Protein sorting and protein traffic

Question 1

Why is it important for proteins to be sorted to the correct compartments?

Answer 1

1. Cells are highly organised and compartmentalised
2. Different organelles contain different sets of proteins-Transcription factors
3. If not sorted correctly chemical chaos would result
4. Some proteins (enzymes) could be harmful to other parts of the cell.

Question 2

Where does protein synthesis occur?

Answer 2

1. In the cytoplasm.

Question 3

What happens to proteins once they are synthesised?

Answer 3

1. They can remain in the cytoplasm
2. They can be targeted into the nucleus
3. they can go to the ER for trafficking
4. be distributed to the mitochondrion.
5. Can produce own ribosomes
6. Go to peroxisomes
7. Go to Chloroplasts/plastids (plants)

Question 4

What are sorting signals?

Answer 4

1. Tell proteins where to go
2. Specific stretches of amino acids in proteins
3. Different sorting signals have different properties depending on destination in protein
   - Nucleus, Mitochondrial or Endoplasmic reticulum

Question 5

What are the three types of sorting signals?

Answer 5

1. Those responsible for sorting to the nucleus
   2, those to the mitochondria
2. those to the ER.

Question 6

What amino acids are important in nuclear targetting?

Answer 6

Lysine and Arginine.

Question 7

How does nuclear targeting work in Nucleoplasmin?

Answer 7

1. It has two stretches of positively charged amino acids that are close to each other when the protein is folded.
2. A positive patch forms that can be recognised by sorting machinery.
3. The stretches could be continuous nuclear localisation sequences or discontinuous nuclear localisation sequences

Question 8

How has the importance of the positively charged amino acids in nuclear targeting been shown?

Answer 8

1. Experiments show that theses sequences are involved in nuclear target
2. T-antigen with a single amino acid change were expressed in cells in culture.
3. Have normal protein
4. Have one with a mutation- one of lysine to threonine
5. Expressed mutated protein in cell- goes to cytoplasm instead of nucleus- no longer contains the correct import signal.

Question 9

What does it mean that the double membrane is contiguous with the endoplasmic reticulum?

Answer 9

The space between the inner and outer membranes is directly connected with the lumen of the endoplasmic reticulum.

Question 10

Where does movement of molecules into and out of the nucleus occur?

Answer 10

1. Nucleus is double membrane with nuclear pore

2. Nuclear proteins are fully folded before they enter the nucleus- they must enter the nucleus through the nuclear pore

Question 11

What is each nuclear pore composed of?

Answer 11

1. A large number of protein subunits.
2. Fibrils protrude from both sides of the complex.
3. Anything going in and out of nucleus has to go through nuclear pore complexes
4. Large molecules are actively transported through nuclear pores
5. Pores are freely permeable to small molecules

Question 12

What is the word for when two subunits are the same?

Answer 12

Heterodimeric.

Question 13

What are the words for when two subunits are different?

Answer 13

Homodimeric.

Question 14

How are proteins imported through the nuclear pore complex?

Answer 14

1. Nuclear import receptors recognise NLS’ (nuclear localisation sequences).
2. The complex of the receptor and cargo protein binds to the fibrils on the cytoplasmic side.
3. Repeats on the fibrils guide the complexes to the nuclear pore.
4. The binding of nuclear protein to the pore opens the pore
5. Active transport into nucleus (together with receptor)

Question 15

What is the conformation of proteins that are imported into the nucleus?

Answer 15

They are fully folded.

Question 16

How is the energy provided for import of proteins into the nucleus?

Answer 16

GTP hydrolysis.

Question 17

What does Ran GTPase do?

Answer 17

It provides directionality of transport, as nuclear export can occur in a similar way to nuclear import.

Question 18

What is RanGEF and how does it work?

Answer 18

1. It is involved in protein import into the nucleus.
2. It swaps GDP for GTP so that Ran becomes GTP loaded (Ran-GTP) which can then replace cargo from nuclear import receptors.
3. Nuclear import receptors can then move out of the nucleus when bound to Ran-GTP.

Question 19

How can nuclear import receptors bind to another cargo protein after leaving the nucleus?

Answer 19

1. They need to release the Ran by hydrolysing the GTP to GDP.
2. This is stimulated by the protein RanGAP.

Question 20

What happens when RanGTP is converted to RanGDP?

Answer 20

1. As RanGDP has a different conformation, it is released from the nuclear import receptor.
2. The process can now repeat over again.

Question 21

Where are mitochondrial targeting sequences found?

Answer 21

1. The N-terminus.

Question 22

How can proteins get into the mitochondrial matrix?

Answer 22

1. They go through protein conducting channels.

Question 23

What conformation do proteins need to be in to get into the mitochondrial matrix?

Answer 23

1. They need to remain unfolded in the cytoplasm- no large nuclear pores in mitochondrial membrane

Question 24

What are the two protein conducting channels in the mitochondrial matrix?

Answer 24

1. TOM (translocator of the outer membrane) and TIM (translocator of the inner membrane).

Question 25

How does Ran-GTP help bring proteins into the nucleus

Answer 25

1. Ran-GTP- has high affinity for nuclear import receptor
2. When protein complex enters nucleus Ran-GTP binds to receptors with higher affinity than cargo protein
3. So protein is released
4. Ran-GTP receptor complex leaves the nucleus
5. Ran-gdp dissociates from receptors- Ran-GDP has lower affinity

Question 26

What are small GTP binding proteins

Answer 26

1. Act like molecular switches
2. Usually either on or off
3. Controlled by whether bound to GTP or not
4. GTP bound- on
5. GDP bound- off

Question 27

How is RAN-GTP switched off

Answer 27

1. i) GTP hydrolysis
2. Controlled by GAP-GTPase activating protein
3. G- proteins on their own have low turnover of GTP
4. GAP protein provides an amino acid residue into the active site which allows GTP hydrolysis to be accelerated
5. Different GAP for different G- proteins
6. As soon as Ran-GTP reaches cytoplasm (where GAP is) GTP is hydrolysed

Question 28

How is RAN-GTP switched on

Answer 28

1. Remove GDP
2. Load with GTP
3. Don’t add phosphate onto GDP
4. Controlled by Guanine nucleotide exchange factor- GEF

Question 29

What is the difference between GAP and GEF

Answer 29

1. GEF- inside nucleus

2. GAP- outside nucleus

Question 30

What does the mitochondrial targeting sequence look like

Answer 30

1. Contain positively charged amino acids that fold up to form amphipathic helix
2. 3.6 Residues per turn
3. Positively charged are 3-4 apart
4. All positively charged on one side of helix
5. Other side is hydrophobic
6. Amphiphatic helix recognised by receptors on surface of mitochondrial membrane
7. Very in size- 20-40 amino acids

Question 31

What is the sequence of events of moving a protein into the mitochondria

Answer 31

1. Mitochondrial import sequence is recognised and positively charged amino acid sequences are attracted across the membrane by membrane potential (proton gradient different to nucleus)
2. Then need transport of protein across two membranes using TOM and TIM - Translocase of inner membrane and outer membrane
3. In matrix chaperone binds- stops proteins moving in opposite direction
4. First chaperone is released by ATP hydrolysis and protein folding can occur
5. Mitochondrial proteins can be in outer membrane, inner membrane, intermembrane space or matrix.
6. Mitochondria also have their own genome, protein synthesis machinery.

Question 32

How is it ensured that the proteins are not folded

Answer 32

1. Bound to by chaperones to stop proteins from folding
2. E.g Hsp70
3. Can bind to proteins and release proteins using ATP hydrolysis