Protein Trafficking Flashcards
Where are cytosolic proteins made?
How?
In the cytosol
On free ribosomes through a ‘simple cycle’
Where are membrane and secretory proteins made?
How?
In the rough endoplasmic reticulum (RER)
Through an SRP cycle
Where are nuclear proteins made?
In the outer nuclear membrane
Where do the proteins made in the RER travel to? (in order)
- Smooth ER
- Golgi
- Secretory vesicles
- Membrane
Why is the RER ‘rough’?
Due to ribosomes
Where does ALL protein synthesis START?
In the cytosol
Describe the SRP cycle
ER signal sequence in the mRNA binds to the signal recognition particle and directs the ribosome to the ER membrane
The mRNA then remains permanently bound to the ER as part of a polyribosome (as the ribsomes translate the mRNA - the protein sequence falls into the ER lumen)
Ribosomes move along the mRNA and are recycled at each round of replication to join the free ribsome pool
Where do proteins travel to after they are synthesised in the RER?
To the edges of the ER where there are no ribosomes - the SER
Where are LIPIDS made?
In the SER
What happens to lipids and proteins in the SER?
They are packaged together in a vesicle - to travel towards the golgi and further on
What happens in the SER?
Lipids are made and packaged with proteins into vesicles
What is used to separate the heavy and light vesicles?
Describe this process
Differential centrifugation:
- Gradient of sucrose concentration
- Spin at high speed
- Low density settles at the bottom
- High density settles at the top
What are the high density vesicles?
Studded ribosomes - ER vesicles that studded with ribosomes
How are proteins sent to the right destination in the cell?
Based on their signal sequence
What are signal sequences?
The first 10-12 amino acids on the polypeptide chain
What type of signal sequence is needed to direct the protein to the nucleus?
What is this sequence different to?
Positive charges
These sequences are different to the signal sequences that destines the proteins to the ER
What does the SRP do?
Attaches to and guides free ribosomes to the ER by binging to the signal sequence on the growing poly peptide chain
What does the SRP consist of?
Cellular proteins
How does the SRP attach to ribosomes?
With its hinge region
What happens after the SRP is bound to the signal sequence?
- Pause in translation
- SRP receptor in the ER membrane binds the SRP-ribosome complex
- Directs the complex to the to a protein translocator in ER membrane
- Signal sequence is captured by the translocator - opening the channel
- Ribosome is passed to the translocator and translation continues
- SRP and receptor are released and recycled
What is the topography of the ribosome on the ER membrane?
Small ribosome subunit facing to cytosome
Large ribosome subunit is attached to the protein translocator in the ER membrane
What removes the signal sequence from proteins in the ER?
ER-based signal peptidases (specific signal peptidases)
What happens if the proteins being synthesised on the ER membrane have a stop-transfer sequence?
The protein becomes embedded in the ER membrane
What happens if the proteins being synthesised on the ER membrane don’t have a stop sequence?
What can this protein then be destined for?
ALL of the protein goes into the ER lumen
Destined for secretion or into the lysosomes
What 2 kinds of proteins are moved from the cytosol to the ER?
1) Water soluble proteins - completely cross the membrane and go into the ER lumen
2) Transmembrane proteins - only partially cross the ER and become embedded
What is the signal sequence that directs the proteins to the ER?
Small hydrophobic amino acids
How can swapping the signal peptide for a lipid anchor in the ER membrane occur?
What does this allow?
- WITHOUT a transmembrane sequence
- Signal peptide removed and a lipid tail is attached
- Allows the continuation of membrane association
Where are GPI anchors found?
In the OUTER leaflet of the lipid bilayer
What is a GPI anchor?
GlycoslyPhosphatidylInositol
What follows the removal of the signal sequence by specific signal peptidases?
What happens during this process?
Conformational maturation:
- Formation of disulphide bridges
- Protein is glycosylated by a standard carbohydrate sugar chain
Where are disulphide bridges formed in the maturation of an ER protein?
What is the function of these bridges?
Between cysteine residues
To ‘solidify’ the protein shape - allowing the protein parts to stay together in a harsh environment (outside the cell or acidic pH inside vesicles)
In what proteins are disulphide bridges important in?
Luminal and extracellular proteins
Why are proteins glycosylated in the maturation of an ER protein?
How does this work?
For quality control:
- If the protein is synthesised into the incorrect shape - sugar cannot be attached to the right amino acid
- Therefore, enzymes inside the ER lumen will recognise these as defective and destroy them
Where do glycosylation and disulphide bridges occur?
Extracellularly or inside the lumen of the ER
Cytosol is a reducing environment
What do ER-resident enzymes carry and why is this important?
KDEL sequences
Important for their return back to the ER - captured by the KDEL receptor and brought back to the lumen
What is KDEL?
Lysine-aspartic acid-glutamic acid-leucine
What 3 things are the addition of carbohydrates to proteins important for?
1) Protein stability in the harsh extracellular environment
2) Cell-cell recognition
3) Cross-species separation
How is the addition of carbohydrates used for cell-cell recognition?
Subtle changes can have major affects
How are humans separated from other animals using the addition of carbohydrates?
Humans use beta-galactose
Other animals use alpha-galactose
What is the difference between N-linked and O-linked glycosylation?
N-linked:
- Sugar attached to asparagine by a terminal nitrogen
O-linked:
- Sugar attached to threonine through oxygen
What does INITIAL addition of carbohydrates occur?
In the ER
What is the INITIAL function of the addition of carbohydrates?
Quality control
Where does FINAL addition of sugars and sorting occur?
In the golgi
What is the ‘protein maturation organelle’?
The golgi
What is different about the cisterns of the golgi?
- Different cisterns have different enzyme number/type with different modifications occurring at each enzyme
How does trimming and growth of carbohydrate chains on protein occur?
- Step-by-step in individual golgi cisternae
What does each glycosylation step require?
Separate golgi compartments (cisternae)
What is the process of glycosylation?
Multi-step:
- Glucose residues removed from the original quality control signal
- Mannose residues are trimmed down
- Sugars expand via the attachment of N-acetylglucosamine
- Then, more complex olligosaccharides added
What are limitations in organ transplant due to?
Simple sugar modification
What is the difference between human and animal cells? (in terms of glycosylation)
Human - beta-galactose
(Hydroxyl at pos. 1 faces UPWARDS)
Animals - alpha-galactose
(Hydroxyl at. pos 1 faces DOWNWARDS)
How can human rejection of animal transplants be overcome?
Genetically modify pigs:
- To lack galactose all together
- By removing the protein responsible for the synthesis of alpha-galactose sugars
How are proteins activated?
Trimmed by proteases
Where does trimming of hormones and enzymes (in order to activate them) occur?
During when?
In immature secretory vesicles (before maturation and secretion)
What amino acids need to be removed from insulin in order to make it function?
LLALLAL (leucinces and alanines)
What is protein trimming also known as?
Protein processing/maturation
Why does insulin travel freely through the trans-golgi network?
No sugars are attached to it
What causes type 1 diabetes?
What does this result in?
Genetically inherited
- Miss folding of proinsulin in the ER
- Protease in secretory vesicle cannot cleave off the C-peptide and mature the protein
- Pro-insulin can’t bind insulin receptors
- Blood glucose concentration increase
In type 1 diabetes, why is there a destruction of the pancreatic cells?
Due to the generation of antibodies against the pancreatic cells that release the deficient pro-insulin
How can the same polypeptide be processed into different hormones?
By different cells
What hormones can opiomelanocortin give rise to?
Where are they released from?
Several hormones with completely different functions:
- ACTH and beta-lipotrophin (from the pituitary gland)
- Beta-endorphin (from neurons)
When is beta-endorphin generated by the neurons?
In response to exercise and stress
What does insulin trigger?
The delivery of vesicles containing glucose transporters to the plasma membrane of muscle cells