Post-translational modifications

Question 1

What are the post-translation protein modifications done in the ER?

Answer 1

Signal peptide cleavage
Glycosylation
Disulfide bond formation
Addition of metal groups
Mutlimerisation
Folding of the amino acid sequence into tertiary structure for function.

Question 2

What is signal peptide cleavage?

Answer 2

The signal peptide is removed by a peptidase.

Question 3

What is glycosylation?

Answer 3

The addition of a 14 sugar oligosaccharide.

Question 4

What is disulfide bond formation?

Answer 4

Covalent bonds between two cysteine amino acids.

Question 5

What is addition of metal groups?

Answer 5

Many enzymes contain iron, zinc or manganese ions as co-factors in their catalytic sites.

Question 6

What is multimerisation?

Answer 6

e.g. in haemoglobin 4 polypeptide chains are brought together to form it.

Question 7

When does glycosylation occur?

Answer 7

Secreted and membrane proteins are glycosylated as the polypeptide chain is translated and threaded through the translocator in the ER membrane.

Question 8

What is added in glycosylation?

Answer 8

A preformed oligosaccharide is transferred from a lipid in the ER membrane to the newly synthesised proteins.

Question 9

What is the structure of the oligosaccharide?

Answer 9

2 N-acetylglucosamine molecules
9 mannose molecules
3 glucose molecules

Question 10

How does glycosylation occur?

Answer 10

The oligosaccharide is attached to a specific amino acid sequence in target proteins.
As soon as the sequence is translated and emerges from the translocator, it is recognised by an enzyme that catalyses the transfer of the oligosaccharide from the lipid dolichol onto the protein.

Question 11

What is the acceptor sequence for glycosylation?

Answer 11

The sequence is a 3 amino acid sequence: Asn-X-Ser-Thr.
X is any amino acid except pro.
The asparagine side chain is modified.

Question 12

What is the enzyme responsible for glycosylation?

Answer 12

Glycosylation is catalysed by an ER membrane protein complex oligosaccharyl transferase (OST).

Question 13

Where is oligosaccharyl transferase?

Answer 13

It has some components that are ER membrane proteins.
Its active site is located on the luminal or internal side of the ER membrane.
So proteins are glycosylated in the ER lumen.

Question 14

Why are proteins glycosylated?

Answer 14

The glycosyl group can be bound by lectins like calnexin and calreticulin, this helps protein folding.
Glycosylation marks newly translated proteins for the ER protein folding machinery.

Question 15

How are glucose sugars removed?

Answer 15

During folding reactions the glucose sugars are removed sequentially.
Proteins that lack the 3 glucose sugars are no longer bound by lectins and are released for onward transport to the Golgi body.

Question 16

What is the function of the Golgi apparatus?

Answer 16

It receives proteins and lipids from the ER, modifies and packages them and then sends them to their final destinations:
The plasma membrane of the cell.
Secreted outside of the cell.
Lysosomes in the cell.

Question 17

What is the function of vesicles?

Answer 17

Transport of proteins from the ER, to the Golgi, between the Golgi stacks and from the Golgi are mediated by membrane-bound vesicles.
These bud off from the ER and capture luminal and membrane proteins from the ER into vesicular intermediates.
They are then carried to the Golgi apparatus and fuse.

Question 18

What is the structure of the Golgi?

Answer 18

The Golgi is made up of a stack of membrane compartments called the cisternae.
These are stacked one on top of another.

Question 19

What is the process of vesicular transport?

Answer 19

Vesicles from the ER will fuse with the first stack on the ER side of the Golgi - the cis face.
Proteins then undergo successive rounds of modifications with distinct activities in each of the cisternae.
Proteins move between the cisternae by the same process of vesicular transport that brought them to the Golgi.

Question 20

What happens in vesicular transport after it modifies one stack of cisternae?

Answer 20

Vesicles then split off from the cisternae and capture proteins from the cisternal lumen and from the membrane.
The vesicle moves on to the next cisternae and the process is repeated until the proteins reach the trans face of the Golgi and are taken to their final destination in vesicles that split off from the trans face.

Question 21

What is N-linked glycosylation?

Answer 21

Glycosylation of asparagine residues as proteins pass through the Golgi.
N-linked because sugar addition occurs at the NH2 group of the asparagine side chain.
H+ is removed from asparagine and OH- from the sugar in a condensation reaction.
Bond is to N on peptide so is N-linked.

Question 22

How are high mannose oligosaccharides generated?

Answer 22

High mannose oligosaccharides are generated when the original N-linked oligosaccharide Is trimmed and further mannose residues are added.

Question 23

How are complex oligosaccharides generated?

Answer 23

When the original N-linked oligosaccharide is trimmed by glycosidases and further sugars are added by glycosyltransferases.

Question 24

What is O-linked glycosylation?

Answer 24

Occurs when sugar residues are added to selected serine or threonine residues.
It is a condensation reaction where H+ is removed from the serine or threonine hydroxyl group, and the OH- group is removed from the sugar.
The bond is to oxygen on the peptide side chain so is O-linked.

Question 25

How are sugars added for O-linked glycosylation?

Answer 25

N-acetylgalactosamine is added first, then a variable number of additional sugar residues are added one at a time by glycosyltransferases.

Question 26

What are glycosylated proteins?

Answer 26

Glycoproteins e.g.
Protein hormones
Digestive enzymes
Extracellular matrix proteins
Channels and transporters
Receptors
Cell adhesion molecules

Question 27

What are proteoglycans?

Answer 27

A type of glycoprotein.
They contain glycosaminoglycan (GAG) chains.
A link tetrasaccharide is added to the serine side chain on the core protein through O-glycosylation.
Glycosyltransferases synthesise the GAG chain one sugar at a time.

Question 28

What is aggrecan?

Answer 28

A 3 megadalton ECM proteoglycan.
3000 amino acid serine rich core protein
100 chonroitinsulfate chains
30 keratin sulfate chains.
It is synthesised and secreted by fibroblasts in connective tissue.

Question 29

What is the purpose of protein glycosylation?

Answer 29

Promotes protein folding by making proteins more soluble and preventing aggregation during synthesis.
Aids cell recognition processes
Are important in the inflammatory response
Protect the cell from chemical and mechanical damage.

Question 30

How are proteins sorted to their final destinations?

Answer 30

Proteins are sorted to their final destinations at the trans Golgi network (TGN).
3 routes:
Lysosomal pathway
Constitutive secretory pathway
Regulated secretory pathway

Question 31

What is the lysosomal pathway?

Answer 31

Proteins are routed to intracellular organelles - lysosomes.
These degrade expired organelles and other materials that allow the cell to recycle molecules.

Question 32

What is the constitutive secretory pathway?

Answer 32

Proteins are transported from the TGN in vesicles then targeted to the cell membrane.
When vesicles fuse with the membrane, their cargos are liberated to the extracellular space, for luminal proteins, or inserted into the cell membrane.
Proteins like the components of the ECM use this pathway where there is continuous movement to, and fusion with the cell membrane.

Question 33

What is the regulated secretory pathway?

Answer 33

Vesicles from the TGN accumulate under cell membrane.
When triggers e.g. nerve impulses are received they fuse with the membrane and liberate their contents to the exterior in regulated bursts.
e.g. for insulin triggered by increased glucose levels.

Question 34

What are key features of constitutive secretion?

Answer 34

Default pathway
Operates continuously
Supplies the plasma membrane with newly synthesised membrane spanning proteins and lipids.
Soluble proteins are continually secreted from the cell.

Question 35

What side of the cell will things be secreted?

Answer 35

What was inside the ER goes to the inside of vesicles and the inside of the Golgi.
When vesicles fuse with the plasma membrane what was inside the vesicle will be on the outside of the cell.
Extracellular domains of cell surface proteins are in the lumen of the ER, vesicles and Golgi and are heavily glycosylated.

Question 36

How are lipids exported by the secretory pathway?

Answer 36

Lipids are synthesised in the SER and carried to the surface via vesicles of the secretory pathway.
Only those lipids on the inner leaflet of the ER, which becomes the extracellular leaflet of the plasma membrane are glycosylated.