Lecture 5 - Protein Trafficking into ER

Question 1

Define the Endoplasmic Reticulum in terms of:
(i) Structure
(ii) Environment
(iii) Subsections (+ Function)

Answer 1

(i) Labyrinth of branching tubules/sacs, which are contiguous with the Nuclear Envelope
(ii) Oxidising Environment (compared to Cytosol), which is rich in Calcium ions (Ca2+ store)
(iii) Smooth ER (Lipid Synthesis) and Rough ER (Lumenal, TM, and secreted proteins)

Question 2

Describe Post-Translational Translocation of proteins into the ER, including how energy requirement is met

(3 Points)

Answer 2

• Following translation, chaperones associate with polypeptide chain to prevent folding
• N-terminal Signal peptide is recognised by Sec62/63 complex, which pass polypeptide to Sec61 translocon
• ER lumenal chaperones (e.g., BIP) bind to nascent polypeptide and utilise ATP hydrolysis to pull it into ER lumen (provides energy)

Question 3

Describe Co-Translational Translocation of proteins into the ER

(Four Points)

Answer 3

• N-terminal signal peptide emerges from ribosome exit tunnel, and is recognised by the signal recognition particle (SRP)
• SRP interacts with ribosome, causing pause in the nascent polypeptide chains elongation
• SR associated with Sec61 translocon binds SRP-ribosome complex, causing dissociation of SRP and the passing of nascent polypeptide to translocon
• Dissociation of SRP resumes translation, with “translational force driving polypeptide through the translocon

Question 4

What is the Structure of the SRP?

Answer 4

• Consists of 7S RNA and 6 different polypeptides
• Structure - consists of:
  1. Translational Pause Domain
  2. S/SRP54 Protein - split into three domains

Question 5

What are the functions of the three SRP54 domains?

Answer 5

• G-domain - Ras-like GTPase fold
• N-domain - four helix bundle
• M-domain - rich in methionine, forms hydrophobic cleft that binds signal peptide

Question 6

Describe the structure of the SRP-receptor (SR), explaining how it causes release of RNC

Answer 6

• Consists of:
  1. Sra - peripheral membrane GTPase, which regulates the targeting of RNC to translocon
  2. SrB - TM protein, which anchors Sra to ER membrane
• SR-SRP interaction activates the GTPase activity of each protein, inducing conformational change that causes release of RNC to Translocon

Question 7

What is the Structure of the Sec61translocon

(2 Points)

Answer 7

Consists of:
* Sec61 Heterotrimer (a/B/y) - forms the aqueous pore
* Sec62/63 Accessory proteins - help recruit ER lumenal chaperones via J-domain

Question 8

Compare the Open and Closed Conformations of the Sec61 Translocon

Answer 8

• Closed Conformation - pore is occluded by a plug domain (a-helix) until signal peptide is recognised
• Open Conformation - recognition of signal peptide displaces plug and causes opening of hinge region, releasing signal peptide into the membrane

Question 9

How is N-terminal Signal Peptide cleaved from nascent polypeptides (soluble/TM)?

Answer 9

• Signal Peptidase enzyme associated with Sec61 complex cleaves signal peptide as protein enters ER lumen

Question 10

By what two methods can single-pass membrane proteins be inserted into the ER, and how can this influence orientation?

Answer 10

1. N-terminal Signal Sequence + Internal Stop Transfer Sequence, which causes lateral release of protein into membrane
2. Internal Signal Sequence, with the orientation of +ve residues adjacent to hydrophobic region determining orientation (due to high Ca2+ in ER lumen)

Question 11

How are Multi-pass membrane proteins generated?

Answer 11

Combinations of Stop and Start Transfer sequences generate multi-pass proteins with different numbers of TM segments

Question 12

How are proteins anchored by C-terminal tails inserted into the ER membrane (Give Example)? How does it work?

Answer 12

• Guided Entry of Tail Anchored Proteins (GET) Pathway
• proteins with no signal sequence (e.g., SNAREs) are inserted post-translationally into the ER membrane

Question 13

What modifications can proteins undergo post-translocationally?

(Five Points)

Answer 13

• Signal Sequence Cleavage (Signal Peptidase)
• Disulphid Bond Formation (Protein Disulphide Isomerase)
• N-linked Glycosylation (Oligosacchryl transferase)
• Folding (Assisted by Chaperones e.g., BIP, Calnexin)
• Further Modifications

Question 14

Why is the ER an ideal site for the formation of Disulphide Bonds?

(2 Points)

Answer 14

• Oxidising Environment allows stable S-S bond formation, stabilising protein structure
• PDI - serves as donor/reducing agent, breaking incorrect/ transient S-S bridges to allow correct disulphide bonds to form

Question 15

1. Why is Glycosylation Important?
2. How can it occur co-translationally in the ER?

Answer 15

1. Glycosylation is essential for both the function and stability/folding of many proteins
2. Oligosacchryl Transferase is an enzyme associated with lumenal face of ER membrane, which can transfer preformed oligosaccharide chains from dolichol lipids in ER membrane onto Asparagine residues of nascent polypeptide as it passes through translocon

Question 16

How can the proper folding of glycosylated proteins be assisted in the ER?

(3 Points)

Answer 16

1. Integral membrane protein Calnexin can bind to misfolded proteins both via their primary sequence, and via the terminal glucose residues on its glycosylations
2. Glucosidase enzyme cleaves the terminal glucose, releasing the polypeptide and giving it the opportunity to refold
3. If it does not refold, glucosyl transferase adds on terminal glucose residues, allowing calnexin to rebind and repeat process

Question 17

What two processes can be activated by the cell to deal with misfolded proteins?

(4 Points)

Answer 17

1. ERAD Pathway - misfolded peptides are translocated back into the cytosol, where they enter the Ubiquitin-Proteasome system
2. Unfolded Protein Response:
   * Misfolded proteins in ER bind to RTKs, causing their dimerisation and subsequent autophosphorylation, exposing ribonuclease domains
   * Ribonuclease activity splices pre-mRNA into mRNA encoding transcription factors, which following translation enter the nucleus and trigger the expression of chaperone proteins that help the misfolded proteins to fold correctly

Question 18

1. How are Proteins trafficked from the ER to the Golgi?
2. How are ER-resident proteins retrieved from the Golgi?

Answer 18

1. Correctly folded proteins enter ER exit sites, where membrane budding occurs due to action of COPII coat
2. ER Resident proteins - contain KDEL motif recognised in Golgi by KDEL receptor, leading to recruitment into COPI coated vesicles that transport protein back to ER

Question 19

Describe the Process of COPII coat formation?

Answer 19

• Sec12 activity (GEF) converts Sar1-GDP to Sar1-GTP, which associates with membrane
• Sar1-GTP recognises cargo export signal, recruiting COPII export machinery (Sec23/24, Sec13/31) which deforms ER membrane to produce a vesicle