CH 12 Transport of Proteins Into Mitochondria and Chloroplasts

A. Is Tim23 an integral component of the inner or outer mitochondrial membrane? Explain your reasoning.

A. Tim23 appears to be an integral component of both mitochondrial membranes. In intact mitochondria, a small portion of Tim23 is digested by the protease, indicating that a segment of Tim23 is exposed outside mitochondria. This result implies that a portion of Tim23 extends through the outer mitochondrial membrane. The digested segment of Tim23 must be at the N-terminus because the remaining portion is still recognized by antibodies specific for the C-terminus (see Figure 12-13B, lane 2). In mitoplasts, a larger N-terminal segment of Tim23 is digested by the protease but the C-terminal portion is still protected, indicating that it is in the inner membrane or inside the mitoplasts (see Figure 12-13B, lane 3). In combination, these results indicate that Tim23 must extend through both mitochondrial membranes.

Mitochondria normally provide cells with most of the ATP they require to meet their energy needs. Mitochondria that cannot import proteins are defective for ATP synthesis. How is it that cells with import-defective mitochondria can survive at all? How do they get the ATP they need to function?

Although it is true that mitochondria normally generate the majority of the cell's ATP, they are not the sole source. Two steps in the glycolytic pathway for glucose catabolism, which occurs in the cytosol, also generate ATP. If glucose is provided, yeast (and many other cells) can survive on the ATP they generate by glucose metabolism. It is this process that allows cells to survive in the absence of oxygen, which is required for ATP production by mitochondria. In the absence of oxygen, or when mitochondria are defective, the end product of glucose metabolism in yeast is ethanol.

matrix space

Central subcompartment of a mitochondrion, enclosed by the inner mitochondrial membrane.

T/F: Import of proteins into mitochondria and chloroplasts is very similar; even the individual components of their transport machinery are homologous, as befits their common evolutionary origin.

False. Although import of proteins is similar, the components of the import machinery in chloroplasts and mitochondria are not related. The functional similarities appear to have arisen by convergent evolution, reflecting the common requirements for translocation across a double- membrane system.

T/F: The two signal sequences required for transport of nucleus-encoded proteins into the mitochondrial inner membrane via the TIM23 complex are cleaved off the protein in different mitochondrial compartments.

False. Only one of the two signal sequences is cleaved. The N-terminal signal is cleaved off the imported protein when it reaches the mitochondrial matrix. The second signal—a very hydrophobic sequence at the new N-terminus—directs the protein to the inner membrane through either the TIM23 complex or the OXA complex. The second signal is not cleaved; it anchors the protein in the inner membrane.

thylakoid

Flattened sac of membrane in a chloroplast that contains the protein subunits of the photosynthetic system and of the ATP synthase.

Are proteins imported into mitochondria as completely unfolded polypeptide chains, or can the translocation apparatus accommodate fully or partially folded structures? That is, is the protein sucked up like a noodle, or is it swallowed whole, as a python devours its prey? It is possible to engineer cysteine amino acids into barnase and then cross-link them to make disulfide bonds either between C5 and C78 or between C43 and C80 (Figure 12-12). Import of N95-barnase (see Problem 12-70) was tested in the presence and absence of disulfide cross-links at these two positions. Its import was unaffected by either cross-link. By contrast, import of N65-barnase was blocked by the C5-C78 cross-link but unaffected by the C43-C80 cross-link. Do these results allow you to distinguish between import of extended polypeptide chains or of folded structures? Why or why not?

If barnase could be imported in its native folded configuration, the cross-links should have no effect on import; however, import of N65- barnase was blocked by the C5-C78 cross-link. On the other hand, if import required a completely unfolded, extended polypeptide chain, the presence of either cross-link should have blocked import of both N65- barnase and N95-barnase. Thus, it seems that the mitochondrial import machinery cannot import completely folded structures, but it doesn't require that the protein be in a fully extended configuration either. The ability of the import machinery to accommodate cross-links indicates that it can pass at least two, side-by-side polypeptide chains. The authors of the original study point out that a key event in mitochondrial import is destabilization of the N-terminus of the imported protein. Thus, import of N65-barnase, which occurs at a low rate, is completely blocked when the N-terminus is stabilized by a cross-link. They also show that while the N95 extension attached to dihydrofolate reductase (DHFR) allows efficient import, import of N95-DHFR is blocked by methotrexate (see Problem 12-67).

To aid your studies of protein import into mitochondria, you treat yeast cells with cycloheximide, which blocks ribosome movement along mRNA. When you examine these cells in the electron microscope, you are surprised to find cytosolic ribosomes attached to the outside of the mitochondria. You have never seen attached ribosomes in the absence of cycloheximide. To investigate this phenomenon further, you prepare mitochondria from cells that have been treated with cycloheximide and then extract the mRNA that is bound to the ribosomes associated with the mitochondria. You translate this mRNA in vitro and compare the protein products with similarly translated mRNA from the cytosol. The results are clear-cut: the mitochondria-associated ribosomes are translating mRNAs that encode mitochondrial proteins. You are astounded! Here, clearly visible in the electron micrographs, seems to be proof that protein import into mitochondria occurs during translation. How might you reconcile this result with the prevailing view that mitochondrial proteins are imported only after they have been synthesized and released from ribosomes?

Import of mitochondrial proteins occurs post-translationally. Normally, translation is much faster than mitochondrial import, so that proteins completely clear the ribosome before interacting with the mitochondrial membrane. By blocking protein synthesis with cycloheximide, you have made the rate of translation artificially slower than the rate of import. Since the signal peptide for protein import into mitochondria resides at the N-terminus, some of the partially synthesized mitochondrial proteins, which are still attached to ribosomes, will be able to interact with the mitochondrial membrane. The attempted import of even one such protein will attach the ribosome and the mRNA (and all other ribosomes translating the same mRNA molecule) to the mitochondrial membrane.

Describe in a general way how you might use radiolabeled proteins and proteases to study import processes in isolated, intact mitochondria. What sorts of experimental controls might you include to ensure that the results you obtain mean what you think they do?

Incubate the radiolabeled proteins with isolated mitochondria under conditions you wish to test, allow a sufficient time for import, and then treat the mixture with a protease. Proteins that are not imported will be digested by the protease. Proteins that have been imported will be resist- ant to the protease. Protease-resistant proteins could be assayed by re-isolating the mitochondria and measuring the counts associated with them. Alternatively, they could be assayed by solubilizing the entire mixture and separating the proteins by gel electrophoresis. Protease-resist- ant proteins would run at the same position as untreated proteins. These analyses assume that proteins are protease-resistant because they are sequestered inside mitochondria, meaning they have been imported. You would need to include several controls before you could make this conclusion. You would need to know that the protease is work- ing, which could be measured by leaving the mitochondria out of the incubation mixture. You would need to know that the protein is stable in the absence of the protease, which you could assay by leaving the protease out of the incubation mixture. You would need to know that protease-resistant proteins are in the mitochondria, which could be assayed by solubilizing the mitochondria with a detergent to show that protease-resistant proteins now become protease-sensitive. Appropriate controls are essential for informative research into any biological problem.

mitochondria

Membrane-enclosed organelles, about the size of bacteria, that carry out oxidative phosphorylation and produce most of the ATP in eukaryotic cells

TOM complex

Multisubunit protein assembly that transports proteins across the mitochondrial outer membrane.

Components of the TIM complexes, the multisubunit protein translocators in the mitochondrial inner membrane, are much less abundant than those of the TOM complex. They were initially identified using a genetic trick. The yeast Ura3 gene, whose product is an enzyme that is normally located in the cytosol where it is essential for synthesis of uracil, was modified so that the protein carried an import signal for the mitochondrial matrix. A population of cells carrying the modified Ura3 gene in place of the normal gene was then grown in the absence of uracil. Most cells died, but the rare cells that grew were shown to be defective for import into the mitochondrial matrix. Explain how this selection identifies cells with defects in components required for import into the mitochondrial matrix. Why don't normal cells with the modified Ura3 gene grow in the absence of uracil? Why do cells that are defective for mitochondrial import grow in the absence of uracil?

Normal cells that carry the modified Ura3 gene make Ura3 that gets imported into mitochondria. It is therefore unavailable to carry out an essential reaction in the metabolic pathway for uracil synthesis. These cells might as well not have the enzyme at all, and they will grow only when uracil is supplied in the medium. By contrast, in cells that are defective for mitochondrial import, Ura3 is prevented from entering mitochondria and remains in the cytosol where it can function normally in the pathway for uracil synthesis. Thus, cells with defects in import into the mitochondrial matrix can grow in the absence of added uracil because they can make their own.

mitochondrial hsp70

Part of a multisubunit protein assembly that is bound to the matrix side of the TIM23 complex and acts as a motor to pull the precursor protein into the matrix space.

You have made a peptide that contains a functional mitochondrial import signal. Would you expect the addition of an excess of this peptide to affect the import of mitochondrial proteins? Why or why not?

Peptides with mitochondrial import signals would be expected to compete with mitochondrial proteins for binding to the translocation machinery. Thus, an excess of such peptides should reduce or abolish import of mitochondrial proteins.

mitochondrial precursor protein

Protein encoded by a nuclear gene, synthesized in the cytosol, and sub- sequently transported into mitochondria

Barnase is a 110-amino-acid bacterial ribonuclease that is often used as a model for studies of protein folding and unfolding. It forms a com- pact folded structure that has a high energy of activation for unfolding (about 85 kJ/mole). Can such a protein be imported into mitochondria? To the N-terminus of barnase, you add 35, 65, or 95 amino acids from the N-terminus of pre-cytochrome b2, all of which include the cytochrome's mitochondrial import signal. N35-barnase is not imported, N65-barnase is imported at a low rate, and N95-barnase is imported very efficiently into isolated mitochondria. None of these N-terminal extensions have any measurable effect on the stability of the barnase domain. If these proteins are denatured before testing for import, they are all imported at the same high rate. How do you suppose that longer N-terminal extensions facilitate the import of barnase?

Since each modified barnase includes an import signal and the length of the N-terminal extension does not affect the stability of the barnase domain, the dependence of import on the length of the extension presumably reflects some process inside mitochondria. The most likely possibility is that only the longer extensions can span both mitochondrial membranes and project into the matrix. There they can be bound by the mitochondrial hsp70, which can use the hydrolysis of ATP to help drive import. Presumably, the 95-amino-acid extension is long enough to be efficiently engaged by hsp70, whereas the 65-amino-acid extension must be less efficiently bound. Hsp70 and the energy of ATP hydrolysis are required for import of barnase because of its extremely stable folded structure. If the protein is first denatured, all three N-terminal extensions can facilitate its import at the same high rate because the unfolded protein does not hinder entry into the matrix.

If the enzyme dihydrofolate reductase (DHFR), which is normally located in the cytosol, is engineered to carry a mitochondrial targeting sequence at its N-terminus, it is efficiently imported into mitochondria. If the modified DHFR is first incubated with methotrexate, which binds tightly to the active site, the enzyme remains in the cytosol. How do you suppose that the binding of methotrexate interferes with mitochondrial import?

The binding of methotrexate to the active site prevents the enzyme from unfolding, which is necessary for import into mitochondria. Evidently, methotrexate binds so tightly that it locks the enzyme into its folded con- formation and prevents chaperone proteins from unfolding it.

stroma

The matrix space of a chloroplast.

inner membrane

The membrane of a mitochondrion that encloses the matrix and is folded into cristae.

B. To the extent the information in this problem allows, diagram the arrangement of Tim23 in mitochondrial membranes.

The pattern of protease sensitivity of Tim23 in mitochondria and mito- plasts suggests that Tim23 is arranged as shown in Figure 12-24. You may have noticed that the hydropathy plot does not predict a membrane-spanning segment at the N-terminus. The authors of the original study noticed the same thing. There is, however, a predicted propensity for β-sheet formation in the N-terminus (not shown). Thus, the authors suggest that Tim23 may span the outer membrane in a β-strand conformation, which is typical of certain outer membrane proteins such as porins.

Why do mitochondria need a special translocator to import proteins across the outer membrane, when the membrane has already has large pores formed by porins?

The pores formed by porins are large enough for all ions and metabolic intermediates, but not large enough for most proteins. The size cutoff for free passage through the pores of mitochondrial porins is roughly 10 kilo- daltons.

T/F: The TOM complex is required for the import of all nucleus-encoded mitochondrial proteins.

True. Regardless of their final destination in the mitochondrion, all proteins that are synthesized in the cytosol (that is, all nucleus-encoded mitochondrial proteins) must first enter the TOM complex. After the TOM complex, the pathways of import diverge as proteins are sorted to their appropriate mitochondrial compartment.