Protein Folding and Processing in the Secretory Pathway (Rademacher)

Question 1

Shortly describe the principle of ribosomal pools. Where do the ribosomal subunits go to from the pool? What is the destiny of the newly translated proteins? Why are some compartments important for protein maturation in comparison to cytosolic environment?

Answer 1

From ribosomal pools:
- cytosolic translocation
- ER bound translocation
- dissociation upon translation completion and retrieval back to the free pool of ribosomal subunits

Proteins after translation go either to:
- ER bound: rER -> sER -> Golgi -> lysosomes / PM
- cytosolic: nucleus / cytosolic / peroxisome / mitochondria

Protein maturation and PTM compartments: different redox milieu (cytosol: more reducing, ER: oxidizing), different pH, concentration of proteins for PTM

Question 2

Describe the translation of protein in ribosomes.

Answer 2

Large + small subunit, RNA binding site: three positions APE
- A: aminoacyl tRNA
- P: peptidyl tRNA
- E: exit

Translation initiation: 5’-UTR bind eIF4E cap, 3’-UTR binds PABPS

1) Growing polypeptide chain-tRNA on P + new AA-tRNA on A
2) Growing polypeptide chain binds new AA-tRNA and moves to P, old tRNA goes on E
3) New AA-tRNA on A and 2) repeats until completion
4) Release factor (UAG) binds A -> hydrolysis, termination, complex dissociation

Synergic movement of small and large ribosomal subunit

Question 3

What is the difference between secreted and membrane bound proteins? Name general examples.

Answer 3

Secreted:
- soluble
- less abundant
- cytokines, chemokines, coagulation factors, hormones. GF, TF
- can be nevertheless retained by GPI anchors or in ER

Membrane-bound:
- more abundant
- trans-membrane or peripherally bound
- PM, ER, Golgi, mitochondria
- receptors, ion channels, transporters, anchoring proteins, enzymes

Question 4

What is the of ER?

Answer 4

rER: ribosome bound, peptides with specific seq. are inserted into the lumen and signaling seq. is cleaved.
- protein folding
- disulphide bridge formation
- protein isomerization
- N-glycosylation

sER: lipid synthesis and metabolism, steroids and hormones synthesis, detoxification

Question 5

What is the role of Golgi?

Answer 5

• O-glycosylation
• N-glycosylation
• phosphorylation
• methylation
• sulfanation
• palmitoylation
• proteolytic cleavage
• protein sorting (TGN)

Question 6

Briefly describe the principle of different Golgi compartment formation.

Answer 6

Cis- middle- and trans-Golgi: compartments are not fixed but rather mature from one to another (cis- becomes middle- and middle- becomes trans-Golgi whereas all the proteins migrate back to their designated compartments - anterograde vs. retrograde movement)

Question 7

Describe the ER translocation in detail.

Answer 7

1) SRP (signal recognition particle) recognizes emerging signal sequence of the polypeptide chain from a ribosome
2) Guides the ribosome to SRPR on ER
3) Recognition of the chain by (closed) Sec61 translocon
4) GTP -> GDP of SRP-SRPR comples -> Sec61 translocon opens
5) Peptide chain is inserted
6) Bip-ATP (T-state): open
7) J-domain of the Sec61 translocon interacts with Bip -> ATP -> ADP
8) Bip-ADP (D-state): closed around the peptide, preventing it from going back
9) Nascent polypeptide inside the lumen
10) Bip-ADP -> Bip-ATP
11) SRP sequence is cleaved by SRP peptidase from the growing polypeptide chain and folded

Question 8

Describe the structure of the sequence for ER translocation recognition. What happens to the sequence upon ER translocation?

Answer 8

SRP sequence consists of:
- positively charged N-term that can penetrate the membrane
- hydrophobic core
- C-term where cleavage of the signalling sequence takes place

1) Sequence is cleaved off by SRP peptidase
2) Sequence is inserted into the membrane, interaction of the positive N-term with negatively charged polar heads of the upper layer of the membrane
3) Degradation

Question 9

Describe the structure of the translocon. How is it primed for insertion?

Answer 9

Sec61 translocon:
- hourglass shape with helices around the pore
- ring of hydrophobic amino acids shielded from the cytosol

Priming:
1) Binding of channel partners: ribosome + Sec62/63 complex + SecA
2) Activation of the complex by peptide recognition and insertion

Question 10

Describe the differences in translation rates of different proteins. How can be achieved that proteins are translated quicker/slower? Why would we want to regulate such thing?

Answer 10

Protein + multiple domains that:
- required independet folding: slower translation, folded domain by domain, use of rare codons that don’t have alot of tRNA
- require coupled folding: faster translation, coupled domain interaction required for proper protein function, use of accessible codons with alot of tRNA

if faster / slower, respectively -> misfolding and aggregation,

Question 11

What is the role of Bip in UPR?

Answer 11

When unfolded protein concetration rises rapidly:
1) Bip bound to IRE1 monomers in the ER membrane
2) Unfolded protein emerges and Bip is released from IRE1
3) IRE1s dimerize in the membrane and trigger the expression of Hac1 TF

Question 12

What other chaperones are there in the ER except Hsps’?

Answer 12

• peptidyl-prolyl isomerase: maintanance of cis- to trans-prolines equilibrium
• protein disulphide isomerase: rearrangement of incorrectly formed disulphide bridges

Question 13

Describe the N-glycosylation of protein in detail.

Answer 13

During translation-translocation process to the ER lumen:
1) Membrane-bound OST (oligosacharyl transferase) en-block adds N-glycan to asparagine (Asn) of the nascent polypeptide (from Asn-X-Ser/Thr or Phe-Y-Asn-X-Ser/Thr as enhanced aromatic (hydrophobic) sequence)

• N-glycan from dolichol bound by two pyrophosphates
• glucose
• mannose
• acetylglucoseamine
• N-glycan assembled on both ER membrane sides

2) Glucosidase 1/2 removes 2 out of 3 glucoses from N-glycan
3) ER p57 and oxidoreductase fold the protein and from dislphide bridges
4) Membrane-bound CNX/CRT (Calnexin/Calreticulin complex) recognizes the sacharides and activates the glucosidase 2 to cleave the remaining 1 glucose

5.1) Only mannose and acetylglucoseamines remain on the folded chain and protein goes to the Golgi via COPII coated vesicles
/
5.2) If the protein is incorrectly folded, UGGT adds 1 glucose back from UDP-glucose donor and the folding takes place once again
/
BUT if the protein still cannot be propperly folded and stuck in an energy minimum:
6) EDEM-1-3 mannosidase removes 2 mannoses and the protein is transported into the cytosol
7) N-glycan is cleaved off by N-glycanase, sequentially degraded and transported back to ER in fragments
8) Protein is targeted for proteosomal degradation

Question 14

What types of vesicles are used for ER-ERGIC-Golgi and Golgi-ER?

Answer 14

ER exit place - ERGIC - cis-Golgi: COPII
Golgi - ER: COPI

Question 15

By what pathway are terminally misfolded proteins transported to cytosol for degradationn?

Answer 15

ER-associated degradation (ERAD): Terminally misfolded
proteins and unassembled oligomers are retro-translocated
to the cytosol and are degraded by the proteasome

Question 16

What is the functional difference between CNX and CRT?

Answer 16

• CNX mainly interacts with N-glycans close to the ER membrane
• CRT binds preferentially to glycoproteins in the ER lumen, large luminal dom.

Question 17

How are ER-resident proteins kept in the ER?

Answer 17

ER-resident proteins have a canonical KDEL (AA) seq. (whith small possible variations) that is recognized by the KDELR.
1) KDELR has a His that is protonated (becomes positive) in the Golgi environment, pH ~6.2
2) KDEL-sequence containing cargo (ER-resident protein) binds this +His
3) COPI-mediated retrieval
4) KDELR His is deprotonated in the ER, pH ~ 7.4

Question 18

Based on what mechanism are Golgi proteins retained in the respective compartments?

Answer 18

Based on the membrane thickness and the length of the membrane domains of proteins. If membrane domain < membrane thickness -> protein not retained.