Synthesis and sorting of mitochondrial proteins in the cytosol

Many mitochondrial proteins are encoded by nuclear DNA, they are translated on ribosomes in the cytosol.

Proteins coming out of a ribosome are unfolded. In order to prevent fatal protein aggregation, and maintain them in an unfolded, transport competent state, they are complexed with Hsc70, a molecular chaperone (see fig. 15.3 and section 14.1 on page 212).

On the N-terminal side of mitochondrial proteins is a signal sequence, which is recognised by a receptor in the outer mitochondrial membrane. This sequence forms an amphipatic helix, with a positively charged hydrophilic, and an uncharged hydrophobic side (see fig. 15.4). This structural motive (rather than the amino acid sequence) is recognised by a specific receptor on the outer mitochondrial membrane. If this motive is transferred to a cytosolic protein by genetic engineering the chimeric proteins gets imported into the mitochondria. If the signal sequence is removed from mitochondrial proteins, they can no longer be imported.

Mitochondria have about 1000 TOM/TIM pairs each (transporter of the outer/inner) membrane, see fig. 15.5). The import of proteins from the cytosol into the mitochondria occurs in two distinct steps.

Figure 15.3. Proteins synthesised on the ribosome are immediately captured by the 70 kDa Heat shock cognate protein. This prevents miss-folding.

Figure 15.4. N-terminal signal sequence for import into mitochondria, here the subunit 4 of cytochrome c oxidase from yeast. The sequence MLSLRQSIRFFKPATRTL-forms an a-helix. Every third or fourth amino acid is positively charged (Lys or Arg), since there are 3.6 amino acids per turn in an a-helix all positively charged amino acids (blue) are in the same quadrant. The remaining quadrants are occupied by hydrophobic (green) or polar (orange) amino acids, also a few aromatic (dark grey) amino acids are present. This type of helix is called amphipatic. Left: Ribbon diagram of the alpha helix, the N-terminal Met is coloured yellow. Turning this helix by 90 degrees around the y-axis results in right: Helical wheel projection. The numbers inside the circle denote the position of the respective amino acid in the sequence. These numbers are coloured red for the first turn, orange for the second, yellow for the third, green for the fourth and blue for the fifth.

Figure 15.5. Mitochondrial proteins are transported from the cytosol into the mitochondria by two membrane transporters, called TIM and TOM respectively. Cytosolic Hsc70 and mitochondrial DnaK are also required. Import occurs only in places where the inner and outer membrane are close together, allowing concurrent transport of the protein across both membranes. The pore through the transporter has a diameter of approximately 2 nm.

Figure 15.5. Mitochondrial proteins are transported from the cytosol into the mitochondria by two membrane transporters, called TIM and TOM respectively. Cytosolic Hsc70 and mitochondrial DnaK are also required. Import occurs only in places where the inner and outer membrane are close together, allowing concurrent transport of the protein across both membranes. The pore through the transporter has a diameter of approximately 2 nm.

First the signal sequence of the protein is bound to a receptor on the outer membrane of the mitochondrium. This signal sequence, as we have seen, has a number of positive charges. The matrix of the mitochondrium is negatively charged with respect to the cytosol (—200 mV, this is equivalent to a field strength of 400000V/cm), because endoxydation leads to the transport of protons from the matrix to the cytosol. As a result the signal sequence is elec-trophoretically pulled through the outer and inner membrane transporter. Experimentally, this step does not require ATP and can proceed at low temperatures. However, uncouplers like 2,4-dinitrophenol (DNP, see page 294), which dissipate the electrical potential difference across the inner membrane, prevent signal sequence transfer.

The rest of the protein is imported in the second step, which depends on ATP-hydrolysis and can occur only at 37, but not at 5 °C. The protein inside the transporters can move either into or out of the mitochondrium, and it will do so randomly (Brownian motion). Thus, in the absence of other factors, no net transport of protein in either direction would occur. However, any stretch of protein that comes into the mitochondrium is immediately bound by mtDnaK. This prevents the protein from slipping back. On the other hand, on the cytosolic side the protein is released from Hsc70, which allows it to move into the mitochondrium. Thus a random movement is used to achieve vectorial transport by the binding and unbinding of 70 kDa heat shock proteins. This mechanism is called molecular ratchet.

Some researchers claim that binding of mtDnaK to the imported protein leads to a change in the way mtDnaK interacts with the inner membrane, allowing it to actively pull the protein through the transporter. Evidence for this additional mode of action of mtDnaK is currently limited, though.

Once the protein has been imported into the mitochondrial matrix, the signal sequence is cleaved of by matrix processing protease (Mpp). Matrix proteins then fold, as mtDnaK dissociates from them. Folding may involve GroEL/GroES chaperonin action.

Proteins of the inner mitochondrial membrane or the intermembrane space are transported to their destination directed by a second signal sequence, which is unmasked once the first has been cleaved of. In other words, these proteins (for example ATP synthase subunits) are transported from the cytosol through both mitochondrial membranes into the matrix, and from there into the inner membrane or intermembrane space, respectively. This is a reminder of the origin of mitochondria as endosymbionts. Other proteins like cytochrome c are small enough that in their unfolded form (e.g. apocytochrome c, without the haem group) they can enter the intermembrane space directly through the outer membrane, presumably through a porin-like channel. Folding of these proteins either around a prosthetic group or by disulphide bond formation prevents them from returning to the cytosol.

Synthesis of proteins encoded by mitochondrial and nuclear DNA is synchronised, but the mechanism is still unknown.

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