11 Protein Targeting

Question 1

How does newly translated polypeptide enter ER originally? (9)

Answer 1

1. mRNA molecule and ribosome
2. Hydrophobic N terminal produced
3. Signal Recognition Particle binds signal sequence
4. Protein synthesis stops
5. SRP directs ribosome to SRP receptor on cytosolic face of ER
6. GTP => GDP + Pi, SRP dissociates, translocon opens
7. Protein synthesis continues and newly formed polypeptide fed into ER via pore in membrane (Translocon)
8. Signal peptidase cleaves signal sequence
9. Ribosome dissociates and is recycled, folded protein forms

Question 2

What modifications occur within the ER?

Answer 2

1. Signal peptidase - cleavage of signal
2. Protein disulphide isomerase - disulphide bonds made
3. N-linked glycosylation - sugar groups added on N of R groups

Question 3

What modifications occur within the golgi?

Answer 3

1. O linked glycosylation - add sugar onto O group of R group
2. Proteolytic processing - removal of pro in proinsulin e.g.

Question 4

How is a polypeptide targeted to nucleus?

Answer 4

1. Sequence of AA form Nuclear Localisation Signal (NLS) Arg+Lys
2. In the cytosol, importin binds cargo protein containing NLS
3. Complex binds to nuclear pore and translocates into nucleus
4. Ran-GTP binds to importin causing a conformation change displacing the cargo protein (which is left in the nucleus)
5. Importin with bound Ran GTP is recycled to cytoplasm

Question 5

How is a polypeptide targeted to mitochondria?

Answer 5

1. Amphiphatic N terminal signal - MTS
2. Signal sequence recognised by proteins TOM on outer membrane
3. Protein, with signal kept unfolded by chaperone proteins, fed through TOM
4. Protein moves through adjacent inner membrane by TIM
5. Mitochondrial peptidase removes N-terminal sequence
6. ATP dependent folding with help from chaperones

Question 6

How is a polypeptide targeted to a lysosome?

Answer 6

1. Addition of mannose 6 phosphate (M6P) in cis golgi
2. On N linked oligosaccharides on protein
3. Recognised by M6P receptor in trans golgi
4. Vesicles buds off, coated in Clathrin coat, to lysosome
5. At lysosome, acid pH causes receptor and protein to dissociate
6. Receptors recycled back
7. Acid phosphatase enzyme cleaves part of signal complex
   - phosphate group from mannose
   - destroys sorting signal and contributes to m6p release

Question 7

How is a polypeptide retained in the ER?

Answer 7

1. Normal translation into ER
2. Leaves ER in coated COP2 vehicle
3. Enters golgi and is processed
4. KDEL sequence at C-terminal sequence (Lys-Asp-Glu-Leu-COOH) recognised by KDEL receptor at golgi (low pH)
5. Protein secreted from golgi coated in COP1
6. COP1 sends vesicle back to ER
7. KDEL receptor lost and returned back to golgi because increase in pH at ER

Question 8

As lysosomal enzyme passes through golgi apparatus, phosphate group added to hydroxyl group of carbon 6 of mannose sugar. What is the name of the two enzymes required for this?

Answer 8

1. N-acetyl glucosamine phosphotransferase

2. Phosphodiesterase

Question 9

Give an inherited condition related to lysosomes?

Answer 9

1. I-cell disease
2. Deficiency in N-acetyl glucosamine phosphotransferase
3. Proteins instead excreted outside cell
4. Lysosomes cannot function without these and become bloated with undegraded material

Question 10

Give a disease occurring by mutation of nuclear localisation signal

Answer 10

1. Swyer syndrome
2. Loss/mutation in NLS in sex determining region
3. XY genotype but outwardly female