Protein Translocation and Protein Orientation in Membranes

Second last lecture before enzyme kinetics... joy :P

Describe the structure of signalling or targeting sequences in proteins

Proteins that need to be moved to a different compartment of the cell have signalling sequences or targeting sequences in their amino acid sequence. These are recognised by other proteins that help move stuff around the cell. While there are no "consensus sequences" as such (structure seems to be more important here), there are some similarities. These sequences tend to be composed mainly of hydrophobic residues, though there is usually at least one positively-charged residue near the amino terminus.

Cotranslational translocation

Cotranslational translocation is the movement of a protein while it is being translated (hence the "co-" in "cotranslational"). This usually happens in the process of bringing a protein to the endoplasmic reticulum.

Cotranslational translocation requires signal recognition particles (SRPs), signal/SRP receptors (SRs), translocons, signal peptidase and some energy in the form of GTP. The signal sequence of proteins that are translocated during translation tends to be located right at the N-terminal of the protein, so it's synthesised first. After it is synthesised, it is recognised by an SRP, which binds to an SR on the membrane of the endoplasmic reticulum with a little help from GTP. This causes a nearby translocon (which is kinda like a little "channel" through which the growing peptide can pass through) to open up, hydrolysing GTP and causing the SRP to dissociate. The peptide can then elongate through the translocon into the lumen of the endoplasmic reticulum. While this is happening, a signal peptidase cleaves off the signal sequence. After elongation is complete, the ribosome dissociates and the translocon closes.

Protein orientation in membranes

As I'm sure you know, a bunch of proteins are transmembrane, which means that they pass through the cell membrane. They tend to have hydrophobic bits located in the membrane and hydrophilic bits on either side. Now, you might be wondering a) how the proteins get embedded in the membrane in the first place and b) how they get embedded in the membrane in the right way (i.e. N-terminal intracellular and C-extracellular, or the other way around?). Well, wonder no more!

Basically, when transmembrane proteins are synthesised, they are synthesised directly into the membrane of the endoplasmic reticulum, and they keep the orientation that they were synthesised in. This process occurs slightly differently for the four types of transmembrane proteins. I'm only going to cover the first three, though.

The first type of transmembrane protein has the N-terminal extracellular and C-terminal intracellular (just like the insulin receptor). They tend to have a signal sequence to allow for cotranslational translocation, which gets cleaved off by a signal peptidase. They also have a special sequence in the middle somewhere called the "stop-transfer" sequence which is hydrophobic and eventually becomes a membrane-spanning α-helix. The synthesis of this stop-transfer sequence within the translocon prevents further elongation into the lumen of the endoplasmic reticulum. Instead, the stop-transfer sequence moves laterally (so that it is next to the translocon). Synthesis continues outside the endoplasmic reticulum, so the end result is that the N-terminal is inside the ER lumen, the stop-transfer sequence is transmembranous and the rest of the protein is outside the ER lumen.

The second type of transmembrane protein is the opposite: C-terminal extracellular, N-terminal intracellular. Unlike Type I, this type doesn't have a cleavable signal sequence. Instead, its special sequence, the signal anchor sequence, serves as both a signal sequence and an anchoring sequence (hence the name). SRPs bind after the signal anchor sequence has been made and takes the growing protein to the endoplasmic reticulum. The signal anchor sequence moves laterally out of the translocon, but the rest of the protein is allowed to grow through the translocon, so that the C-terminal ends up inside the lumen of the ER while the N-terminal remains outside.

The third type is similar to type I in that it has the N-terminal extracellular and C-terminal intracellular, but in this case the N-terminal bit is really really short and there is no stop-transfer sequence. Instead, rather like type II transmembrane proteins, type III has a signal anchor sequence located very soon after the N-terminal. The short N-terminal ends up in the lumen of the ER, and after the lateral movement of the signal anchor sequence, the rest of the protein keeps growing in the cytosol.

Just so you know, type IV is the type that has multiple transmembrane domains, just like G-protein coupled receptors. They're obviously more complex, which is why we don't need to go into them just yet.

Posttranslational translocation

Posttranslational translocation, as the name implies, is translocation after translation. Once again, signal sequences or targeting sequences are important here. Sometimes, proteins might need multiple targeting sequences to get to their destination, kinda like how you might need two tickets to get to your final destination if you're transiting somewhere.

We're going to focus on how proteins move into the mitochondria, because mitochondria are cool. They have two membranes that proteins have to pass through. For ease of movement, proteins tend to pass through where these two membranes come into contact, in places that are very originally called "contact sites." The membranes themselves have translocons called TOMs and TIMs (Translocons of the Outer Membrane and Translocons of the Inner Membrane). Targeting sequences of proteins that pass through here tend to be amphipathic helices, with hydrophobic proteins on one side and positively charged proteins on the other.

The first step in translocation into the mitochondria is ensuring that the protein isn't folded up. These TOM/TIM complexes only have little channels, so a great hulking ball of protein won't get through too easily. Since proteins naturally want to fold up, they're going to need some assistance and energy to stay unfolded. This assistance comes in the form of chaperone proteins like Hsc70, and the energy comes in the form of ATP.

The next steps involve stuff binding and getting moved. Basically the protein to get transported binds to an import receptor on the mitochondrial membrane, and is then transferred to the general import pore of the TOM complex. This allows for translocation through the outer membrane via the TOM complex. Next the protein is translocated again via the TIM complex. All of this translocation is helped along by the movement of protons across the inner membrane (or at least that's my understanding) and ATP hydrolysis by Hsc70 ATPase located inside the mitochondria. Once across, the targeting sequences are cleaved off by matrix processing proteases and the protein finally gets to fold. Yay!