Protein Targeting - Nucleus, Mitochondria  and Chloroplasts.

1. Targeting signals

All proteins have a common beginning. Synthesis of all proteins, regardless of their final destination is initiated is initiated on free ribosomes in the cytoplasmic compartment of the cell.  Any differences in the fate of proteins is a consequence of targeting signals contained within the amino acid sequence of the protein itself.

Figure 14-5

Key concept: Each protein is destined by its amino sequence to go to certain places and to do certain things.  Different proteins contain different addressing information that enables them to be routed to different specific destinations. See Fig 14-5. Changes in amino acid sequence (mutations) often result in improper transport and processing of proteins so that the abnormal polypeptides do not appear in the normal position for the protein.

There are three major types of transport systems

• Nuclear pores
• Translocation across membranes.
• Vesicle transport

The key problem is how to get largely hydrophilic proteins across the hydrophobic barrier presented by the membrane core. -- It is done in the same way that transporters for smaller hydrophilic molecules work, by creating a controlled  hydrophilic passage through the membrane for specific molecules.

Signal information is the key. It corresponds to the address on a letter and is contained in the amino acid sequence of the protein. It allows the protein to be regognized and processed by other proteins.

In general, the signal sequences are located at or near the amino-terminal end of the polypeptide. Notice in the table above that the exception is the ER retention signal, which is located at the C-terminal end of the protein.

Each of the signal sequences has a characteristic amino acid motif: for example, in the nuclear localization signal the key motif is lys-lys-lys-arg-lys.

The signal sequences are critical for proper targeting of proteins. 

Key experiment: Take the signal peptide sequence from a protein that is normally targeted to the lumen of the ER and put it onto the N-terminal end of a protein that normally is found in the cytosol: The modified protein is now located in ER. Conversely, if the signal sequence is removed from the protein that is normally targeted to the ER lumen, it does not enter the ER, but stays in the cytosol.

Figure 14-6 experimental test of the role of signal sequences. IMPORTANT

2. Entering the nucleus. 

Good link for nuclear pores. Lots of material

Entry of large proteins into the nucleus requires:

1. Nuclear Localization Signal (NLS) lys-lys-lys-arg-lys.  inside the protein, near but not at the N-terminal end of protein.
2. NLS region of protein binds to a cytosolic nuclear import receptor protein (NIR)
3. NIR with attached NLS-protein then binds to the nuclear pore. A GTP driven reaction results in a change in the configuration of the pore that results in the translocation (movement) of the NIR-NLS-protein complex into the nucleus
4. When there, the NIR proteins dissociate from the NLS-protein and are returned to the cytosol.
5. As the NLS is an internal localization signal it is not removed by a specific peptidase  as are ER, mitochondrial and chloroplast localization signals that are located at the N terminal end of the protein.

Figure 14-9 Import of proteins into the nucleus. Notice the role of the nuclear import receptor protein (blue). Link to animation.

3. Entry to a mitochondrion, chloroplast or peroxisome

Key concept: Protein containing signal sequence is recognized, bound, unfolded on entry and refolded on the organellar side of the membrane. See Fig 14-10 (left). This requires a set of special organellar chaperone proteins.

The protein recognition signal is quite large (See table). The entry site to mitochondria or chloroplasts is a site where the inner and outer membranes touch. This is referred to as a contact site.

1. The protein containing the signal sequence is synthesized in the cytoplasm.
2. Signal sequence binds to a receptor in the organelle membrane
3. Receptor - protein complex diffuses within membrane to a contact site.
4. Protein is unfolded, moved across the membrane, and refolded. These operations are carried out by the protein transporter complex and its associated chaperone proteins. Remember chaperones from earlier discussion of protein processing? The signal sequence is the first part of the protein to enter the organelle.
5. Once inside, the signal sequence is cleaved off by a specific peptidase.