outer membrane to the matrix face of the inner membrane at these contact sites is consistent with the length of an unfolded spacer sequence required for formation of a stable translocation intermediate. Moreover, stable translocation intermediates can be chemically cross-linked to the protein subunits that comprise the translocation channels of both the outer and inner membranes. This finding demonstrates that imported proteins can simultaneously engage channels in both the outer and inner mitochondrial membrane, as depicted in Figure 16-26. Since roughly 1000 stuck translocation intermediates can be observed in a typical yeast mitochondrion, it is thought that mitochondria have approximately 1000 general import pores for the uptake of mitochondrial proteins.

Three Energy Inputs Are Needed to Import Proteins into Mitochondria

As noted previously and indicated in Figure 16-26, ATP hydrolysis by Hsc70 chaperone proteins in both the cytosol and the mitochondrial matrix is required for import of mitochon-drial proteins. Cytosolic Hsc70 expends energy to maintain bound precursor proteins in an unfolded state that is competent for translocation into the matrix. The importance of ATP to this function was demonstrated in studies in which a mito-chondrial precursor protein was purified and then denatured (unfolded) by urea. When tested in the cell-free mitochon-drial translocation system, the denatured protein was incorporated into the matrix in the absence of ATP. In contrast, import of the native, undenatured precursor required ATP for the normal unfolding function of cytosolic chaperones.

The sequential binding and ATP-driven release of multiple matrix Hsc70 molecules to a translocating protein may simply trap the unfolded protein in the matrix. Alternatively, the matrix Hsc70, anchored to the membrane by the Tim44 protein, may act as a molecular motor to pull the protein into the matrix (see Figure 16-26). In this case, the functions of matrix Hsc70 and Tim44 would be analogous to the chaperone BiP and Sec63 complex, respectively, in post-translational translocation into the ER lumen (see Figure 16-9).

Cytosol

Outer membrane

Intermembrane space ilSfl

COO-

Unfolded DHFR

ytpj-

Cleaved Mitochondrial targeting matrix sequence

COO-

Unfolded DHFR

ytpj-

Cleaved Mitochondrial targeting matrix sequence

Spacer sequence

(b) Bound methotrexate inhibitor rnn-

Folded DHFR "

WllllWlll lillSlMl«

rnn-

Folded DHFR "

Translocation intermediate

Cleaved targeting sequence

Outer Intermembrane membrane space

Inner membrane

Mitochondrial matrix

▲ EXPERIMENTAL FIGURE 16-27 Experiments with chimeric proteins show that a matrix-targeting sequence alone directs proteins to the mitochondrial matrix and that only unfolded proteins are translocated across both membranes. The chimeric protein in these experiments contained a matrix-targeting signal at its N-terminus (red), followed by a spacer sequence of no particular function (black), and then by dihydrofolate reductase (DHFR), an enzyme normally present only in the cytosol. (a) When the DHFR segment is unfolded, the chimeric protein moves across both membranes to the matrix of energized mitochondria and the matrix-targeting signal then is removed. (b) When the C-terminus of the chimeric protein is locked in the folded state by binding of methotrexate, translocation is blocked. If the spacer sequence is long enough

The third energy input required for mitochondrial protein import is a H+ electrochemical gradient, or protonmotive force, across the inner membrane (Chapter 8). In general, only mitochondria that are actively undergoing respiration, and therefore have generated a proton-motive force across the inner membrane, are able to translocate precursor proteins from the cytosol into the mitochondrial matrix. Treatment of mitochondria with inhibitors or uncouplers of oxidative phosphorylation, such as cyanide or dinitrophenol, dissipates this proton-motive force. Although precursor proteins still can bind tightly to receptors on such poisoned mitochondria, the proteins cannot be imported, either in intact cells or in cell-free systems, even in the presence of ATP and chaperone proteins. Scientists do not fully understand how the proton-motive force is used to facilitate entry of a precursor protein into the matrix. Once a protein is partially inserted into the inner membrane, it is subjected to a transmembrane potential of 200 mV (matrix space negative), which is equivalent to an electric gradient of about 400,000

to extend across the transport channels, a stable translocation Intermediate, with the targeting sequence cleaved off, is generated in the presence of methotrexate, as shown here. (c) The C-terminus of the translocation intermediate in (b) can be detected by incubating the mitochondria with antibodies that bind to the DHFR segment, followed by gold particles coated with bacterial protein A, which binds nonspecifically to antibody molecules (see Figure 5-51). An electron micrograph of a sectioned sample reveals gold particles (red arrowhead) bound to the translocation intermediate at a contact site between the inner and outer membranes. Other contact site (black arrows) also are evident. [Parts (a) and (b) adapted from J. Rassow et al., 1990, FEBS Letters 275:190. Part (c) from M. Schweiger et al., 1987, J. Cell Biol. 105:235, courtesy of W. Neupert.]

V/cm. One hypothesis is that the positive charges in the am-phipathic matrix-targeting sequence could simply be "elec-trophoresed, " or pulled, into the matrix space by the inside-negative membrane electric potential.

Multiple Signals and Pathways Target Proteins to Submitochondrial Compartments

Unlike targeting to the matrix, targeting of proteins to the intermembrane space, inner membrane, and outer membrane of mitochondria generally requires more than one targeting sequence and occurs via one of several pathways. Figure 16-28 summarizes the organization of targeting sequences in proteins sorted to different mitochondrial locations.

Inner-Membrane Proteins Three separate pathways are known to target proteins to the inner mitochondrial membrane. One pathway makes use of the same machinery that is

Imported protein

Alcohol dehydrogenase III

Cytochrome oxidase subunit CoxVa

Location of imported protein

Matrix

Inner membrane (path A)

Locations of targeting sequences in preprotein

Cleavage by matrix protease

Cleavage by matrix protease

Matrix-targeting sequence

Mature protein

Matrix-targeting sequence

Mature protein

Cleavage by Hydrophobic stop-

matrix protease transfer sequence

< FIGURE 16-28 Arrangement of targeting sequences in imported mitochondrial proteins. Most mitochondrial proteins have an N-terminal matrix-targeting sequence (pink) that is similar but not identical in different proteins. Proteins destined for the inner membrane, the intermembrane space, or the outer membrane have one or more additional targeting sequences that function to direct the proteins to these locations by several different pathways. The designated pathways in parentheses correspond to those illustrated in Figures 16-29 and 16-30). [See W. Neupert, 1997, Ann. Rev. Biochem. 66:863.]

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