17. Summary

8.17 Summary

Synthesis of all proteins starts on ribosomes that are "free" in the cytosol. In the absence of any particular signal, a protein is released into the cytosol when its synthesis is completed. Proteins that are imported by post-translational transfer into mitochondria or chloroplasts possess N-terminal leader sequences that target them to the organelle envelope; then they are transported through the double membrane into the matrix. Translocation requires ATP and a potential across the inner membrane. The N-terminal leader is cleaved by a protease within the organelle. Proteins that reside within the membranes or intermembrane space possess a signal (which becomes N-terminal when the first part of the leader is removed) that either causes export from the matrix to the appropriate location or which halts transfer before all of the protein has entered the matrix. Control of folding, by Hsp70 and Hsp60 in the mitochondrial matrix, is an important feature of the process. Requirements for export from bacteria also show strong dependence on control of protein conformation.

The N-terminal region of a secreted protein provides a signal sequence that causes the nascent protein and its ribosome to become attached to the membrane of the endoplasmic reticulum. The protein is translocated through the membrane by co-translational transfer. The process starts when the signal sequence is recognized by the SRP (a ribonucleoprotein particle), which interrupts translation. The SRP binds to a receptor in the ER membrane, and transfers the signal sequence to the Sec61/TRAM receptor in the membrane. Synthesis resumes, and the protein is translocated through the membrane while it is being synthesized, although there is no energetic connection between the processes. The channel through the membrane provides a hydrophilic environment, and is largely made of Sec61.

A secreted protein passes completely through the membrane into the ER lumen. For type I integral membrane proteins, the N-terminal signal sequence is cleaved, and transfer through the membrane is halted later by an anchor sequence. The protein becomes oriented in the membrane with its N-terminus on the far side and its C-terminus in the cytosol. Type II proteins do not have a cleavable N-terminal signal, but instead have a combined signal-anchor sequence, which enters the membrane and becomes embedded in it, causing the C-terminus to be located on the far side, while the N-terminus remains in the cytosol. The orientation of the signal-anchor is determined by the "positive inside" rule that the side of the anchor with more positive charges will be located in the cytoplasm. Proteins may have multiple membrane-spanning regions, with loops between them protruding on either side of the membrane. The mechanism of insertion of multiple segments is unknown.

Bacteria have components for membrane translocation that are related to those of eukaryotes, but translocation often occurs by a post-translational mechanism. SecY/E provide the translocase, and SecA associates with the channel and is involved in inserting and propelling the substrate protein. SecB is a chaperone that brings the protein to the channel.

Nuclear pore complexes are massive structures embedded in the nuclear membrane, and are responsible for all transport of protein into the nucleus and RNA out of the nucleus. Whether nuclear pore complexes are heterogeneous remains to be established. Each nuclear pore complex contains a central pore, which forms a channel of diameter <10 nm. Additional channels are present round the periphery. The central channel can be opened to a diameter of ~20 nm to allow passage of larger material, some of which may need to undergo conformational changes to fit.

Proteins that are actively transported into the nucleus require specific NLS sequences, which are short, but do not seem to share common features except for their basicity. Nuclear entry is a two stage process, involving docking followed by an ATP-dependent translocation. The docking reaction is undertaken by the importin complex, which has subunits that bind to the substrate protein and to a nucleoporin protein in the pore, respectively. The direction of translocation is controlled by Ran. The presence of Ran-GDP in the cytosol destabilizes export complexes. The presence of Ran-GTP in the nucleus destabilizes import complexes. This ensures release of substrate on the appropriate side of the nuclear envelope.

Proteins that are exported from the nucleus have specific NES sequences, which share a pattern of leucine residues; they may bind to nucleoporins. Some nucleoporins are required specifically for RNA export from the nucleus.

The major system responsible for bulk degradation of proteins, but also for certain specific processing events, is the proteasome, a large complex that contains several protease activities. It acts upon substrate proteins that have been conjugated to ubiquitin through an isopeptide bond, and upon which a polyubiquitin chain has formed.