L24 - Protein Trafficking

Question 1

Where are cytosolic proteins made?

Answer 1

Cytosol

Question 2

Where are membrane and secretory proteins made?

Answer 2

RER

Question 3

Where are nuclear proteins made?

Answer 3

Outer nuclear membrane

Question 4

Protein pathway overview - membrane protein synthesis method

Answer 4

1. RER/cytosol - synthesis starts
2. SER - lipids and vesicles form
3. Proteins directed to right destinations based by their signal sequence
   - small hydrophobic amino acids
4. Ribosome binds signal sequence
5. SRP guides ribosome-signal sequence to ER membrane
6. SRP binds signal sequence to new polypeptide as it emerges
7. SRP-ribosome complex binds SRP receptor in ER membrane
8. SRP released allowing attachment of ribosome to protein translocator channel
9. Polypeptide chain goes through protein translocator into ER lumen
10. ER-based signal peptidase removes signal sequence from secretory proteins
11. Transmembrane proteins stay anchored in ER membrane
    12 Golgi - glycosylation and sorting
12. Proteases trim and activate enzymes during maturation of vesicles

Question 5

What is differential centrifugation used for?

Answer 5

Used to separate heavy and light vesicles
Lighter vesicles remain on top of the tube
ER vesicles are heavier and drop to the bottom

Question 6

What does electron microscopy reveal about ribosomes?

Answer 6

Reveals real topography of ribosomes on ER membranes

Smaller ribosome unit faces the cytosol

Question 7

What does swapping the signal peptide for a lipid anchor in the ER cause?

Answer 7

This allows the continuation of membrane association

Question 8

What is an example of a lipid anchor?

Answer 8

Glycosylphosphatidylinositol anchor (GPI anchor)
Allows the protein to stay in the membrane even without transmembrane sequence

Question 9

What is the role of the ER in protein maturation?

Answer 9

The signal sequence is removed by a specific signal peptidase
This is followed by conformational maturation
Then disulfide bridges are formed between cysteine residues to solidify protein shape
The protein is glycosylated by a standard carbohydrate chain

Question 10

What is the purpose of protein glycosylation?

Answer 10

Quality control – if protein is synthesised in wrong shape the sugar cannot attach correctly
Enzymes in the lumen will destroy the protein

Question 11

Why do ER resident enzymes carry KDEL sequences?

Answer 11

Important for their return to the ER lumen
KDEL: lysine-aspartic acid-glutamic acid-leucin
- Marks the proteins so they can be captured by KDEL receptors

Question 12

Why is the addition of carbohydrate/sugar to proteins important?

Answer 12

Protein stability in the harsh extracellular environment
Cell-cell recognition – tagging 
Cross-species separation
- Humans use beta-galactose
- Other animals use alpha-galactose

Question 13

What are the two types of glycosylation?

Answer 13

N-linked
- Sugar attached to NH terminal of asparagine
O-linked
- Sugar attached to O terminal of threonine

Question 14

How are carbohydrate chains added to proteins?

Answer 14

Starts in the ER
Occurs via asparagine residue
Multi-step process
- Proceeds step-by-step in individual Golgi cisternae
- Each step requires separate Golgi cisternae to keep specific glycosylation enzymes away from each other

Question 15

What is the structure of the quality control signal?

Answer 15

2 acetylglucosamine residues
Many mannose residues
3 glucose residues

Question 16

Why are there limitation in organ transplantations?

Answer 16

Due to a simple sugar modification
Human cells make beta galactose while other animals make alpha-galactose
Animal-derived organs are rejected by the human organism
- Humans produce antibodies against alpha-galactose and thus reject transplants

Question 17

What technique made organ transplants possible?

Answer 17

Genetically modified pigs lacking alpha-galactose
The alpha-galactose was knocked out via gene targeting
Donor cells used to produce cloned GE pigs

Question 18

Once made how is the protein activated?

Answer 18

By trimming/maturation - proteolytic processing

1. Takes place prior to secretion
2. Proteases trim and activate hormones and enzymes during maturation of vesicles
   - E.g. insulin

Question 19

How do you make insulin?

Answer 19

To make insulin functional you need to remove a particular middle sequence

1. Preproinsulin translation, signal cleavage and proinsulin folding
2. Proinsulin is transported to Golgi
3. Proinsulin is cleaved to produce mature insulin and C-peptide

Question 20

What does insulin trigger?

Answer 20

Insulin triggers delivery of glucose transporters into the plasma membrane of muscle cells

Question 21

What causes diabetes?

Answer 21

Dysfunctional glucose uptake into muscle cells

Question 22

Method of what causes type 1 diabetes

Answer 22

1. Misfolding of Proinsulin in the ER due to a mutation
2. Protease in secretory vesicle cannot cleave off the C-peptide
3. Secretion of dysfunctional pro-insulin instead of insulin
4. Generation of antibodies against the pancreatic cells destroying them
5. Increase in blood glucose concentrations

Question 23

How can different cells can process the same polypeptide into different hormones?

Answer 23

Cleavage of opiomelanocortin can give rise to several hormones

• ACTH and β-lipotropin - secreted by the pituitary gland
• β-endorphin - generated by neurons in response to exercise and stress

Question 24

Which two proteins are moved from cytosol to ER?

Answer 24

1. Water soluble proteins cross the ER membrane and are released into the lumen
2. Transmembrane proteins only partially cross the ER and get embedded in the membrane