Protein targeting

Question 0

What is the consituitive secretory pathway? Name a protein in this pathway?

Answer 0

• Continual exocytosis of protein packed vesicles by unregulated fusing with the membrane
• Albumin
• Collagen

Question 1

What are the three possible outcomes for proteins synthesised?

Answer 1

• Secreted
• Membrane bound
• Free in cytosol

Question 2

What is the regulatory secretory pathway? Name a protein secreted in this way

Answer 2

• Proteins packed into vesicles and released in response to a stimulus
• eg insulin

Question 3

Describe the secretory pathway up until the golgi

Answer 3

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

Question 4

List some modifications which occur within the ER and golgi

Answer 4

• Glycosylation
• Hydroxylation
• Folding and assembly
• Trimming
• DSB formation
• Signal cleavage

Question 5

Describe N-linked glycosylation

Answer 5

• 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

Question 6

Describe O-linked glycosylation

Answer 6

-Sugar is added to the hydroxyl group of serine or threonine by glycosyl transferase

Question 7

Describe insulin synthesis

Answer 7

• 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

Question 8

Why is C peptide important in diabetics?

Answer 8

-Can be used as a marker for exogenous insulin production

Question 9

What are the three main features of collagen fibres?

Answer 9

• Non-extensible
• Non-compressible
• High tensile strength

Question 10

What is the basic structural unit of collagen and what is it’s structure?

Answer 10

• Tropocollagen

- (Glycine-X-Y)n

Question 11

Which a’a are usually found at the X and Y positions?

Answer 11

• Proline

- Hydroxyproline

Question 12

Why is proline important in the structure of collagen?

Answer 12

-Allows the non-extensible conformation and encourages no other conformation

Question 13

What is the advantage of hydroxyproline in collagen?

Answer 13

-Allows more H bonds to be made stabilising the structure

Question 14

What position is glycine in collagen?

Answer 14

-Every 3rd position

Question 15

How is hydroxyproline made?

Answer 15

-Hydroxylation of proline via prolyl hydroxylase

Question 16

What is the structure of collagen?

Answer 16

-3 left handed a-chain polypeptides which form a right-handed helix stabilised by hydrogen bonds

Question 17

What does prolyl hydroxylase require to function?

Answer 17

• Vitamin C

- Fe2+

Question 18

What happens in scurvy?

Answer 18

• 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

Question 19

Describe the synthesis of collagen

Answer 19

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

Question 20

What catalyses the formation of aldehyde derivatines from lysine?

Answer 20

-Lysyl oxidase with cofactors Vit B6 and Cu2+

Question 21

What disease occurs when lysyl oxidase is deficient?

Answer 21

-Ehlers-Danos syndrome

Question 22

Why does collagen appear striated?

Answer 22

-Tropocollagen subunits are staggered and when they align they exclude stains

Question 23

Describe nuclear targeting

Answer 23

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

Question 24

Describe mitochondrial targeting

Answer 24

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

Question 25

Describe lysosomal targeting

Answer 25

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

Question 26

Name a disease caused by a defect in lysosomal targeting

Answer 26

-Chondrodysplasia

Question 27

What is I cell disease?

Answer 27

• Lack of m6p addition onto lysosomal hydrolases

- Hydrolases mistargeted for secretion

Question 28

What mechanism keeps resident ER proteins within the ER?

Answer 28

• 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