Lecture 11. ER Events Flashcards by Ben Hallam

What is ER targeting?

Starts in the cytosol
The ER is NOT a major site of protein manufacturing, the cytosol is
ER targeting requires proteins to be made in the cytosol initially and is a later step

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What happens in co-translational targeting?

Proteins are made but the first part of the protein is extruded (N-terminus)
N-terminal acts as a signal peptide and directs the nascent protein into the ER lumen (engages with translocon): and from there other parts of the secretory pathway can be accessed

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What happens in the secretory pathway during protein secretion?

From the cytosol, proteins cross (translocate) the ER membrane and exit the ER in vesicles
These vesicles then fuse with the Golgi, and the proteins are transported through the Golgi into secretory vesicles (ultimately fuse with the outside of the cell)
After fusion of the last vesicles with the plasma membrane, the protein content (cargo) is secreted to the outside of the cell

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What happens to some secretory pathway proteins that are directed from the Golgi that don’t go to the plasma membrane?

Some secretory pathway proteins are nucleus directed from the Golgi to the late endosomes/lysosomes, and some are retained in membranes

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What is the entry point for the secretory pathway?

The endoplasmic reticulum (ER)

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What does entry into the ER require?

A signal peptide (part of the primary sequence of the protein)
mRNAs that encode secretory pathway proteins encode a 5’ signal sequence (SS) in frame to the sequence encoding the mature secretory protein: this signal sequence encodes an N-terminal signal peptide (SP)
SP removed by proteolytic cleavage to provide mature protein

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What do ER signal peptides look like?

Don’t all look the same
Variable length of ~15-30 amino acids
All SPs can be recognised due to overall similarities between them

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What do all ER signal peptides tend to have?

A +ve amino acid (Lys, Arg) towards the N’ terminus and a hydrophobic stretch that is recognised by signal recognition particle (SRP). The cleavage site usually follows a small amino acid
No sequence specificity for a single peptide

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How does a SP ensure that the translating ribosome binds the ER membrane?

Because the SP is recognised by signal recognition particle ( SRP) in the cytosol – and this targets the SP to the translocon

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How does a protein enter into the ER?

An emerging SP captures SRP (recognition step) and binds the α subunit of an SRP receptor (targeting step), recruiting a closed translocon (protein-conducting channel)
Translocon opens, allowing entry of the SP into the channel. The SRP cycle SRP/SRP receptor complex is dismantled and SRP is recycled
Signal peptidase (SPase) cleaves the SP from the protein, and the SP leaves the translocon through a lateral gate
Signal peptide peptidase (SPPase) cleaves the SP into two, and translation continues until termination

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What influences the specificity of ER targeting?

Depends upon broad substrate specificity in the recognition step
The folding of the SP into a hook shape by the SRP
Which allows signal peptidase to cleave the signal within the translocon
Releasing the SP into the ER membrane
Which causes problems downstream, because SPs disrupts the ER membrane, requiring their removal by chopping with signal peptide peptidase (although Saccharomyces lacks SPP)

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How are ER targeted nascent proteins formed?

Formed in locally crowded conditions
Nascent membrane-bound polyribosomes tend to crowd on ER lumen, so ER chaperons are required for efficient folding to allow the proteins to enter the ER

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What are the two major ER modifications that can happen to folding proteins?

Folding proteins may become N-glycosylated and/or disulphide-bonded (only forms between two adjacent cysteines in oxidising cellular environments, i.e not the cytosol) depending on whether they have the right amino acid sequence motifs for these events

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What happens if a protein does not fold properly in the ER lumen?

It will fail a quality control check carried out by ER chaperones. Misfolded proteins are not usually allowed to proceed through the secretory pathway
Instead, misfolded proteins are ejected from the ER in a process called retro-translocation (dislocation), and are then degraded in the cytosol

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In the Hsp70 family , what are the three chaperones involved with maintaining client solubility?

Hsc70 and Hsp 70 in the cytosol
BiP (GRP78) - binding protein (glucose regulating protein of 78 kDA) in the ER

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In the Hsp90 family , what are the three chaperones involved with assembly?

Hsp90 in the cytosol
GRP94 in the ER

What are the specialised ER chaperones that govern disulphide bond formation in the ER lumen?

PDI family
PDI and ERp57

What are the specialised ER chaperones that govern N-glycan specific interactions/N-glycosylation in the ER lumen?

CRT family
CRT and CNX (N-glycans are trees of sugar attached to asparginine residues)

What is N-glycosylation of proteins?

The covalent addition of an oligosaccharide tree of sugars (core N- glycan) from a lipid carrier to a target protein by the membrane-integral OST (oligosaccharyltransferase)
As the protein moves through the secretory pathway the core N-glycans are modified to make complex N-glycans

Can archaea and bacteria N-glycosylate proteins?

Many archaea can, but only a few bacteria can do this

What is the core N-glycan?

The core N-glycan is attached covalently to the N atom of the side chain of an asparagine residue in a specific context – the N-glycosylation signal NXS/T

What is the N-glycosylation signal?

NXS/T
N = Asp, asparaginyl (residue of an asparginine( - recognised as attachment point for tree of sugars
X = residue of any amino acid except ptroline
S/T = seryl/threonyl (residue of either a serine of a threonine)

What are the functions of N-glycosylation?

N-glycans act as flags for folding and ER Quality Control
Increase protein solubility
Influence folding rates and final protein conformation

How do N-glycans act as flags for folding and ER quality control?

The removal of the two terminal Glc residues from the core N-oligosaccharide in the ER allows interactions with ER chaperones (e.g. calreticulin) required for efficient folding of N-glycosylated proteins (Glc-1 interacts with lectin chaperones). As the protein proceeds through the folding steps, the final Glc residue and a particular Man residue are removed, removing the protein from the folding environment and signalling that the protein is now deemed fit to be passed to the Golgi

How do N-glycans increase protein solubility?

A core N-glycan is very large (~19 times larger than the asparagine residue to which it is attached) and made of hydrophilic sugars. N-glycans therefore increase protein solubility and can reduce aggregation problems during folding in the ER

How do N-glycans influence folding rates and final protein conformation?

N-glycans are bulky and therefore constrain the α-carbon backbone of the polypeptide Because the final conformation of the protein may be constrained, N-glycans may influence the activity of a protein (e.g. enzymes) or its interaction with other N-glycans molecules (e.g. the interaction of an antibody with effector molecules)

How do disulphide bonds form?

Do not form readily in reducing conditions (the cytosol): this requires oxidising conditions (the ER in eukaryotes or the bacterial periplasm) They form where 2 cysteine residues are brought close together during protein folding

What are the biological catalysts of disulphide bond formation?

Protein disulphide isomerase (PDI) and its relatives PDIs can make, break and shuffle disulphide bonds

What do covalent S-S bonds contribute to?

The stability of protein tertiary structures (of secreted proteins, not cytosolic proteins) Many are essential for the activity of proteins

How does PDI (protein disulphide isomerase) shuffle disulphide bonds?

Reduced PDI recognises unstable proteins, binds as a chaperone, and forms a mixed disulphide bond (proteins and chaperon bond and become one complex - PDI only binds to unstable proteins) Isomerisation of disulphide bonds continues until the client protein reaches a stable conformation, when it is released from PDI

Besides shuffling disulphide bonds, what other roles does PDI have?

PDI can make and break disulphide bonds (oxidised PDI can oxidise reduced client and vice versa) PDI can join and separate two proteins (oxidised PDI binds to to reduced clients and fuses them into a singular, oxidised client and vice versa)

How do we know PDI is involved in joining and separating proteins?

In vitro recapitulation E.g separating a disulphide-bonded heterodimer (ricin) into constituent A and B chains (RTA and RTB) required produced PDI Proteins can only be split in reduced conditions and the process is completely reversible

Because ER protein folding processes do not work in isolation and co-ordinated, how does ER protein folding occur (in MHC Class I assembly)?

1. BiP maintains solubility of MHC Class I HC (heavy chain) until β2 microglobulin binds 2. N-glycosylation allows entry into a folding environment provided by the chaperon calreticulin and its PDI-related partner ERp57 which is disulphide-linked to tapasin (tapasin is found in the transmembrane) 3. This allows assembly and folding of Class I molecules , providing a groove for insertion of a peptide fed in from the cytosol by the TAP transporter that is recruited by tapasin 4. The peptides are derived from proteosomal degradation

What happens to an ER protein that fails quality control (e.g an MHC Class I HC that has failed to find β2m)?

1. If the MHC Class I HC has a long residence time with ER chaperons (e.g BIP) this allows access of a mannosidase that removes one particular mannose removing this protein from the folding environment 2. The protein is unfolded and fed through a multisubunit discolon (narrow channel) and is ubiquitylated during dislocation, targeting it to the proteasome where it is deubiquitylated and degraded

What is ERAD?

ER-associated protein degradation Misfolded ER proteins are destroyed in the cytosol

What is O-linked glycosylation?

Attachment of sugars t oxygen atoms of amino acids Occurs in the ER/Golgi apparatus in eukaryotes. It also occurs in Archaea and Bacteria O-N-acetylgalactosamine (O-GalNAc) may be attached to serine or threonine residues - the high concentration of carbohydrates gives mucus its "slimy" feel O-mannose can be attached to serine and threonine residues in secretory pathway proteins. O-mannosylation is common to both prokaryotes and eukaryotes. O-fucose and O-glucose can be added to cysteine residues

What is proteolytic cleavage in terms of secretory system modifications?

ER proteins being activated by cleavage just before the end of the secretory pathway

What enzymes does proteolytic activation occur for?

Active digestive enzymes (e.g., trypsin) Hormones (e.g., glucagon and insulin) Neurotransmitters (e.g., enkaphalin) Proteins that would otherwise aggregate (e.g., mature collagen)

What is the addition of lipids to permit/maintain membrane targeting in terms of secretory system modifications?

GPCRs are polytopic membrane proteins that have a myristic acid lipid anchor GPI-linked proteins have a glycophosphatidylinositol membrane anchor linked via sugars to the protein