Lecture 5 ER targeting Flashcards
What are the general steps in protein targeting from the cytosol to the ER and vice versa?
Either post or co translational mechanisms, signal peptides, signal peptide recognition particle, translocon, transfer of soluble and membrane proteins, folding in ER, glycosylation and ER export and transport to the Golgi apparatus.
What is the ER?
A labryinth of branching tubules and sacs contiguous with the outer nuclear membrane (rough ER). It accounts for 50% of the surface area of cell membranes.
What is the ER environment?
Oxidizing environment (substances lose electrons to promote oxidation reactions). It is a major Ca2+ store.
What occurs at the ER?
Phospholipid (smooth ER) and membrane protein (rough ER) synthesis and protein folding.
What are the common elements involved in protein translocation into the ER?
Ribosome, mRNA, nascent polypeptide, signal sequence (16-30 residues, hydrophobic core flanked by positive residues) translocon.
What is the SRP (signal recognition particle)?
A key molecular complex involved in protein targeting to the ER.
What is the composition of SRP (signal recognition particle)?
7S RNA (a ncRNA) and six different polypeptides. One of the most conserved proteins in SRP is SRP54. Plays a role in recognizing and binding signal sequences of nascent polypeptides.
What are the 3 conserved domains of SRP54?
- G-domain (Ras-like GTPase fold): Involved in GTP binding and hydrolysis, which is critical for SRP function.
- N-domain (4-helix bundle): Provides structural stability and helps in interactions.
- M-domain (methionine-rich domain): Contains a hydrophobic cleft that binds the signal sequence of proteins.
What are some important functional regions in the SRP?
Translational pause domain which temporarily halts protein synthesis. Hinge provides flexibility to the SRP structure. Signal-sequence binding pocket located within the M-domain where the signal sequence of a nascent protein binds.
Why does the signal peptide particle pause translation of the nascent polypeptide at the ribosome?
To facilitate protein targeting to the ER lumen.
What are the steps to SRP pausing translation?
A signal sequence emerges from the ribosome and SRP binds to the signal sequence. This causes a pause in translation. This prevents the protein from fully synthesizing in the cytosol. The SRP-bound ribosome complex interacts with the SRP receptor on the ER membrane. The hinge structure in SRP provides flexibility helping guide the ribosome to the receptor. This interaction ensures the growing protein is properly directed into the ER lumen.
Why is this process important?
This translation pause prevents premature folding of the protein in the cytosol. It ensures that proteins destined for secretion or membrane localisation enter the ER for proper processing and modification.
What is the SRP receptor (SR)?
Rough ER membrane localised heterodimer that binds ribosome-nascent chain-SRP complex (RNC).
What are the two types of SRP receptor?
SRalpha: 70kDa peripheral membrane GTPase regulates targeting of RNC to translocon. Interaction with SRP/RNC triggers GTPase activity of SR and SRP leading to the release of RNC to the translocon.
SRbeta: 25kDa transmembrane protein anchors SRalpha at the ER membrane.
What is the translocon?
A complex of proteins associated with the translocation of polypeptides across membranes.
What is the translocon machinery composed of?
Composed of Sec61alpha beta and gamma which form a heterotrimer. Sec61alpha lines the aqueous pore of translocon. Sec62/63 accessory protein with J domains (fold Hsp70) assist with import- activation of Hsp70-like BiP- may assist in post translational translocation.
What is Hsp70-like BiP?
Binding immunoglobin protein. Localised in the ER. Helps folding of newly synthesised proteins in ER.
What are the steps to co-translational translocation into the ER?
Once the SRP-ribosome complex is formed it is directed to the SRP receptor located in the rough ER membrane. The growing peptide is guided to Sec61. The SRP and its receptor dissociate allowing the ribosome to dock onto the translocon. The signal sequence is inserted into the translocon and SRP is released. The ribosome continues translating the polypeptide. Protein is threaded into ER lumen. Here it may undergo folding, modifications and cleavage of signal sequence. SRP is recycled.
What are the steps to post translational translocation to the ER?
Translation fully completes in the cytosol. The synthesized protein contains an N-terminal signal sequence that directs it to the ER. Delivered to sec61. No ribosome is attached. Cytosolic chaperons Hsp70 etc prevent premature folding instead. Unfolded protein is threaded through Sec61 translocon in an ATP dependent manner. Since there is no driving force from a ribosome additional chaperones are required: Sec62,63 BiP. BiP binds to prevent sliding back into cytosol. BiP hydrolyzes ATP undergoing confirmational changes to pull the protein into the ER lumen.
What is the difference between single pass membrane and multi pass membrane proteins?
Single pass proteins only pass through the membrane once.
What are the steps to the translocation of a soluble lumenal polypeptide into the ER lumen?
Signal peptide directs polypeptide to translocator. Translocator activates and threads polypeptide into ER lumen. Signal peptidase cleaves SP (signal peptide) into 9 amino acid fragments released from membrane-roles in immune regulation.
What are the steps to the translocation of a single pass membrane protein?
The N-terminal signal peptide directs the ribosome to ER membrane. Polypeptide enters translocator and starts moving into ER lumen. A hydrophobic stop transfer sequence halts translocation. This sequence anchors the protein in the membrane. Translocator’s lateral gate releases the signal peptide and stop-transfer sequence into the membrane. The signal peptidase cleaves the N-terminal signal peptide. The mature transmembrane protein is now embedded in the ER membrane. The N-terminal remains in the cytosol while the C terminal is in the ER lumen.
What are internal signal sequences on single pass membrane proteins?
Can orient polypeptides in translocon to generate different membrane orientations.
A. Positive residues preceding hydrophobic (stop) signal: type II.
B. Positive residues follow hydrophobic (stop) signal.
Why are these charge distributions favoured?
Positive Residues Preceding a Hydrophobic Signal Sequence (Start-Transfer Sequence):
When the N-terminal signal sequence is recognized, the positive residues before the hydrophobic region promote the orientation where the N-terminus remains in the cytosol.
This ensures that the hydrophobic transmembrane domain inserts correctly into the membrane.
Positive Residues Following a Hydrophobic Signal (Stop-Transfer Sequence):
When a stop-transfer sequence is encountered, translocation stops, and the polypeptide is laterally released into the membrane.
If positive residues are present after the hydrophobic stop-transfer sequence, they favor positioning the C-terminal side in the cytosol, reinforcing the orientation.
How are multi pass membrane proteins generated?
Combinations of start and stop transfer sequences.
How are tailored anchored proteins (proteins with a single transmembrane domain at the C-terminus. They lack an N-terminal signal sequence so cant use SRP-Sec61) transported to ER?
The GET pathway (guided entry of tailed anchored proteins). Get3 ATPase binds the hydrophobic C terminal anchor of the TA protein. Get3A-TA complex binds to the ER receptor Get1-Get2. ATP hydrolysis drives TA protein insertion into the membrane. Get3 is released and reused. E.g., SNARE proteins.
How does the position of an N-link affect oligosaccharide processing in further compartments?
A protein may top being processed and be a mannose oligosaccharide or proceed to a complex oligosaccharide in further compartments.
