Lecture 2 - Protein Maturation, Sorting + Turnover Flashcards
1
Q
Outline the role of chaperons in protein maturation.
A
- Some proteins require the assistance of chaperons to fold correctly, these can:
- Stabilise folding intermediates.
- Maintain proteins in the unfolded state to allow passage through membranes.
- Help unfold + refold misfolded segments.
- Prevent the formation of misfolded intermediates.
- Prevent aggregation + inappropriate interactions with other proteins.
- Chaperons bind reversibly to hydrophobic regions of nascent proteins in an intermediate state of folding.
2
Q
Protein Sorting: Where are proteins synthesised by free cystolic ribosomes destined for?
A
- Cytosol
- Nucleus
- Mitochondria
- Other organelles
3
Q
Protein Sorting: Where are protein synthesised by membrane bound ribosomes destined for?
A
- Cell membrane
- Exportation from the cell
- These proteins have an N-terminal hydrophobic signal sequence added to them by mRNA
4
Q
Outline the process of initiation + elongation (10).
A
- Initiation + elongation starts on the free cytosolic ribosomes + as the signal peptide emerges from the ribosome, it binds to a signal recognition particle (SRP).
- SRP = 6 different proteins + a small 7S RNA molecule.
- Translation halts → complex moves to the ER.
- SRP binds to an SRP receptor on the cytosolic surface of the ER membrane.
- Ribosome is transferred to a translocon – a ribosome receptor that crosses the membrane.
- SRP is released → translocon pathway opens to allow transit of nascent protein.
- Concomitant translation + extrusion through the translocon into the ER lumen.
- Signal peptide is excised by a signal peptidase.
- Protein folds processed further if necessary through the Golgi.
- Protein packed into secretory vesicles → delivered to the cell membrane
5
Q
Post-translational protein modifications: Outline N-linked glycosylation (8).
A
- The attachment of an oligosaccharide to a protein through the amine nitrogen of asparagine (Asn).
- Usually occurs at available Asn-X-Thr sequences (X = any amino acid residue except Pro or Asp).
- Starts with a lipid-linked dolichol-phosphate at the cytosolic surface of the ER membrane – serves as a glycosyl acceptor N-acetyl glucosamine (GlcNAc).
- A branched (GlcNAc)2-(Man)5-pyrophosphoryl-dolichol is formed by the stepwise addition of mannose molecules (Man).
- Intermediate is reoriented into the luminal surface of the ER membrane.
- 4 addition Man and 3 glucose residues are added using enzyme Gtfs.
- Complete oligosaccharide is transferred to an Asn residue of the polypeptide as it emerges into the ER lumen.
- Is a co-translational process: it occurs as the protein is synthesised + can affect its folding.
6
Q
Post-translational protein modification: Outline O-linked glycosylation (3).
A
- Oligosaccharides attached to proteins through hydroxyl groups of serine (Ser) or Threonine (Thr) residues.
- Occurs after the folded protein has reached the Golgi apparatus → a post-translational process.
- Does not require specific amino acid sequences, can also occur at available Ser or Thr residues on the protein surface.
7
Q
What varies the amount of glycosylation?
A
- The amount of glycosylation varies depending on the types and amounts of glycotransferases in different cells, hence identical proteins may exhibit variable glycosylation, giving rise to protein heterogeneity.
- The same protein differently glycosylated will have different properties.
8
Q
Outline the events of the protein from the ribosome to the cell membrane (5).
A
- Complete folded N-glycosylated protein is released into the ER lumen were glycosylase enzymes remove the glucose residues.
- In the Golgi, glycosyl transferase enzymes link one or two GlcNAc-Phosphate residues to the oligosaccharide.
- Glycosylase enzyme removes the GlcNAc residues to leave behind phosphorylated Mannose residues.
- Phosphorylated glycoprotein binds to the mannose 6-phosphate receptor embedded in the Golgi membrane → buds out to form the lysosome which is transported to the membrane.
- At the membrane, the lysosome will fuse and either donate the protein to the membrane or it is released from the cell.
9
Q
What is the cause of inclusion-cell disease (2) and what are the symptoms/consequences (5)?
A
Causes:
- Patients lack the GlcNAc-P glycotransferase à cannot transfer GluNAc-P to the high-mannose type oligosaccharides of proteins destined for lysosomes à cannot mark lysosomal enzymes for their destination + secretion from the cell.
- Fibroblasts from patients show abnormal inclusion bodies + lack of lysosomal enzyme i.e. they aren’t synthesised in the first place.
Symptoms/consequences:
- Psycomotor retardation.
- Skeletal abnormalities.
- Restricted joint movement.
- Coarse facial features.
- Death by age 8.
10
Q
Outline the process of importing protein into the mitochondria (5).
A
- Most mitochondrial proteins are synthesised by ribosomes in the cytosol + must be imported in.
- N-terminal pre-sequences mark proteins for this.
- Targeting signal is not a specific sequence but a +ve charged amphiphilic a-helix recognised by a mitochondrial receptor.
- Protein is simultaneously transported/unfolded with the aid of chaperon translocators through both membranes (energy-dependent).
- Proteases in the mitochondrial matrix remove the presequence either completely (matrix protein) or partially (retargeted back to membrane).
11
Q
Outline the process of importing protein into the nucleus (4).
A
- Imports proteins synthesised in the cytosol.
- Targeted for transport by nuclear localisation signals (NLS) which include clusters of basic amino acid sequences.
- NLSs interact with carrier proteins (importins) that transport them through cylindrical nuclear pore complexes.
- GTP hydrolysis releases the protein from the carrier protein which then returns to the cytosol.
12
Q
Name the 10 post-translational protein modifications.
A
- Hydroxylation.
- Sulfation.
- Methylation.
- Phosphorylation.
- Glycosylation.
- Acyl lipidation.
- Acetylation.
- Proteolysis.
- Prenylation.
- Disulphide bonds.
13
Q
Compare and contrast the function of sulfatases normally and in sufferers of multiple sulfatase deficiency (4+5).
A
Normally:
- Sulfatases are activated by a post-translational modification.
- Cysteine residue → Ca-formylglycine.
- Essential for their enzymatic activity.
- Most sulfatases are located in lysosomes.
Sufferers:
- Rare lysosomal deficiency means sufferers cannot catalyse the Cys modification to activate their sulfatases.
- Sulphated molecules cannot be de-sulphated leading to sulphate accumulation in the body:
- Physical deformities.
- Neurological deficiencies.
- Death by age 10.
14
Q
Protein turnover: How can proteins be damaged? (5)
A
Common:
- Oxidation
- Proteolysis
- Denaturation
- Irreversible modifications
Rare:
- Errors during translation and/or folding gives a non-functional protein.
15
Q
How are ‘trash proteins’ removed?
A
Proteolysis in the proteasome system.
