Cellular Protein Trafficking and Secretion

Yes, yet another revision post :( Still a few more to go...

1. Outline how proteins with distinct localisation signals are delivered to the nucleus and mitochondrion.

Proteins have localisation signals in their amino acid sequence that indicate where they will be needed in the cell.

Proteins that are needed in the nucleus have a nuclear localisation signal (NLS) in their amino acid sequence. This sequence can be located anywhere in the protein and is not removed during translation. To be imported into the nucleus, the NLS binds to a protein called importin, or a nuclear import receptor. Once the bound complex enters the nucleus, Ran-GTP binds to importin, releasing the protein. Importin with Ran-GTP bound can then exit the nucleus, where a GAP (GTP activating protein) hydrolyses GTP, resulting in Ran-GDP, which dissociates from importin, leaving it free to import another molecule.

Export of proteins from the nucleus can also occur in a similar way. Some proteins have nuclear export signals. They bind to a special nuclear export receptor along with Ran-GTP. The entire complex travels out of the nucleus, where Ran-GTP has its GTP hydrolysed to GDP. Ran-GDP then dissociates along with the protein being carried out.

Proteins that are needed in the mitochondria travel there a little differently. Once again, they require a localisation signal, but in this case the localisation signal must be at the end that is translated first. They are translated in the cytoplasm, and then their folding is blocked by hsp70 as a folded protein would be too bulky to travel into the mitochondria. The mitochondrial membrane is somewhat harder to cross than the nuclear membrane and as such there are TOM and TIM protein translocators in the mitochondrial membrane that make this job easier. The localisation signal first binds to the TOM (transporter outer membrane complex) where it is fed through to the TIM (transporter inner membrane complex). From there, it is dragged into the mitochondrion. Energy for this process is provided by the electron transport chain, which is provided through oxidation of food molecules (if I understand correctly). Once safely inside the mitochondrion, the localisation signal is cleaved off.

This isn't super relevant, but it was in the lecture slides, so I'll include it anyway. Aside from the TOM and TIM complexes, there are also complexes that serve to help anchor proteins into the membrane. The SAM complex helps anchor proteins in the outer membrane, while the OXA does the same for the inner membrane.

2. 3. Be able to list the steps involved producing a secreted protein. Outline the functions of vesicles.

Like proteins that are needed in the mitochondria, proteins that will eventually be secreted have a special signal at their N-terminus. These proteins are first translated on free ribosomes in the cytosol. Once the signal has been translated, however, signal recognition particles (SRPs) recognise the signal, pause translation, and take the protein to SRP receptors on the endoplasmic reticulum. The ribosome and peptide are inserted onto a translocator, where translation continues.

Translated proteins are released into the inside of the endoplasmic reticulum. The endoplasmic reticulum can then "bud off" into vesicles which can carry proteins around to different areas of the cell. For example, proteins can go to the Golgi apparatus, where post-translational modifications are made, or to the cell membrane, where they can be excreted. I'll talk a bit more about vesicles in the next part...

4. Identify the roles of coat proteins, Rabs, and SNAREs.

Coat proteins are required for formation of vesicles. Cargo receptors in the endoplasmic reticulum bind to their cargo on the inside of the cell, and to coat proteins on the outside of the cell (at least, that's my understanding of it). Coat proteins form a sort of "case" around the budding vesicle. They are discarded before the vesicle fuses to another membrane. There are several types of coat proteins: clathrin assembles on vesicles moving between the Golgi and plasma membrane, COPI forms around vesicles moving through the Golgi or moving from the Golgi to the ER, and COPII forms around vesicles moving from the ER to the Golgi.

Rabs are proteins which act as a sort of address tag on the protein. They bind to Rab effectors, which are motor or tether proteins located on the outside of other membranes in the cell.

SNARE proteins mediate fusion of vesicles to other membranes. SNAREs located on the vesicle are known as vSNAREs, while those on the target membrane are known as tSNAREs.

5. Describe the difference between regulated and unregulated exocytosis in secretion. 

In a nutshell, vesicles in regulated exocytosis require a signal before their contents are secreted, while vesicles in unregulated, or constitutive, exocytosis do not. As far as I know, only the latter type have special proteins on the outside of their vesicles that probably act as some kind of signal, telling the cell to target them and move them to the membrane for secretion right away.