L24 - Protein Trafficking Flashcards
Where are cytosolic proteins made?
Cytosol
Where are membrane and secretory proteins made?
RER
Where are nuclear proteins made?
Outer nuclear membrane
Protein pathway overview - membrane protein synthesis method
- RER/cytosol - synthesis starts
- SER - lipids and vesicles form
- Proteins directed to right destinations based by their signal sequence
- small hydrophobic amino acids - Ribosome binds signal sequence
- SRP guides ribosome-signal sequence to ER membrane
- SRP binds signal sequence to new polypeptide as it emerges
- SRP-ribosome complex binds SRP receptor in ER membrane
- SRP released allowing attachment of ribosome to protein translocator channel
- Polypeptide chain goes through protein translocator into ER lumen
- ER-based signal peptidase removes signal sequence from secretory proteins
- Transmembrane proteins stay anchored in ER membrane
12 Golgi - glycosylation and sorting - Proteases trim and activate enzymes during maturation of vesicles
What is differential centrifugation used for?
Used to separate heavy and light vesicles
Lighter vesicles remain on top of the tube
ER vesicles are heavier and drop to the bottom
What does electron microscopy reveal about ribosomes?
Reveals real topography of ribosomes on ER membranes
Smaller ribosome unit faces the cytosol
What does swapping the signal peptide for a lipid anchor in the ER cause?
This allows the continuation of membrane association
What is an example of a lipid anchor?
Glycosylphosphatidylinositol anchor (GPI anchor) Allows the protein to stay in the membrane even without transmembrane sequence
What is the role of the ER in protein maturation?
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
What is the purpose of protein glycosylation?
Quality control – if protein is synthesised in wrong shape the sugar cannot attach correctly
Enzymes in the lumen will destroy the protein
Why do ER resident enzymes carry KDEL sequences?
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
Why is the addition of carbohydrate/sugar to proteins important?
Protein stability in the harsh extracellular environment Cell-cell recognition – tagging Cross-species separation - Humans use beta-galactose - Other animals use alpha-galactose
What are the two types of glycosylation?
N-linked
- Sugar attached to NH terminal of asparagine
O-linked
- Sugar attached to O terminal of threonine
How are carbohydrate chains added to proteins?
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
What is the structure of the quality control signal?
2 acetylglucosamine residues
Many mannose residues
3 glucose residues
Why are there limitation in organ transplantations?
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
What technique made organ transplants possible?
Genetically modified pigs lacking alpha-galactose
The alpha-galactose was knocked out via gene targeting
Donor cells used to produce cloned GE pigs
Once made how is the protein activated?
By trimming/maturation - proteolytic processing
- Takes place prior to secretion
- Proteases trim and activate hormones and enzymes during maturation of vesicles
- E.g. insulin
How do you make insulin?
To make insulin functional you need to remove a particular middle sequence
- Preproinsulin translation, signal cleavage and proinsulin folding
- Proinsulin is transported to Golgi
- Proinsulin is cleaved to produce mature insulin and C-peptide
What does insulin trigger?
Insulin triggers delivery of glucose transporters into the plasma membrane of muscle cells
What causes diabetes?
Dysfunctional glucose uptake into muscle cells
Method of what causes type 1 diabetes
- Misfolding of Proinsulin in the ER due to a mutation
- Protease in secretory vesicle cannot cleave off the C-peptide
- Secretion of dysfunctional pro-insulin instead of insulin
- Generation of antibodies against the pancreatic cells destroying them
- Increase in blood glucose concentrations
How can different cells can process the same polypeptide into different hormones?
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
Which two proteins are moved from cytosol to ER?
- Water soluble proteins cross the ER membrane and are released into the lumen
- Transmembrane proteins only partially cross the ER and get embedded in the membrane
