Protein Targetting Flashcards
When is a protein synthesised by a ribosome attached to the ER membrane?
If the protein is destined for the membrane or secretory pathway via co-translational insertion
What happens to the proteins as they are synthesised when the ribosome is attached to the ER membrane?
They are inserted into the lumen
When are proteins synthesised to completion in the cytoplasm?
If the protein is destined for the cytosol, or post-translational import into organalles
What organelles may receive proteins from post-translational import?
- Mitochondria
- Nucleus
- Peroxisomes
What are the requirements for protein sorting?
- Signal
- Receptor
- Translocation machinery
- Energy
Where is the signal for protein sorting?
Intrinsic to the protein
What is the purpose of the receptor in protein sorting?
Recognises the signal, and directs it to the correct membrane
What is the energy needed for in protein sorting?
To transfer the protein to it’s new place
Where is the signal usually located if the protein target is the ER?
Usually at the N-terminus
What eventually happens to the signal if the protein target is the ER?
It is removed
What is the signal when the protein target is the ER?
6-12 hydrophobic amino acids, often preceded by 1 or more basic amino acids
Where is the signal usually located when the protein target in the mitochondrial matrix?
At the N-terminus
What eventually happens to the signal if the target is the mitochondrial matrix?
It is removed
What is the signal when the target is the mitochondrial matrix?
An amphipathic helix of 20-50 residues with alternating R/K and hydrophobic sites
Where is the signal if the target is a peroxisome?
C-terminus
What eventually happens to the signal if the target is a peroxisome?
It remains
Usually, what is the signal if the target is a peroxisome?
S-K-L
Where is the signal if the target is the nucleus?
Internal- can be anywhere in the protein, but has to be exposed on the surface
What eventually happens to the signal when the target is the nucleus?
It remains
What is the signal when the target is the nucleus?
Either one cluster of 5 basic amino acids, or 2 smaller basic clusters separated by about ~10 amino acids
How is a protein imported into the mitochondrial matrix?
- Protein with signal kept unfolded by chaperones
- Signal binds receptor
- Receptor delivers to important pore
- Protein fed through pore in outer membrane
- Protein moves through channel in adjacent inner membrane
- Targeting signal cleaved, allowing protein to fold
What is required for chaperone proteins to bind to mitochondrial proteins?
ATP hydrolyssi
What does a pyruvate dehydrogenase deficiency result in?
A built up of lactic acid, and neurological problems
What causes pyruvate dehydrogenase deficiency?
- Mutation at codon 10 in N-MTS of PDH E1å. subunit, resulting in an arg →pro substitution
- Receptor can’t recognise targeting sequence
- Reduced uptake into mitochondria
Why does the base substitution mean that the receptor can’t recognise the targeting sequence in PDH?
The helix breaking proline destabilises the helical N-MTS, and there is a loss of one basic residue on the hydrophobic face of the amphipathic helix
How does cargo get imported into the nucleus?
- In the cytosol, importin binds cargo containing a nucleur localisation signal
- Can then travel through nuclear pore
- In nucleus, Ran-GTP binds to importin, causing a conformational change that displaces the cargo
- Importin with bound Ran-GTP recycled to the cytoplasm
What is required to make Ran-GTP and release importin?
GTP hydrolysis
What can mutation of nuclear localisation signals lead to?
- Swyer syndrome
- Leri-Weill dyschondrosterosis
- Langer mesomelicdysplasia
What causes Swyer syndrome?
A loss/mutation of NLS in sex determining region Y (SRY) protein, leading to an XY genotype, but outwardly female, as SRY type needed for testis differentiation
What causes Leri-Weill dyschondrosterosis and Langer mesomelicdysplasia?
R173C mutation of NLS of SHOX transcription factor, as SHOX required for skeletal development
How doesLeri-Weill dyschondrosterosis and Langer mesomelicdysplasia present?
Short stature
Describe the process of PTS-1 directed import of peroxisomal matrix proteins
- In cytosol, peroxisomal import receptor binds cargo with PTS
- Peroxisomal protein remains folded, and receptor integrates into translocon, thereby opening it, so whole peroxisomal protein can enter through channel
- Peroxisome targeting sequence dissociates from the receptor
- Receptor is returned to the cytosol
What is required for the returning of the PTS-1 directed import receptor to the cytosol?
ATP hydrolysis
What is meant by co-translational transport?
Proteins delivered as they’re being synthesised
Where does protein synthesis occur when the protein is destined for the ER/secretory pathway?
Bound ribosomes
Are proteins synthesised for the ER/secretory pathway soluble or membrane bound?
Can be either
Where are proteins synthesised on bound ribosomes synthesised into/across?
The ER membrane
What happens once a protein has been synthesised into/across the ER membrane?
- Budding and fusion of ER-to-Golgi vesicles containing proteins to form cis-Golgi
- Cisternal progression form cis-Golgi → medial-Golgi → trans-Golgi
What happens to proteins required by the ER?
There can be retrograde transport from the Golgi to the ER, and from later to earlier Golgi cisternae
Where does sorting occur?
In the trans-Golgi network
How can proteins be secreted?
- Regulated secretion
- Constitutive secretion
What can happen to proteins not secreted?
Sorted into lysosomes
How are proteins sorted into lysosomes?
They bud off from the trans-Golgi network in a transport vesicle, then fuse with an endocytic vesicle to form a late endosome, which then turns into a lysosome
What is in endocytic vesicles?
Material to be broken down
What kind of cells tend to carry out regulated secretion?
Specialised cells;
- Endocrine cells
- Exocrine cells
- Neurocrine cells
What do endocrine cells secrete?
Hormones
What do exocrine cells secrete?
Digestive enzymes
What do neurocrine cells secrete?
Neurotransmitters
Give an example of a polarised secretory cell
The pancreatic acinar
What is true of a polarised secretory cell?
It is very organised
How is a polarised secretory cell organised?
Golgi next to nucleus, secretory vesicles near apical surface of cell
Why is it important that products are secreted at apical surface, not basal, in a polarised secretory cell?
Because the cell is often secreting digestive enzymes
What are proteins with signal peptides attached termed?
Pre-
What happens once a pre- protein is cleaved?
It looses the term pre-
What cleave signal sequences?
Specific enzymes called signal peptidases
What do signal peptidases recognise?
Charged residues adjacent to signal sequences
What is a signal recognition particle (SRP)?
A multi-domain riboprotein
What does a SRP do?
Mediates 3-way association with SRP-receptor in ER, the ribosome and the signal peptide
What does a SRP particle consist of?
A single RNA molecule of ~300 bases long, in complex with 6 different proteins
How are the proteins in SRP named?
P(number), based on the proteins molecular weight
What does P54 in SRP do?
Binds to signal sequences present in secretory proteins
What do other proteins, for example P9 and P14, in SRPs do?
Important for interacting with ribosomes
Describe the process of synthesis of secretory proteins and their translocation across the ER membrane using SRPs
- As protein synthesised, signal sequence protrudes from ribosome- is exposed.
- SRP recognises protruding signal sequence, stopping translation
- SRP binds to signal sequence, and is therefore attached to the ribosome
- SRP is recognised by receptor present in ER membrane
- Hydrolysis of GTP bound to the receptor and the SRP opens the translocon
- Signal peptidase cleaves signal sequence in ER
- Translation continues into ER lumen until finished
Why does the SRP stop translation?
To prevent synthesis in the cytoplasm, as it should be inserted into the secretory pathway
What is the receptor that binds to SRP composed of?
α and ß unit sub unit
What happens when a sub-unit of the receptor and a SRP has GTP bound?
It increases the affinity of a ribosome-SRP complex to receptor, and so binds easier
Why must the translocon remain closed when idle?
Otherwise important things such as calcium ions may leak out
What happens once translation is finished?
The ribosome detaches, the translocon closes and the folded protein is in the lumen
What is insertion into the ER membrane required for the delivery of?
Membrane proteins destined for plasma membrane or internal membrane of secretory pathway
What is the mechanism for delivery of N-terminal signal sequence to translocator the same as?
For a secretory pathway
What exists in the case of a type 1 membrane protein?
A second hydrophobic sequence
What is the purpose of the second hydrophobic sequence in a type 1 membrane protein?
To anchor the protein in the membrane, and prevents further transfer into the ER lumen
What is thesecond hydrophobic sequence in a type 1 membrane protein called?
A stop transfer anchor sequence
What happens once a stop transfer anchor sequence has been implanted into the membrane?
Synthesis continues in cytoplasm until a stop codon is reached
What are the functions of the endoplasmic reticulum?
- Insertion of proteins into membranes
- Specific proteolytic cleavage
- Glycosylation
- Formation of S-S bonds
- Proper folding of proteins
- Assembly of multi-subunit proteins
- Hydroxylation of selected Lys and Pro residues
What is glycosylation of proteins important?
- Correct protein folding
- Protein stability
- Facilitates interactions with other molecules
What may happen to protein stability without glycosylation?
Half life may be reduced
What can happen if theres deficiencies in N-linked glycosylation?
Severe human diseases
Where are sugars added in N-linked glycosylation?
Sugars are added on a asparagine side chain
What does the glycosylation reaction involve?
An amino group
Can every asparagine be glycosylated?
No, needs to be [X]-[Ser/Thr]
Why does the asparagine need to be[X]-[Ser/Thr] to be glycosylated?
To be recognised by the enzyme that transfers oligosaccharides
How are oligosaccharides made?
Assembled in ER on lipid carrier, then transferred to asparagine on growing protein
What does the oligosaccharide consist of?
3 glucose, 9 mannose and 2 N-acetylglucosamine
Where does glycosylation occur?
In ER
Where is the oligosaccharide preassembled?
A lipid carrier (dolichol)
What happens once the oligosaccharide has been transferred onto the protein?
Extensive modification in the ER and Golgi, by trimming and addition of further sugars
What do peptidyl-prolyl isomerases do?
Accumulate the interconversion of cis and trans isomers of proline residues
Why is proline different from other amino acids?
It has a rigid structure
Where can proline rotate?
Around the peptide bond
What is the result of the ability of proline to rotate around the peptide bond?
Gives rise to cis and trans forms
When does the interconversion of cis and trans proline need to occur?
During the folding reactions in a number of proteins, particularly IgGs
Why are peptidyl-prolyl isomerases useful industrially?
They greatly facilitate rapid protein folding
What is formed when 2 thiol side chains are oxidised?
Disulphide bonds
Give the equation for the formation of disulphide bonds?
-SH + HS- → -S-S- + 2e - + 2H +
Where is disulphide bond formation important?
Holding sub-units together, and correct folding of proteins
What catalyses the formation of disulphide bonds?
Protein disulphide isomerase (PDI)
What does the oxidised PDI have?
A disulphide bond in it’s active site
How does PDI work?
It can transfer electrons from reduced substrate protein to that disulphide bond to generate a disulphide bond in the substrate protein, producing reduced PDI and the oxidised substrate
What will have happened to proteins with disulphide bonds?
They will have passed through the ER
What kind of proteins tend to have disulphide bonds?
Extracellular or secreted proteins
What does the KDEL receptor do?
Forms a transmembrane link between ER-resident proteins and the COPI-coat
How does a soluble ER resident protein differ from secreted proteins?
By KDEL sequence before the -COOH
What is the KDEL sequence?
Lys-Asp-Glu-Leu
What does the KDEL sequence do?
Bind to the KDEL receptor
Where are soluble ER resident proteins synthesised?
ER lumen
What happens to the soluble ER resident protein once it’s been synthesised?
- Vesicles bud off from ER, containing the protein, and transported to cis-golgi
- In the cis-golgi, KDEL containing proteins are recognised by specific receptors, causing it to bud off, and be returned to RER
Why must soluble ER resident proteins be returned to ER after going to cis-golgi?
Don’t want it to get any further and get secreted
How is the KDEL cycle regulated?
Regulated binding due to pH
How does KDEL binding regulation due to pH work?
Cis-Golgi is more acidic that the ER, so under these conditions, KDEL containing proteins have a higher affinity for the receptor, enabling it to bind and then be released when it returns to the ER
What happens if there are folding problems?
- Protein may be trapped in mis-folded conformation
- Protein may be incorrectly associated with other sub-units
What attempts to correct folding problems?
ER chaperone proteins
Give 3 examples of chaperone proteins?
- BiP
- Calnexin
- Calreticulin
What does BiP do?
Binds to exposed amino acid sequences that would normally be buried in the interior of a folded protein
What does calnexin and calreticulin do?
Binds to oligosaccharides on incompletely folded proteins
Where are chaperone proteins important?
For quality control
What are the functions of chaperones?
- Retain unfolded proteins in the ER to prevent secretion
- Acts as sensors to monitor the extent of protein misfolding
- Mediate increased transcription of chaperones
- Mediate reduction in translation
What happens if misfolding can’t be corrected?
NAME?
What are the two mechanisms by which protein misfolding can cause disease?
- Mutations resulting in disposal
- Misfolding causing retention
How can misfolding causing retention cause disease?
- Gain of toxic function
- Loss of function
What occurs in the cis-Golgi network?
NAME?
What occurs in the cis cisterna of Golgi stack?
Removal on Man
What occurs in the medial cisterna of Golgi stack?
NAME?
What happens in the trans cisterna of the Golgi stack?
- Addition of Gal
- Addition of NANA
What happens in the trans Golgi network?
- Sulfation of tyrosines and carbohydrates
- Sorting → lysosome, plasma membrane or secretory vesicle
What do the lysosomes need to have?
The correct components of enzymes to carry out degradative functions
What does delivery of lysosomal enzymes to the lysosome require?
A mannose-6-phosphate signal
What happens as a lysosomal enzyme passes through the Golgi apparatus?
It’s recognised by enzymes, and a phosphate group is added to the hydroxyl group on carbon 6 of a mannose sugar
What enzymes does the addition of a phosphate group to the mannose sugar on lysosomal enzymes require?
- N-acetylglucosamine phosphotransferase
- A phosphodiesterase
What does the glucosamine phosphotransferase do?
Recognises a signal patch (not a linear sequence)
What does the phosphodiesterase do?
Removes N-acetylglucosamine, to leave phosphate in 6-position on mannose sugar
Give the overall equation for to addition of a phosphate group to lysosomal enzymes
UDP-GlcNAc + lysosomal enzyme → UMP + GlcNAc + M6p-tagged lysosomal enzyme
What is a I-cell disease?
A fatal inherited condition whereby there is a deficiency in N-actyl glucosamine phosphotransferase
What happens in I-cell disease?
The lysosomes become bloated with undegraded material
What does the mannose-6-phosphate receptor mediate?
The delivery of lysosomal enzymes from the trans-Golgi network to the lysosomes
Where are lysosomal enzymes modified?
In the trans-Golgi network
What happens when lysosomal enzymes get to the trans-Golgi network?
It binds to the M6P receptor
What happens when the M6P receptor binds to lysosomal enzymes?
It initiates the formation of the clathrin coat, which causes the vesicles to pinch off
What happens when the vesicles with lysosomal enzymes have pinched off?
It undergoes receptor-dependent transport, until it fuses with the late endosome
What happens when the vesicle has fused with the late endosome?
The enzyme dissociates from the receptor at the acidic pH
What happens to the receptor when the enzyme has dissociated?
It pinches off into the transport vesicle, and is recycled back to the trans golgi network
How is a mature lysosomal hydrolase formed?
The phosphate group is removed from the enzyme
What is formed once the enzyme becomes a mature lysosomal hydrolase?
The late endosome becomes a lysosome
What is O-linked glycosylation?
Attachment of sugar to -OH group
Where does O-linked glycosylation occur?
In the Golgi
Where is the sugar attached in O-linked glycosylation?
To hydroxyl group of serine and threonine
Where is O-linked glycosylation important?
In proteoglycans
What are proteoglycans?
A component of the extracellular matrix and mucus secretions
How may proteins be delivered to the plasma membrane?
Via a regulated or constitutive pathway
What happens to proteins destined for either unregulated secretion of constituent secretion?
Vesicles bud off from the trans-Golgi network containing the proteins
How do proteins for constitutive secretion enter the extracellular space?
Unregulated membrane fusion
Other than proteins for constitutive secretion, what else uses the same vesicles?
Newly synthesised plasma membrane proteins and lipids
What happens to proteins for regulated membrane fusion?
They bud off into secretory vesicles, and remain there until a signal such as a hormone or neurotransmitter are released into the intracellular signalling pathway. The proteins are then released into the extracellular space by regulated membrane fusion
What is the most abundant protein in the body?
Collagen
Where is collagen found?
- Tendons
- Ligaments
- Cartilage
- Bone
- Loose connective tissue
What is the purpose of collagen in loose connective tissue?
It provides structure to internal organs
What produces collagen?
By fibroblasts in connective tissue
What is the basic unit of collagen fibres?
Tropocollagen
Describe the shape of tropocollagen?
300nm rod-shaped protein
What does tropocollagen consist of?
3 polypeptides (α-chains), each ~1000aa long
What is the important feature of the primary structure of collagen?
There is a glycine at every 3rd position along each α-chian
Where is the Gly-X-Y repeat important?
In assembly of collagen polypeptide
What shape to the α-chains of collagen form?
Triple helix
What does the glycine at every 3rd position allow in collagen?
The 3 α-chains the assemble together into the triple helix, as glycine is a very small amino acids
How is the collagen triple helix stabilised?
Hydrogen bonding between different α-chains
What are the features of tropocollagen’s structure?
- Non-extensible
- Non-compressible
- High tensile strength
What does tropocollagen have at the X and some Y positions?
Mostly proline or hydroxyproline
What is the result of proline being in the tropocollagen structure?
The secondary structure can’t be an α-helix, as proline as a helix breaker
What do different α-chains assemble to make up?
Different collagens in different parts of the body
What does different combinations of α-chains produce?
Collagens with different functions
What appearance does collagen have?
Regular, repeating grooves
What produces the regular repeating grooves in collagen?
The assembly of tropocollagen within collagen
How is collagen synthesised and modified in the ER?
- Chain synthesised and enters lumen of ER
- Signal peptide cleaved as it enters the ER lumen
- Selected proline and lysine residues are hydroxylated
- N-linked oligosaccharides added
- Addition of galactose to hydroxylysine residues
- Chain alignment
- Formation of triple-helical procollagen from C- to N- terminus
- Completion of O-linked oligosaccharide chains by addition of glucose
- Procollagen buds off into transport vesicle
- Vesicle fuses with plasma membrane
- Protein released into extracellular space, N and C terminal propeptides removed
- Lateral association of collagen molecules followed by covalent cross-linking
- Aggregation of fibrils to make collagen fibre
What happens when the signal peptide of tropocollagen is cleaved in the ER?
Propro α-chains are converted into pro α-chains
What catalyses the hydroxylation of proline and lysine residues?
Prolyl hydroxylase
What does prolyl hydroxylase require for function?
- Vit C, as cofactor
- Fe 2+
What is prolyl hydroxylase associated with?
PDI in ER
What does prolyl hydroxylase allow?
H-bonding to stabilise triple helix
How does prolyl hydroxylase allow increased H bonding?
Adding extra hydroxyl groups on proline that are able to form H bonds
What is scurvy due to?
Weak tropocollagen triple helices
What is galactose added to hydroxylysine residues involved in?
Interchain interactions
Where is chain alignment stabilised?
At C terminal domain
How is the chain alignment stabilised?
By formation of disulfide bonds
How much of the procollagen doesn’t form the triple helix?
- 150 N-terminal amino acids
- 250 C-terminal amino acids
What are the extra N- and C- terminal amino acids that don’t form the triple helix important for?
Assembly of the tropocollagen unit
What happens when the vesicle containing procollagen fuses with the plasma membrane?
It undergoes exocytosis
How is procollagen converted into tropocollagen?
By action of procollagen peptidases
What will happen when the N and C terminals of procollagen are removed?
Tropocollagen will be able to assemble with other units
What is the purpose of covalent cross-linking of tropocollagen molecules?
For stability
Why is it important that aggregation of tropocollagen fibrils is tightly regulated?
Because if cleavage of N and C terminal peptides too early, could assemble in cell
How are tropocollagen’s cross linked?
Two lysine residues in different tropocollagen units are covalently linked
How are two lysine residues covalently linked?
By action of lysyl oxidase and addition of oxygen, which forms two aldehyde derivatives, which spontaneously link to form a aldol cross link
Where is lysyl oxidase found?
Extracellular
Why is it important that lysyl oxidase is extracellular?
Prevents covalent cross linking of collagens within the cell
What does lysyl oxidase require for activity?
- Vitamin B6
- Cu 2+
What causes Ehlers-Danlos syndrome?
A mutation in collagen type V or a lysyl oxidase deficiency
How is insulin secreted?
As proinsulin
How many polypeptides is proinsulin made up of?
1
What must happen to proinsulin in order for it to fold properly?
Form 3 disulphide bonds- 2 interchain, one internal
Where does the formation of disulphide bonds in proinsulin occur?
In the ER
What must happen for proinsulin to become activated?
The connecting C peptide is removed, leaving complete 2 chain insulin molecule
Where does proinsulin activation occur?
Post-Golgi
What enzymes does proteolytic processing of insulin require?
- PC3 endoprotease
- PC2 endoprotease
- Carboxypeptidase
What can proteolytic processing yield?
Different products
How can proteolytic processing yield different products?
Because there are different amounts of processing enzymes in different cell locations
Why is proteolytic processing so common in secretory pathways?
- Can give rise to very small products that would be too short to enter ER via co-translational mechanism
- Some secreted proteins would be destructive if activated inside the cell
- Multiple bioactive products can be produced from some polypeptides
- Avoids activation of insulin receptor in the secretory pathway
