Intracellular Protein Trafficking Flashcards
What are the signal sequences for nuclear import and where are they located on the protein?
The nuclear localisation signals (NLS) are usually a sequence of positively charged amino acids: lysine and arginine rich. These can either be located in a patch within the protein or at the end of a polypeptide chain.
Explain the process of nuclear import.
The soluble receptors (importins) bind to proteins with NLS and subsequently interact with the F-G repeats of the nucleoporin proteins of the nuclear pore and allow the protein to cross the membrane. The concentration of Ran-GTP is kept high in the nucleus by Ran-GEF, and the concentration of Ran-GDP high in the cytosol by Ran-GAP (by phosphorylation/dephosphorylation). This drives directional transport through nuclear pores, as once inside the cell Ran-GTP binds to importin and displaces the nuclear protein, and importin bound to Ran-GTP will leave the nucleus and be dephosphorylated.
What are the nuclear export signals. How does nuclear export occur?
The signal is usually a string of leucine residues, and proteins with this signal in the nucleus bind to exportin proteins, which Ran-GTP binds to. The triple complex moves out of the nucleus through nuclear pores by interaction of the exportin with F-G repeats of the nucleoporins. Outside the nucleus, Ran-GTP is hydrolysed and the exportin released (and transported back into nucleus).
What is the signal sequence for protein import into the mitochondria?
It is often an amphipathic alpha helix, usually at the N-terminus of a protein.
Explain the process of protein import into the mitochondrial matrix.
The unfolded protein (Hsp70 binds in cytosol to prevent folding) with the signal sequence binds to the import receptor within the TOM complex on the outer mitochondrial membrane. Using the energy from the hydrolysis of ATP, it is threaded through the TOM complex and into the IMS, and then using the membrane potential across the inner membrane is threaded through the TIM22 complex on the inner membrane. Hsp70 in the matrix may wither actively pull the protein through the TIM22 complex, or simply prevent it from sliding back in. Once in the matrix the signal sequence is cleaved by signal peptidase, and the protein assisted to fold by Hsp60 to produce the mature protein.
How is a protein inserted into the outer membrane of the mitochondria?
The protein goes into the TOM complex but then is prevented from travelling further as chaperone proteins bind to it and cause it to begin to fold (needs to be unfolded to travel though TOM and TIM complexes). The protein is then passed back through the SAM complex in the outer membrane, and a fully folded membrane protein forms.
What are the 3 ways of targeting a protein to the inner mitochondrial membrane?
1) Passes through TOM and TIM and has signal sequence cleaved and so hydrophobic region stays imbedded in the inner membrane
2) Passes through TOM and TIM23 and then cleavage reveals a second signal sequence, which allows the protein to be inserted into the OXA complex in the inner membrane where it can fold
3) Binds to chaperone proteins in IMS and is inserted into TIM22 complex using energy from membrane potential, and hydrophobic regions stay in membrane and become transmembrane domains
How is a protein targeted to the intermembrane space.?
Protein translocated across TOM and the signal sequence goes into TIM, and in TIM cleavage of signal sequence means that the protein stays in the IMS.
What type of transport is the majority of transport into the ER? What is the signal sequence?
The majority of transport is cotranslational. The signal sequence is a positively charged residue (arginine) followed by 6-12 hydrophobic residues, always at the N-terminus of the protein.
Describe the process of import into the ER.
As soon as part of the nascent chain is extended from the ribosome it is recognised by a SRP molecule (soluble receptor), which causes a pause in translation. This takes the ribosome and nascent chain to the SRP receptor on the ER membrane where the complex binds. Translation continues and translocation begins: the protein travels into the ER via the Sec61 complex. The SRP and SRP receptor are displaced and recycled. If the protein is soluble the signal sequence is cleaved by signal peptidase and the protein remains in the ER lumen. For a membrane protein the signal sequence is cleaved in the Sec61 complex and hydrophobic residues become embedded in the membrane.
What other way can a protein be transported into the ER?
By post-translational import, whereby the protein is fully synthesised before translocation but is kept unfolded by Hsp70 (similar to mitochondrial). A Sec complex in the membrane recognises the signal sequence and the protein is passed through this, and Bip in the ER lumen is thought to either prevent the protein sliding back into the channel or pulls it through using hydrolysis of ATP.
How does N-linked glycosylation in the ER occur?
It occurs at N-X-S or N-X-T sequences, with the sugars added to the asparagine residue by oligosaccharyl transferase membrane protein that binds to Sec61, the ribosome, and the asparagine residue. The sugars added are N-actylglucosamine, mannose and glucose. This happens when the protein is being transported into the ER.
What is ERAD?
ER-associated degradation. Chaperone proteins bind the misfolded protein and transport it out of the ER, where ubiquitin is added that targets it for degradation in the proteasome.
