Post-translational Processing of Proteins - Protein Targeting

Question 1

What are some post-translational modifications? Explain them briefly.

Answer 1

Proteolytic cleavage where peptide bonds are broken to remove parts of the protein.
Chemical modification where there is an addition of a functional group to amino acid residues.

Question 2

Why is proteolytic cleavage important?

Answer 2

Because we might only want the protein to be activated once it is secreted.

Question 3

What proteins are synthesised on free ribosomes?

Answer 3

Proteins that are destined for the cytoplasm or imported into organelles.

Question 4

What proteins are synthesised by ribosomes on the rough ER?

Answer 4

Proteins that are destined for the membrane or destined for secretory pathways to the exterior.

Question 5

What steps are required for protein sorting?

Answer 5

A signal
A receptor that recognises the signal and directs it to the correct membrane
A translocation machinery like a channel or vesicle
Energy to transfer the protein to its new place

Question 6

What types of proteins are there in protein targeting for secretion? Give examples of each

Answer 6

Extracellular proteins (Insulin, pepsin)
Membrane proteins (integral and channel proteins)
Vesicular proteins (lysosomes, endoscopes and peroxisomes etc.)

Question 7

What types of secretions are there?

Answer 7

Constitutive secretion

Regulated secretion

Question 8

What is constitutive secretion?

Answer 8

A slow continuous secretion that happens all the time

Question 9

What is regulated secretion?

Answer 9

A secretion that is based on a signal/stimulus. Involves energy.

Question 10

Why would there be a lot more rough endoplasmic reticulum in a cell in the islets of langerhans than in a muscle cell e.g.?

Answer 10

Because beta and alpha cells in the islets of langerhans secrete hormones. Rough endoplasmic reticulum have ribosomes which makes proteins which are destined for secretion.

Question 11

What is a signal sequence? Outline some features.

Answer 11

It has a specific N-terminal aa sequence which is the signal. It is around 5-30 amino acids long.
The central region of the signal sequence is rich in hydrophobic residues. and it is able to form an alpha helix.
The signal sequence is removed afterwards.

Question 12

What is the ‘pre’ part in preproalbumin?

Answer 12

It defines the signal sequence which is the removed during processing.

Question 13

What is a signal recognition particle?

Answer 13

A riboprotein which recognises the signal sequence and binds to it on the amino acid chain. This signal recognition particle (SRP) then also binds to a receptor that is located on the ER membrane.

Question 14

Briefly explain synthesis of secretory proteins and their translocation across the ER membrane.

Answer 14

A mRNA with a ribosome which is making a polypeptide chain starts off with making the signal sequence at the N-terminal. A SRP recognises the signal sequence and binds to it. The SRP then binds to a SRP receptor on the ER membrane. This brings down the polypeptide chain and the ribosome to the ER membrane. The translocon which is a channel protein now opens on the ER membrane and the polypeptide chain is being made and fed into the channel protein. The signal sequence which is largely hydrophobic gets stuck in the channel protein and is the cut off with by signal peptidase. Proalbumin is now made into the ER lumen and starts folding. It is no longer preproalbumin.

Question 15

Briefly explain the mechanism of synthesis of a type 1 membrane protein at the ER.

Answer 15

Same as in secretory protein with SRP and SRP receptor. However somewhere in the production of the polypeptide chain there is a hydrophobic part which gets stuck in the channel protein. This works as a stop-transfer anchor sequence. Soon after this the ribosome stop translating and the hydrophobic part is transferred over to the phospholipid bilayer. The N-terminal is in the lumen and the C-terminal is in the cytoplasm.

Question 16

What are some functions of the ER?

Answer 16

Insertion of proteins into membranes
Proteolytic cleavage
Glycosylation
Formation of S-S bonds
Proper folding of proteins
Assembly of multisubunit proteins (quaternary)
Hydroxylation of selected Lys and Pro residues (like in collagen)

Question 17

What is N-linked glycosylation?

Answer 17

When sugars are added on an asparagine side chain. It is a reaction involving an amino group.

Question 18

Why is glycosylation important?

Answer 18

It enables correct protein folding
It makes the protein more stable and less prone to damage by organelles in the cytoplasm
Facilitates interactions with other molecules. (most important outside the cell or on the cell membrane)
Deficiencies in N-linked glycosylation lead to severe inherited human diseases: Congenital disorders of glycosylation.

Question 19

Why is formation of S-S bonds important?

Answer 19

Since proteins can be fragile extracellularly S-S bonds make the protein more rigid and less prone to damage. It also aids folding.

Question 20

What are ER chaperones?

Answer 20

Proteins that help folding occur correctly.

Question 21

What are consequences of mis-folding not being able to be corrected?

Answer 21

Proteins may be returned to the cytoplasm for degradation.
However the proteins may accumulate/build up to toxic levels in the ER resulting in disease. This can be a cause of a single mutation.

Question 22

What are steps of the Golgi complex?

Answer 22

Phosphorylation
Removal of Man
Addition of GlcNAc
Addition of Gal
Addition of NANA
Sulfation of tyrosine and carbohydrates.

Question 23

What is the general concept happening in the Golgi complex?

Answer 23

Modification of proteins and they usually come out mature.

Question 24

Give an example of a protein that is not mature when it has been through the Golgi complex.

Answer 24

Collagen fibres. (Tropocollagen)

Question 25

Outline some features of tropocollagen.

Answer 25

The basic unit of collagen fibres.
Rod-Shaped protein
3 polypeptide alpha chains.
Glycine is in every three position along each alpha chain. (Gly-X-Y)n
It is formed in a triple helix this means that three alpha chains are wrapped around each other.

Question 26

Why is glycine so important to tropocollagen?

Answer 26

Glycine is the smallest amino acid and because of this it can fit inside the triple helix.

Question 27

What happens if there is another amino acid in the middle of the tropocollagen?

Answer 27

A larger bulkier protein will deform the tropocollagen, this will make it weaker.

Question 28

What are the most common amino acids to find in the X and Y positions of tropocollagen?

Answer 28

Proline and hydroxyproline.

Question 29

Why is hydroxyproline important?

Answer 29

Because it can form H-bonds between the alpha chains to stabilise the structure.

Question 30

What makes proline form hydroxyproline?

Answer 30

Prolyl hydroxylase, oxygen, iron and ascorbate (Vitamin C)

Question 31

What’s the difference between procollagen and tropocollagen?

Answer 31

Procollagen has 150 extra N-terminal amino acids that do not form triple helix
Procollage also has 250 extra C-terminal amino acids which do not form triple helix.

Question 32

Where does procollagen turn into tropocollagen? By what?

Answer 32

It’s converted into tropocollagen by proteases by proteases (proteolytic cleavage) extracellularly.

Question 33

Why is it important that tropocollagen does not form inside the cell?

Answer 33

Because then tropocollagen would aggregate to form collagen fibres inside the cell and the cell would explode.

Question 34

Where do chemical modifications of procollagen occur?

Answer 34

In the rough ER.