Endoplasmic Reticulum

Question 1

Describe the endoplasmic reticulum?

Answer 1

This is the first step of the secretory pathway
Protein translation and translocation into the endoplasmic reticulum
Protein folding and glycosylation

A labyrinthine-like membrane bound organelle
It has interconnecting tubules and flattened sacs that interconnect
ER lumen - a continuous sheet enclosing a single internal space (or ER cis-internal space)

Major site of protein synthesis and assembly - that make up 1/3 of the cellular proteome
It makes most of the lipids for mitochondrial and peroxisomal membranes
It also acts as a store of Ca2+, sequestering it from the cytosol - used in many cell signalling responses

Question 2

How do we isolated the endoplasmic reticulum to study it?

Answer 2

Homogenise
Forms smooth and rough microsomes - the ER breaks into fragments which reseal into vesicles

Microsomes are separated in sucrose density centrifugation
Smooth microsomes - low density = higher up
Rough microsomes - high density = lower down

Question 3

How are proteins recognised for protein translocation?

Answer 3

Proteins targeted to the ER have N-terminal signal sequences
Signal sequences are short hydrophobic sequences that function as “molecular postcodes” that tell the cell to deliver the protein to the ER
Removing/adding can help with transport if you want something to move into the ER
Signal sequences are typically cleaved by signal peptidase upon insertion into the ER, but are retained in some proteins where they act as transmembrane domains (signal anchors)

The two types of protein the ER captures: transmembrane proteins and water-soluble proteins
Transmembrane protein contain: stop-transfer signals - so they remain in the membrane

Question 4

What happens during protein translocation into the ER?

Answer 4

During protein translation the signal sequence is the 1st part of the protein to appear from the ribosome (N-terminus)
Signal sequences are recognised by signal recognition particle (SRP) - and therefore translocated a protein into the ER lumen
SRP has 6 polypeptide chains bound to a small RNA molecule
It can bind to so many signal sequences as it has a large hydrophobic pocket lined with unbranched methionines

SRP “delivers” the ribosome and the protein to the ER membrane
SRP binds to SRP receptor (SRPR) on the ER membrane
SRP is displaced and the ribosome docks onto SEC61
On binding to the SEC61 translocator translation resumes
The SEC61 translocator forms an aqueous pore across the ER membrane - allowing the peptide in

Protein translocation is co-translational (synthesised as it is being translocated across the membrane)

Question 5

What does the signal recognition particle do?

Answer 5

SRP pauses translation preventing proteins with signal sequences from folding in the cytosol
The SRP54 subunit binds to the signal sequence, whereas the SRP RNA’s Alu domain binds the ribosome elongation factor binding site - blocking it
This prevents synthesising in the cytosol, then disassembly to get into the ER, to then be reassembled again
Therefore enough time for the ribosome to bind to the ER before completion of the peptide

Question 6

What are some elements of protein folding and modification in the ER?

Answer 6

Chaperones
Di-sulfide bond formation
N-linked glycosylation

Question 7

Describe chaperones in the ER?

Answer 7

Chaperone - BiP (Binding immunoglobulin protein) is an ER luminal HSP70 heat shock chaperone
BiP is required for protein translocation into the ER and binds to proteins as they are inserted into the ER
Prevents them from moving back into the cytosol
BiP binds to unfolded regions of proteins and facilitates their folding via an ATP dependent mechanism

Molecular chaperones most commonly interact with exposed, hydrophobic portions in soluble polypeptide chains - preventing protein aggregation
They can also provide an energetically favourable environment

Question 8

Describe di-sulfide bond formation?

Answer 8

The ER is an oxidising environment that favours disulphide bond formation that link different parts of the protein
Proteins can have many cysteine residues, but protein folding requires that the correct pairs of cysteines form disulphide bonds
Correct disulphide bond formation between Cys residues is promoted by protein disulphide isomerases (PDIs)
They normally fold in domains not exposed to the cytosol due to the environment

Question 9

Describe N-Linked glycosylation?

Answer 9

Most proteins produced in the ER are glycosylated
An oligosaccharide chain is added by Oligosaccharyltransferase (OST) to asparagine residues - via a high energy pyrophosphate bond
The oligosaccharide precursor is held by a dolichol in the ER before addition to a peptide

OST is associated with the SEC61 translocon and glycosylates proteins as they are translocated
The oligosaccharide chain is comprised of 2 N-acetylglucosamines, 9 mannoses and 3 glucoses

Question 10

How are proteins monitored using glycosylation?

Answer 10

First the N-linked oligosaccharide is trimmed leaving a single glucose residue
The protein is then recognised by the chaperone calnexin and the associated protein ERp57, which binds to the oligosaccharide and free cysteines respectively and prevent unfolded proteins aggregating together
When the protein is released by calnexin/ERP57 the remaining glucose is trimmed - until folding has completed

If the protein is properly folded it can exit the ER
If the protein fails to fold - a glucose is added to the oligosaccharide by a UDP-glucosyl transferase - it renters the cycle until completely folded

Question 11

How does lipid synthesis occur in the ER?

Answer 11

Phospholipid synthesis occurs on the cytosolic side on the ER membrane
Scramblase catalyses the flipping of phospholipid molecules to allow symmetric growth of both halves of the bilayer

Question 12

How can complex transmembrane structures be formed?

Answer 12

By using start-transfer signals and stop-transfer signals through a peptide, a protein can be dynamically integrated in different ways across the membrane to produce the ‘correct’ structure for the transmembrane protein

Question 13

What is used for incorrectly folded proteins in the ER?

Answer 13

ER-associated degredation (ERAD)
This removes missfolded proteins from the ER for degredation
ERAD helps prevent the accumulation of misfolded proteins in the ER which could otherwise aggregate - they can be very toxic to cells

Question 14

Describe the ERAD mechanism?

Answer 14

Mannosidases (EDEMs) catalyse the slow removal of mannose residues from N-linked oligosaccharide chains
This stops re-addition of glucose but marks the protein for degradation:

Adaptor proteins recognise Mannose trimmed N-glycans and the protein is ubiquitinated by a E3-ubiquitin ligase complex
Adaptors can be chaperones or lectins e.g. Hsp70 chaperone
E3-ubiquitin has to recognise the adaptor, expose the substrate to the cytosolic face of the ER membrane and ubiquitinate it

The ubiquitinated peptide is retrotranslocated into the cytosol out of the ER and then de-ubiquitinated before being recognised by a cytosolic proteasome
This hydrolyses the protein into short peptides - ATP driven by p97 complex

Question 15

What is a stress induced pathway in the ER?

Answer 15

Pre-emptive quality control (pQC)
Some proteins are blocked in their initial translocation into the ER lumen and instead routed directly for proteosomal degradation - during rapidly induced stress

Question 16

What is ERAD involved in, within a human disease?

Answer 16

Cystic Fibrosis
Human Cytomegalovirus (HCMV)

Question 17

Describe cystic fibrosis?

Answer 17

Cystic fibrosis is a hereditary disease caused by mutations that result in the loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR)
There are >1,000 mutations in the gene that encodes CFTR that are associated with cystic fibrosis - most common is ΔF508

Cystic fibrosis affects multiple organs
It is characterised by thick mucous secretions in lungs and intestines
Infections and inflammation impair lung function and a lung transplant becomes the only option
Affected individuals often die before reaching 30

Question 18

What is the physiological function of CFTR?

Answer 18

Cystic fibrosis transmembrane conductance regulator (CFTR)

CFTR is a plasma membrane channel that transports Cl- ions across the membrane
ATP binding and hydrolysis gates the channel opening
CFTR is expressed in epithelial cells where it transports Cl- ions into the mucous, this increases water movement into the mucus making it less viscous

Question 19

What is the CFTR ΔF508 mutant?

Answer 19

CFTR ΔF508 is an autosomal recessive mutation that results in the loss of residue 508, a Phenylalanine (F508) in nucleotide binding domain 1 (NBD1)
CFTR ΔF508 retains some capacity to transport Cl- ions, it is all degraded by ERAD pathway thus preventing the mutant protein from trafficking to the plasma membrane

Question 20

What is a method of helping the CFTR ΔF508 mutant?

Answer 20

Lumcaftor is a chemical chaperone that increases ER exit of CFTR DF508 and Cl- ion transport in cells, but alone it has limited clinical benefit
It works by increasing the trafficking of CFTR proteins to the outer cell membrane
Lumcaftor in combination with the channel opener Ivacaftor, does provide some minor improvement in lung function in clinical trials (Orkambi)

Question 21

What is Human Cytomegalovirus (HCMV)?

Answer 21

This hijacks the ERAD pathway in order to escape immune recognition
US2 and US11 are ER localised membrane proteins and both can cause MHC class I molecules to enter the ERAD pathway
HCMV infected cells can therefore escape detection by cytotoxic T cells