Genes VII

Mitochondria and chloroplasts synthesize only some of their proteins. Mitochondria synthesize only ~10 organelle proteins; chloroplasts synthesize ~50 proteins. The majority of organelle proteins are synthesized in the cytosol by the same pool of free ribosomes that synthesize cytosolic proteins. They must then be imported into the organelle.

Many proteins that enter mitochondria or chloroplasts by a post-translational process have leader sequences that are responsible for primary recognition of the outer membrane of the organelle. As shown in the simplified diagram of Figure 8.11, the leader sequence initiates the interaction between the precursor and the organelle membrane. The protein passes through the membrane, and the leader is cleaved by a protease on the organelle side.

The leaders of proteins imported into mitochondria and chloroplasts are usually hydrophilic, consisting of stretches of uncharged amino acids interrupted by basic amino acids, and they lack acidic amino acids. There is little other homology. An example is given in Figure 8.12. The lack of homology among leader sequences implies that features of the secondary or tertiary structure, or the general nature of the region, must be involved in recognition.

The leader sequence contains all the information needed to localize an organelle protein. The ability of a leader sequence can be tested by constructing an artificial protein in which a leader from an organelle protein is joined to a cytosolic protein. The experiment is performed by constructing a hybrid gene, which is then translated into the hybrid protein.

Several leader sequences have been shown by such experiments to function independently to target any attached sequence to the mitochondrion or chloroplast. For example, if the leader sequence given in Figure 8.12 is attached to the cytosolic protein DHFR (dihydrofolate reductase), the DHFR becomes localized in the mitochondrion.

The leader sequence and the transported protein represent domains that fold independently. Irrespective of the sequence to which it is attached, the leader must be able to fold into an appropriate structure to be recognized by receptors on the organelle envelope. The attached polypeptide sequence plays no part in recognition of the envelope (for review see Baker and Schatz, 1991; Schatz and Dobberstein, 1996).

Hydrolysis of ATP is required both outside and inside for translocation across the membrane. It may be involved with pushing the protein from outside and pulling from inside. In the cases of mitochondrial import and bacterial export, there is also a requirement for an electrochemical potential across the inner membrane to transfer the amino terminal part of the leader.

Some mitochondrial proteins have internal sequences that are responsible for targeting to the organelle or for controlling localization within it, but less is known about these sequences.

What restrictions are there on transporting a hydrophilic protein through the hydrophobic membrane? An insight into this question is given by the observation that methotrexate, a ligand for the enzyme DHFR, blocks transport into mitochondria of DHFR fused to a mitochondrial leader. The tight binding of methotrexate prevents the enzyme from unfolding when it is translocated through the membrane. So although the sequence of the transported protein is irrelevant for targeting purposes, in order to follow its leader through the membrane, it requires the flexibility to assume an unfolded conformation (Eilers and Schatz, 1986).

There are different receptors for transport through each mitochondrial membrane. They are called TOM and TIM, referring to the outer and inner membranes, respectively.

The TOM complex consists of ~9 proteins. many of which are integral membrane proteins. A general model for the complex is shown in Figure 8.13. The TOM aggregate has a size of >500 kD, with a diameter of ~138 Å, and forms an ion-conducting channel. A complex contains 2-3 individual rings of diameter 75 Å, each with a pore of diameter 20 Å.

Tom40 is deeply imbedded in the membrane and provides the channel for translocation. It contacts preproteins as they pass through the outer membrane. It binds to three smaller proteins, Tom,5,6,7, which may be components of the channel or assembly factors. There are two subcomplexes that provide surface receptors. Tom20,22 form a subcomplex with exposed domains in the cytosol. Most proteins that are imported into mitochondria are recognized by the Tom20,22 subcomplex, which is the primary receptor and recognizes N-terminal sequences. Tom37,70,71 provides a receptor for a smaller number of proteins that have internal targeting sequences.

When a protein is translocated through the TOM complex, it passes from a state in which it is exposed to the cytosol into a state in which it is exposed to the intermembrane space. However, it is not usually released, but instead is transferred directly to the TIM complex. It is possible to trap intermediates in which the leader is cleaved by the matrix protease, while a major part of the precursor remains exposed on the cytosolic surface of the envelope. This suggests that a protein spans the two membranes during passage. The TOM and TIM complexes do not appear to interact directly (or at least do not form a detectable stable complex), and they may therefore be linked simply by a protein in transit. When a translocating protein reaches the intermembrane space, the exposed residues may immediately bind to a TIM complex, while the rest of the protein continues to translocate through the TOM complex (for review see Neupert, 1997).

There are two TIM complexes in the inner membrane.

The Tim17-23 complex translocates proteins to the lumen. Substrates are recognized by their possession of a positively charged N-terminal signal. Tim17-23 are transmembrane proteins that comprise the channel. Figure 8.14 shows that they are associated with Tim44 on the matrix side of the membrane. Tim 44 in turn binds the chaperone Hsp70. This is also associated with another chaperone, Mge, the counterpart to bacterial GrpE. This association ensures that when the imported protein reaches the matrix, it is bound by the Hsp70 chaperone. The high affinity of Hsp70 for the unfolded conformation of the protein as it emerges from the inner membrane helps to "pull" the protein through the channel.

A major chaperone activity in the mitochondrial matrix is provided by Hsp60 (which forms the same sort of structure as its counterpart GroEL). Association with Hsp60 is necessary for joining of the subunits of imported proteins that form oligomeric complexes. An imported protein may be "passed on" from Hsp70 to Hsp60 in the process of acquiring its proper conformation (Ostermann et al., 1989).

The Tim22-54 complex translocates proteins that reside in the inner membrane.

How does a translocating protein finds its way from the TOM complex to the appropriate TIM complex? Two protein complexes in the intermembrane space escort a translocating protein from TIM to TOM. The Tim9-10 and Tim8-13 complexes act as escorts for different sets of substrate proteins (Leuenberger et al., 1999). Tim9-10 may direct its substrates to either Tim22-54 or Tim23-17, while Tim8-13 directs substrates only to Tim22-54. Some substrates do not use either Tim9-10 or Tim8-13, so other pathways must also exist. The pathways are summarized in Figure 8.15.

What is the role of the escorting complexes? They may be needed to help the protein exit from the TOM complex as well as for recognizing the TIM complex. Figure 8.16 shows that a translocating protein may pass directly from the TOM channel to the Tim9,10 complex, and then into the Tim22-54 channel

A mitochondrial protein folds under different conditions before and after its passage through the membrane. Ionic conditions and the chaperones that are present are different in the cytosol and in the mitochondrial matrix. It is possible that a mitochondrial protein can attain its mature conformation only in the mitochondrion.

This section updated 2-2-2000