Lecture 8 - Mitochondria 2

Question 1

How is the precursor protein guided to TOM40 in the TOM complex?

Answer 1

TOM20 interacts with N-terminal precursor which is hydrophobic and forms hydrophobic interactions. THe positive side of amphiphillic presequene is exposed and this positive charge can interact with TOM22 which is a very negatively charged receptor which interacts with positive charge of precursor.

Question 2

How does TIM50 allow precursors to interact with TIM23?

Answer 2

TIM50 contains a binding groove with a negatively charged groove which can interact with the positively charged N-terminal presequence.

Question 3

What is the function of TIM22?

Answer 3

TIM22 allows transfer of metabolites into the matrix across inner membrane. It does this by allowing carrier proteins to enter the inner membrane.

Question 4

What are the components of the TIM22 complex?

Answer 4

composed of:

TIM22 (pore)
TIm54 (accessory subunit)
TIM18(accessory subunit)

TIM12 TIM9 TIM10 small chaperone proteins of TIM22

Question 5

What is an example of a carrier protein found on the inner membrane?

Answer 5

ATP/ADP carrier is an example of a carrier protein

Question 6

What do carrier protein molecules look like?

Answer 6

6 transmembrane (alpha helical) domains and both N and C termini are found on intermembrane space.

Question 7

Where do carriers bind to the TOM complex before entering the mitochondria?

Answer 7

Carrier proteins bind to TOM70 rather than TOM20 of the TOM complex. Release from chaperones requires ATP hydrolysis.

Question 8

What are the steps in carrier protein transport into inner membrane?

Answer 8

1) Chaperone binds precursor
2) Chaperone + precursor bind to TOM70 and ATP is hydrolysed.
3) TIM chaperones guide precursor to TIM22.
4) precursor inserts onto TIM22 in loop conformation.
5) After release into inner membrane the proteins dimerize.
* Inner membrane potential is essential for this process to take place.

Question 9

How are carrier proteins isolated at different stages of import into inner membrane?

Answer 9

Carrier protein is tagged with 35S methionine.

In one tube apyrase is added to deplete ATP along with ionophore (dissipates inner membrane potential)

Tube 2: - +

Tube 3: - -

• First one is apyrase and second is ionophore

These tubes are added to a BN-PAGE

Question 10

How does TIM23sort pathway differ to the TIM23motor pathway?

Answer 10

STOP transfer signal is highly hydrophobic and stays in the inner membrane and if protein is cleaved within the IM space the protein can be released into the IM space.

Question 11

What proteins does TIM23sort associate with?

Answer 11

TIM23sort associates with complex 3 and 4 of inner membrane.

Question 12

How was the interaction of TIM23sort with complex 3 and 4 discovered?

Answer 12

TIM18 is tagged with protein A. TIM21 is tagged with protein A. IgG is used in a gel to isolate tagged protein.

The proteins that are associated with the tagged protein are then isolated. Complex 3 and 4 were as a result found next to the TIM23sort complex.

Question 13

Why is there a close association between complex 3+4 and TIM23sort?

Answer 13

This interaction increases strength of membrane potential which is essential to drive the release of inner membrane proteins into the inner membrane

Question 14

What does MIA stand for?

Answer 14

Mitochondrial Intermembrane space Assembly protein

Question 15

Where in the mitochondria is there an abundance of cysteine rich proteins?

Answer 15

The inter membrane space.

Question 16

What is the function of MIA?

Answer 16

biogenesis or import of small cysteine rich inner membrane proteins.

*CX3 CX9C motifs

Question 17

What is MIA40’s function?

Answer 17

MIA40 is a receptor for cysteine rich proteins.

Question 18

What is the characteristic of the intermembrane space that allows TIM22 to import cysteine rich proteins into the inner membrane of the mitochondria?

Answer 18

Intermembrane space of mitochondria is an oxidative environment.

Question 19

What is the chaperone that allows entry of TIM10?

Answer 19

TIM9

Question 20

What cell environments are oxidising?

Answer 20

The mitochondria

The ER of eukaryotes

The periplasm of the prokaryotic cell

Question 21

What are the steps of the Mia40-Erv1 reaction cycle?

Answer 21

1) Precursor enters intermembrane space via TOM40.
2) Precursor forms temporary intermolecular disulife bond with Mia40.
3) Precursor is oxidised and can fold into tertiary structure.
4) Mia40 is reduced as a result of previous reaction and so Erv1 reoxidises it.

Question 22

What type of assembly does the TIM9/10 complex have?

Answer 22

typically exist in hexameric assembly of alternating TIM9/TIM10 units.

Question 23

What kind of disulfide bonds exist within TIM 9/10 complexes?

Answer 23

In all TIM9/10 complexes one outer and one inner disulfide bond.

Question 24

What is the function of URA3?

Answer 24

URA3 is essential for uracil production. It becomes toxic with the addition of 5-FOA

Question 25

How many Cysteines do TIM 9 and TIM10 have and where are they located?

Answer 25

TIM9 has 4 cysteines. C39-C55 C35-C59

TIM10 has 4 cysteines.
C40 C44 C61 C65

1 internal and external disulfide bonds.

Question 26

How can the disulfide bonds of TIM9/10 be done in the lab without compromising entry of TIM9/10 into the intermembrane space?

Answer 26

Disruption of TIM9/10 disulfide bonds can be done via changing a cysteine into a serine.

Question 27

How does plasmid shuffling work?

Answer 27

wild type genotype is expressed with URA3. mutant is expressed on another plasmid with His3 which produces histidine. Expressing the second plasma allows cells to grow on a histidine lacking medium they will grow. The cells are grown in 5-FOA which makes the product of the URA3 toxic. As a result of this the URA3 containing plasmid is slected against and is destroyed. All that remains is the second plasmid with the HIS3 tag and if the cells survive after that then the protein that was mutated is not essential for survival. If the opposite occurs then it is.

Question 28

How important is the structure of TIM10 for its function?

Answer 28

No TIM10 = death

wild type TIM10 = growth

TIM10 with broken disulfide bonds = death

TIM10 needs at least one disulfide bond to work.

Question 29

How does the mitochondria regulate the intermembrane space and remove misfolded proteins?

Answer 29

Mitochondria have quality control proteins in their inner membrane known as AAA proteins.

Question 30

Which side of the membrane is the i-AAA

Answer 30

Active site of i-AAA is in intermembrane space of mitochondria

Question 31

What is the MISS signal?

Answer 31

Leucine and Valine next to the 1st cysteine is vital to function of protein. This signal is known as the MISS signal.

Question 32

Why does the non-reducing SDS page provide interactions between Mia40 and cysteine rich proteins?

Answer 32

Reducing agents break disulfide bonds. When disulfide bonds aren’t broken Mia40 and precursor interact.

Question 33

What happens to the ability of Mia40 to interact with precursor proteins if a mutation occurs?

Answer 33

intermediate structure between precursors and Mia40 can be made easily provided the MISS signal is not mutated.