Protein targeting Flashcards
What is the consituitive secretory pathway? Name a protein in this pathway?
- Continual exocytosis of protein packed vesicles by unregulated fusing with the membrane
- Albumin
- Collagen
What are the three possible outcomes for proteins synthesised?
- Secreted
- Membrane bound
- Free in cytosol
What is the regulatory secretory pathway? Name a protein secreted in this way
- Proteins packed into vesicles and released in response to a stimulus
- eg insulin
Describe the secretory pathway up until the golgi
1) Free ribosome initiates translation from a mature mRNA molecule
2) Hydrophobic N-terminal sequence is produced and protrudes out of ribosome
3) Signal sequence of newly formed protein is recognised and bound by signal recognition particle
4) Protein synthesis stops
5) SRP directs the ribosome towards the SRP receptors on the cytosolic face of the ER
6) SRP binds to SRP receptor and the newly formed polypeptide is translocated into ER lumen;SRP dissociates
7) Protein synthesis continues
8) Signal sequence is removed by peptidase once the polypeptide is full produced
9) Polypeptide is modified and packaged for secretion to the golgi
List some modifications which occur within the ER and golgi
- Glycosylation
- Hydroxylation
- Folding and assembly
- Trimming
- DSB formation
- Signal cleavage
Describe N-linked glycosylation
- a lipid carrier protein which is embedded in the membrane has phosphate group protruding, to which an oligosaccharide is attached
- Oligosaccharide is transferred from the phosphate to the amide asparagine by oligosaccharide-protein transferase
Describe O-linked glycosylation
-Sugar is added to the hydroxyl group of serine or threonine by glycosyl transferase
Describe insulin synthesis
- Insulin synthesised as preproinsulin by ribosome; one polypeptide chain with a N terminal signal sequence
- Translocaiton to rER and cleavage of the signal sequence peptide, and formation of 3 DSB to ensure correct folding produces proinsulin
- Proinsulin is transferred to the golgi
- Proinsulin is then further cleaved by specific endonucleases to remove C peptrides and remnants produce two separate peptides, held together by 2 DSB with another DSB on the a-chain for stability
Why is C peptide important in diabetics?
-Can be used as a marker for exogenous insulin production
What are the three main features of collagen fibres?
- Non-extensible
- Non-compressible
- High tensile strength
What is the basic structural unit of collagen and what is it’s structure?
- Tropocollagen
- (Glycine-X-Y)n
Which a’a are usually found at the X and Y positions?
- Proline
- Hydroxyproline
Why is proline important in the structure of collagen?
-Allows the non-extensible conformation and encourages no other conformation
What is the advantage of hydroxyproline in collagen?
-Allows more H bonds to be made stabilising the structure
What position is glycine in collagen?
-Every 3rd position
How is hydroxyproline made?
-Hydroxylation of proline via prolyl hydroxylase
What is the structure of collagen?
-3 left handed a-chain polypeptides which form a right-handed helix stabilised by hydrogen bonds
What does prolyl hydroxylase require to function?
- Vitamin C
- Fe2+
What happens in scurvy?
- Lack of vitamin C
- Prolyl hydroxylase does not function properly
- Proline residues not hydroxylated
- Reduction in H bonds between polypeptide chains of Collagen
- Produces weak collagen
Describe the synthesis of collagen
1)Preprocollagen synthesised in ribosome
2)Translocated to ER and signal sequence cleaved producing procollagen
3)Hydroxylation of selected proline/lysine residues
4)N-linked Glycosylation and galactose added to hydroxylysine
5)Alignment of procollagen subunits and DSB formation at C terminal initiates folding of helices into procollagen helix
6)C and N terminal peptides not incorporated in helix to prevent aggregation of subunits inside cell
7)Translocation to the golgi
8)O linked glycosylation and modifying
9)Packaged into vesicle
10)secreted via constitutive pathway
11)Procollagen peptidases cleave C and N terminal peptides producing tropocollagen units
12)Lysine residues converted to aldehyde derivative allowing crosslinks between subunits
130 Tropocollagen units assemble into collagen fibrils stabilised by H bonds and cross-links and bundles of fibrils form collagen fibres
What catalyses the formation of aldehyde derivatines from lysine?
-Lysyl oxidase with cofactors Vit B6 and Cu2+
What disease occurs when lysyl oxidase is deficient?
-Ehlers-Danos syndrome
Why does collagen appear striated?
-Tropocollagen subunits are staggered and when they align they exclude stains
Describe nuclear targeting
1) Fully folded protein has signal sequence (about 4-8a’a) known as nuclear localisation signal
2) Importin recognises NLS and binds to protein
3) Protein:importin complex is translocated through nuclear pore
4) GTPase ran displaces protein and binds to importin causing translocation back to the cytosol
5) GTP hydrolysis causes Ran to dissociate from importin and re-enter the nucleus
Describe mitochondrial targeting
1) Unfolded protein has an ampipathic N-terminal signal sequence and is stabilised by a chaperone protein
2) Signal sequence is recognised by receptor located on outer mitochondrial membrane (TOM) resulting in import through a pore
3) Proteins destined for inner matrix are recognised by TIM and enter through a pore
4) If destined for embedding and not entering matrix, protein has a signal sequence to stop entering the matrix
5) Signal sequence is cleaved and the protein takes its fully native folded form
Describe lysosomal targeting
1) lysosomal hydrolases have mannose-6-phosphate added to an N-linked oligosaccharide in golgi
2) M6P recognised at trans golgi and packaged into vesicle with receptor
3) Vesicle pinched off for transport to lysosome
4) Vesicle receptor recognised by lysosome forming a pore and acid pH causes receptor differentiation and protein release
Name a disease caused by a defect in lysosomal targeting
-Chondrodysplasia
What is I cell disease?
- Lack of m6p addition onto lysosomal hydrolases
- Hydrolases mistargeted for secretion
What mechanism keeps resident ER proteins within the ER?
- Proteins which are supposed to be resident in the ER, such as protein disulphide isomerases, can sometimes be pinched off in vesicles and transported to the golgi
- These proteins have a KDEL sequence recognised by KDEL receptors in the golgi
- This causes their translocation back to the ER, with the KDEL receptor returning back to the golgi, enhanced by the pH gradient between the golgi and ER
