Protein targeting to mitochondria and chloroplasts

Protein Targeting and Organelle Biogenesis

MITOCHONDRIA:
The genes for most mitochondrial proteins are located where? (the nucleus). So they are transcribed in the nucleus and translated where? In the cytosol on free polysomes.. That is, some mitochondrial proteins are made on ribosomes that are free in the cytosol, not on ribosomes bound to a membrane. But they need to cross, or be inserted into, a membrane in order to function. (And other mitochondrial proteins are made inside the mitochondrion itself--see Table 17-2). How does that happen?
The current belief is that there is a long targeting sequence, or more precisely several similar kinds of sequences, on mitochondrial proteins that helps them get into the appropriate areas of the mitochondrion after they are synthesized. There appear to be specific receptor proteins on the outer mitochondrial membrane that bind these targeting sequences. Over the years people have altered their . thinking about what the "targeting sequence" on mitochondrial proteins is; as more mitochondrial proteins have been identified and their amino acid sequence determined, there seem to be relatively few things in common that might be the targeting signal. It's now believed that a group of positively charged amino acids at the N terminus of the protein, interspersed with some hydrophilic aas like serine and threonine, is likely to contain the necessary information to allow the protein to bind to the mitochondrial receptor complex (Table 17-1). It's thought that the receptor is associated with a long pore that extends through both the outer and inner mitochondrial membrane. This allows the protein to pass into the matrix, where a signal peptidase, an enzyme that cuts the peptide backbone of a protein in only one place, cuts off the targeting peptide, essentially trapping the protein inside the mitochondrion. If the protein is destined to become an outer membrane protein, like porin, then it presumably has a hydrophobic sequence so that it gets stuck in the membrane as it is being inserted; we'll discuss this membranej-insertion process in more detail when we consider protein targeting into the ER. There is also evidence that some proteins are first passed all the way into the matrix, and then reinsert into the inner mitochondrial membrane, presumably because they contain a second targeting sequence, but little is known about the details of construction of the inner mitochondrial membrane (Fig. 17-2). (Recall that some of the proteins of the inner membrane are encoded by DNA in the mitochondrion itself, and some are encoded by genes in the nucleus. Once the nuclear-encoded proteins get into the matrix, presumably both they and the proteins made inside the matrix are inserted into the inner membrane in the same--unknown--way). This model raises at least as many questions as it answers and the answers aren't known. First, it's not clear what distinguishes inner and outer mitochondrial membrane proteins. That is, how come some proteins (e.g., porin) stick in the outer membrane, while most come to reside in the inner membrane? What determines the orientation of the proteins in the membrane, what distinguishes proteins that get inserted into the membrane on their first pass, vs. those that get in on a subsequent pass? And so forth.
As I mentioned last time, mitochondrial proteins on the other hand pass through the membrane postranslationally. That is, the whole protein is made before it gets to the pore through the membrane. These pores are very narrow, and it appears that the only way a protein can pass through them is strung out in a long skinny line, like a piece of thread through the eye of a needle. But a protein usually folds up into a complex ball once it is synthesized as you know. A protein that is all folded up will not be able to pass through a pore unless it is unfolded first. That seems like a terrible waste of energy--first folding the protein, then unfolding it, then folding it again--and of course it is a terrible waste. So cells evolved a method to get around this wasteful folding and unfolding problem, by preventing mitochondrial proteins from folding up before they get to the pore. The way they do this is with a group of proteins that bind to other proteins as they are being synthesized and thereby keep them in an extended conformation, not a folded up ball. These proteins are now generally called chaperone proteins, or chaperonins. The idea is that they attach themselves to new mitochondrial proteins and escort them--or chaperone them--to the mitochondrion and align them with the pore. Somehow the chaperonins peel off as the protein passes through the pore--it takes breakdown of ATP for the chaperonins to unbind, but there are often other chaperonins in the matrix of the mitochondrion that bind to the protein as it comes out the other side. This second binding of chaperonins may help pull the protein through the pore. (There appear to be chaperonins in the lumen of the ER also that may bind and pull newly formed proteins through the pore).
The area of mitochondrial protein import is one of intense research and rapid progress. What's now know, or suspected about the process, is shown in the figure17-4 from Lodish et al. In the cytosol, mitochondrial proteins are bound by chaperone proteins, typically hsp70 or else mitochondrial-import stimulating factor (MSF) which prevents their folding up completely, and that hydrolyzes ATP in order to accomplish this. The positively charged signal sequence on the mitochondrial protein then binds to a receptor protein complex on the outer mitochondrial membrane, allowing the protein to interact with the transmembrane pore. The binding part of the complex is made up of two proteins called Tom (Transport across the Outer Membrane) 70 and 37 (for their molecular weights), and they then transfer the protein to two other proteins, Tom 22 and 20, which in turn feed it into a channel made up of the Tom 40 protein (thus at least 5 separate proteins make up the binding site and outer membrane pore of the transfer complex). In the inner mitochondrial membrane is another protein complex that forms a pore, made of similar but distinct proteins called Tim (Transport across the Inner Membrane). The energy of the mitochondrial proton gradient is somehow necessary to drive the protein through the pore, during which the hsp70 protein is displaced. On the matrix side of the pore, a different but very similar hsp70 chaperone binds the protein and keeps it from folding up, again at the expense of ATP. Proteins that will remain in the matrix, like all the enzymes of the TCA cycle are folded up with the help of a barrel shaped protein complex made of the hsp60 (erroneously called Hsc 70 in the picture, but correctly named in the legend) chaperone protein (which forms one of those barrel-shaped folding chambers for protein) again using ATP to provide energy. The signal sequence is removed by a mitochondrial signal peptidase as it enters the matrix.
How do we know about this scheme? Several different kinds of experiments support this idea. First, it’s possible to use molecular genetics to introduce mutations in the suspected signal sequences. Removal of the positively charged residues or addition of negatively charged amino acids prevents the protein from entering the mitochondria while addition of a mitochondrial signal sequence to a cytosolic protein redirects it into the mitochondria. Second one can disrupt the mitochondrial proton gradient with agents like cyanide, azide, or DNP; this blocks uptake of mitochondrial protein through the pore, but not binding of the protein to the receptor/pore proteins, implying that the proton gradient supplies the energy for transport across the membrane. Third, yeast genetics have supplied many important clues for the process. Yeast cells can grow both aerobically (making ATP in mitochondria), or anaerobically, using only glycolysis to make ATP (and CO2--for breadmaking and beer making). So mutations that disrupt mitochondrial function in yeast are not usually fatal, but they slow the growth of the yeast cells. Analysis of yeast mitochondrial mutants has established the existence and function a variety of outer membrane receptor proteins and inner membrane proteins, the Toms and Tims.
As this model indicates, chaperone proteins here, and elsewhere in the cell, seem to conduct two separate, though related functions. On the one hand, they keep proteins from folding up completely; that is, they keep immature proteins or proteins not yet in their final functional location, in a partially extended state. But they are also essential for folding up the proteins properly when they are mature. It’s thought that this is accomplished by sequential activity of several chaperone proteins. In mitochondria, these are largely the hsp70 and hsp 60 proteins, but elsewhere in the cell it can be different proteins.
Finally, there are different compartments within the mitochondrion, including the matrix, the intermembrane space, and the inner and outer membranes. How do proteins get sorted to appropriate locations within the mitochondrion? It’s thought that different proteins contain additional signal sequences that specify different locations, such as the membrane or intemembrane space. (Fig. 17-5). The thought is that these additional targeting sequences either prevent the protein from passing through the inner membrane, trapping it in the intermemrane space (left, as is the case for cytochrome b₂) or cause the protein to bind to a different transporter complex in the inner mitochondrial membrane, which allows it to pass back through the inner membrane into the intermembrane space (right; e.g. cytochrome c₁). Proteins that lack these secondary signals are thought to remain in the matrix. Again people have worked out these details by creating modified proteins. If one removes the intermembrane-space targeting sequence from cytochrome c₁, for example, it remains in the matrix, and doesn't get to the intermembrane space. If one inserts the targeting sequence into a matrix protein, it gets misdirected into the intermembrane space.
CHLOROPLASTS:
Not as much is known about how protein import into chloroplasts works, partly because there are no organisms that have been found analogous to yeast where it is easy to do genetics and isolate chloroplast mutants. However, it’s felt that the process of protein import is similar in chloroplasts to the process in mitochondria. That is, there are likely to receptors and channels in the chloroplast outer and inner membranes, signal sequences on nuclear-encoded chloroplast proteins, hsp70-type chaperones that prevent premature folding, and stromal chaperones that promote folding. Some of the components of the outer and inner mitochondrial membrane required for uptake have been identified and named Tocs (Transport across the Outer Chloroplast membrane) and Tics. The general scheme for uptake and assembly of Rubisco in chloroplasts is illustrated in Fig. 17-7. The small subunit of rubisco is synthesized in the cytosol, while the large subunit is made in the chloroplast stroma. The small subunit is imported through the two membranes using both ATP and GTP, and it's N-terminal targeting sequence (whose nature is not well established), is cleaved off. Then internal chaperonins help the small and large subunits fold correctly and combine to form functional protein.
In addition to the inner and outer membranes, the stroma and the intermembrane space (all similar to the compartments inside mitochondria), chloroplasts have one additional compartment absent from mitochondria--the thylakoid membranes and the thylakoid space. Proteins destined for those areas must be able to cross or be incorporated into the thylakoid membrane. It appears that the thylakoid targeting sequence is a group of about 25 hydrophobic amino acids, often preceded by some positively charged amino acids, that allow interaction with a poorly characterized receptor complex or complexes in the thylakoid membrane. As in the mitochondria, the charge gradient across the thylakoid membrane, created by electron transport during photosynthesis, is necessary for uptake of the proteins, suggesting that the energy from transport is supplied by the electrochemical proton gradient. As in other compartment,s, the targeting peptide is cleaved off by an enzyme inside the thylakoid, and chaperone proteins assist the thylakoid proteins to fold correclty.
I mentioned earlier that both mitochondria and chloroplasts can synthesize some of their own protein constituents. Mitochondria make relatively few, and have genes for only about 20 proteins; most mitochondrial proteins are thus made in the cytosol and then imported. Chloroplasts can make more of their own proteins because they contain genes that encode about 120 proteins, but they still must import most of the essential proteins that they use.