Protein Targetting Flashcards
How do most proteins achieve their final conformation?
Themselves
State 2 additional post-modifications of proteins after translation, and what they entail.
- proteolytic cleavage - breaking peptide bonds to remove part of the protein
- chemical modification - addition of functional groups to amino acid residues
What is the peroxisome targeting sequence? What does addition of this mean?
SKL (serine - lysine - leucine) - addition of this will target a protein to the peroxisome
What is the peroxisome targeting sequence? Which end of a protein is this sequence added?
SKL (serine - lysine - leucine) - this sequence is added to the C-terminus end of a protein (towards the end)
Once tagged, how does a protein transverse the peroxisome membrane?
Through a transport channel comprised of 13 PEX proteins - this transfer requires ATP hydrolysis
What must the protein-targeting sequence bind in order to move the protein through the PEX transport channel in the peroxisome membrane?
A PEX5-cargo complex
Failure to target proteins to peroxisomes can lead to abnormal peroxisomes - what biological affects may this have? List 1 disease associated with abnormal peroxisomes.
Peroxisomes break down fatty acids - if this function is disturbed fatty acids may accumulate which can impair neuronal function - long bone shortening may occur as a result of abnormal protein targeting to peroxisomes - Zellweger syndrome is associated with abnormal protein targeting to peroxisomes
What is constitutive secretion?
Secretion that is continual (occurs all the time)
Describe the conformation and composition of the sequence that is added to molecules that require secretion from the cell.
The sequence is added to the N-terminus of the protein, and is around 5-30 amino acids in length - the central part of this signalling sequence is composed mainly of hydrophobic residues, which form an alpha helix in solution
What is required to target a protein to the ER for secretion? How is this molecule composed?
The signal recognition particle recognises the targeting sequence and moves the targeted molecule to the ER - it is composed of 6 subunits and a small stretch of RNA
What organelle does the signal recognition particle direct a protein with a target sequence and an associated ribosome towards?
The ER
Does binding of the signal recognition peptide instigate or inhibit translation of the RNA molecule associated to a ribosome?
The signal recognition particle prevents translation, while it directs the free ribosome to the surface of the ER
How does a type I membrane protein, designed to reside in the membrane of the ER, stop itself from being completely excluded into the lumen?
It contains a stop-transfer anchor sequence - an alpha-helical membrane-spanning sequence
List 7 functions of the endoplasmic reticulum.
- proteolytic cleavage
- insertion of proteins into membranes
- glycosylation
- formation of disulphide (s-s) bonds
- proper folding of proteins
- assembly of multi-subunit proteins
- hydroxylation of selected lysine and proline residues
What is N-linked glycosylation?
The addition of sugars (a glycan) to the amide nitrogen of an asparagine residue of a protein
Give 3 reasons why glycosylation is important.
- correct folding of proteins
- give a protein stability
- facilitates interactions with other molecules
Between what 2 residues does a disulphide bond form?
Between 2 cysteine residues
What is the role of protein disulphide isomerase?
Protein disulphide isomerase ensures that the correct disulphide bonds are formed - it can remove wrong ones and correct them
What is O-linked glycosylation? Where specifically does this occur?
O-linked glycosylation is the attachment of a sugar molecule to the O of an OH- (hydroxyl) group of a serine or threonine residue - this occurs specifically in the Golgi apparatus
Describe the processing of preproinsulin.
The N-terminal signal sequence is cleaved as the protein enters the ER lumen - a pair of disulphide bonds form between the a & c chain within the ER lumen, causing the b chain to bend - the b chain is then cleaved in the Golgi apparatus, to form insulin
In which molecules is O-linked glycosylation an important characteristic?
Glycoaminoglycans (GAG’s)
What is the opposite of constitutive secretion? Give 3 examples.
Regulated secretion:
- endocrine secretion
- exocrine secretion
- paracrine secretion
What cell type secretes collagen?
Fibroblasts
What is the basic unit of collagen? Describe its structure.
The basic unit of collagen is procollagen - it is a 300nm rod-shaped protein, consisting of 3 alpha chains, which are each around 1000 amino acids long - a glycine residue is present every 3rd amino acid along each alpha chain
What is special about the properties of glycine that it features so prominently in procollagen/collagen?
Glycine is the amino acid with a side chain small enough to fit into the middle of the helix form by the 3 alpha subunits
What other amino acids are associated with the structure of procollagen/collagen? How are these important?
Proline and/or hydroxyproline - these allow the formation of hydrogen bonds between the alpha subunits, which give procollagen structural stability
What molecule brings procollagen to the ER? Where is the signal peptide then cleaved?
The signal recognition particle binds the signal sequence of pre procollagen and brings it (and its associated ribosome) to the ER - the signal sequence is then cleaved off within the lumen of the ER
Where does the hydroxylation of certain glycine and proline residues occur on a procollagen polypeptide?
In the lumen of the RER
What type of glycosylation does procollagen undergo in the ER?
Procollagen undergoes N-linked glycosylation in the RER
What is scurvy the result of?
Scurvy is the result of unstable connections between the alpha subunits the comprise a collagen helix - this is due to poor prolyl hydroxylase activity, which catalyses increased hydrogen bonding between the 3 alpha subunits
What is required by prolyl hydroxylase in order to function properly?
Iron ions and vitamin C
How does prolyl hydroxylase function?
Prolyl hydroxylase catalyses hydrogen bonding between the triple helix, meaning that collagen is more stable
What happens with a lack of prolyl hydroxylase?
Proline isn’t hydroxylated, so less hydrogen bonds form between the alpha subunits, meaning the helical structure of collagen is much weaker
Where and how are procollagen subunits first linked? What then forms to further increase their stability?
The 3 procollagen subunits are first linked in the lumen of the ER, via the formation of disulphide bridges at the C-terminal ends of the structures - hydrogen bonds are then formed along the chain increasing the stability of the triple helix
What type of glycosylation does procollagen undergo in the Golgi apparatus?
Procollagen undergoes O-linked glycosylation in the Golgi apparatus
Where is the next destination of procollagen after the Golgi apparatus?
Procollagen is endocytosed to the extracellular matrix in a vesicles from the Golgi apparatus
What defines the conversion of procollagen to tropocollagen?
The cleavage of the N-terminus and C-terminus - this leaves only the triple helix of tropocollagen
How do individual tropocollagen molecules combine to form collagen?
Tropocollagen fibrils aggregate to form collagen - lysine molecules cross-link to form aldol cross-links
Why is procollagen secreted before the final stage of processing occurs?
If collagen were constructed within the cell it would destroy it
What enzyme is responsible for the aldol cross-link formation between tropocollagen subunits? What is required for this enzyme activity?
Lysyl oxidase - this requires copper ions and vitamin B6 for optimal activity
What disorder may a deficiency in lysyl oxidase result in?
Elhers-Danlos Syndrome
Why must oxygen bad transporters to be transported around the body?
It is not very soluble in water
When transferred into the ER, what cleaves the signal sequence off a protein?
A signal peptidase
What happens to proteins that enter the default pathway of the secretory pathway?
They are packaged and stored in granules until the are stimulated for release
How do proteins designed to stay in the ER remain there, and not secreted?
A lys-Asp-Glu-Leu amino acid at the C-terminus of a protein ensures it remains within the lumen of the ER
What signal is used to direct a molecule to a lysosomes? Describe how and where this signal is added?
Mannose-6-phosphate - this is added to the 6th carbon of a mannose sugar on the protein within the Golgi apparatus
What 2 enzymes act to add a phosphate to the 6th position of a mannose sugar? Where will this post-translational modification target the protein?
N-acetyl glucosamine phosphotransferase & phosphodiesterase - the addition of mannose-6-phosphate targets a protein to a lysosome
What receptor transfers a protein tagged with mannose-6-phosphate to a lysosome? How do these dissociate once within the lysosome?
M6P receptor transfers a tagged protein to the lysosome - the acidity of the lysosome is enough to remove the receptor from the mannose sugar, releasing the protein
What is I-cell disease?
This results from a defective phosphotransferase - here the enzyme can no longer transfer phosphate to the 6th hydroxyl group of mannose, and so the protein isn’t targeted to a lysosome - without this signal, the proteins are instead excreted outside the cell - lysosomes cannot function without the majority of these proteins - substances build up within the lysosome (as they cannot be degraded) resulting in the characteristic I-cells
Describe the signal that is added to a molecule to target it to the mitochondria.
It is an amphipathic (one side is positively charged while the other is hydrophobic) alpha helical sequence added to the N-terminus
What keeps a protein unfolded while it is transported to a mitochondria? What else is required?
Hsp70 - ATP hydrolysis is also required
Describe how a protein targeted to a mitochondria is moved from the cellular cytoplasm to the mitochondrial matrix.
Chaperones (such as Hsp70), using ATP hydrolysis, move the protein to the mitochondria in its unfolded form - the signal then binds an import receptor on the mitochondria - the protein is passed through the TOM, and then secondly through the TIM - within the matrix, Hsp pull the protein through, again with the use of ATP - once in the matrix, the signal sequence has served its purpose, and is cleaved
Describe the signal that targets a protein to the nucleus.
A positively charged amino acid sequence consisting of arginine and lysine residues on the protein
How is a targeted protein moved from the cytosol to the nucleus?
The protein is bound by importin, which transfers it through the nuclear pore
Once in the nucleus, how is a molecule unloaded from importin?
A molecule called Ran-GTP configures a conformational change in importin causing it to release the targeted protein
Name 2 diseases that are the result of defects in targeting a protein to the nucleus.
- Swyer Syndrome
- Langer Mesomelic Displasia
How is a protein targeted for the ER retirement there from the Golgi apparatus? What mechanism allows for this protein to dissociate from its transporter?
A protein is coated in a COPI1 coat - this returns it to the ER - the here possesses a PLZ LOOK UP
