Lecture 7 - Mitochondria 1

Question 1

What are the 2 modes of translocation?

Answer 1

2 modes of translocation; co and post translational

Question 2

How are mitochondrial and ER proteins imported?

Answer 2

majority of mitochondrial proteins are imported post-translationally and majority of ER proteins are transported co-translationally (SRP)

Question 3

What are the main principles governing protein translocation?

Answer 3

Target signals

For translocation to take place proteins must be partially unfolded.

Cytosolic chaperones assist delivery of proteins to the correct organelles. They prevent agreggation and clumping of proteins.

Membrane receptors recognize precursor that is being translocated

GTP/ATP hydrolysis is ofteen required

Question 4

What is the function of the N-terminal presequence? What happens if a stop-transfer signal is added?

Answer 4

N-terminal presequence guides proteins to mitochondrial matrix. With a stop-transfer signal it stops at the inner membrane or the intermembrane space.

Question 5

Where do beta signals target proteins?

Answer 5

beta signal targets proteins to outer membrane.

Question 6

Where do MISS signals target proteins?

Answer 6

MISS signal targets proteins to inner membrane

Question 7

What proteins go through the TOM complex?

Answer 7

All proteins transported into the mitochondria go through the TOM complex

Question 8

What is the size of the TOM complex?

Answer 8

440 kda

Question 9

What is the function of TOM20 & TOM70?

Answer 9

They are peripheral receptors that engage with the incoming precursor.

Question 10

What is the function of TOM22?

Answer 10

TOM22 is a central receptor that is essential for the stability of the TOM complex. It passes precursors to the TOM20 and TOM70.

*Without TOM22 the complex breaks into 100kda subunits.

Question 11

What is the function of TOM5, 6, and 7?

Answer 11

Maintain TOM complex dynamics.

Question 12

What is the function of the TOM40 complex?

Answer 12

Pore though which the proteins traverse the membrane

Question 13

What are the steps of performing affinity chromatography? (use TOM22 as the example)

Answer 13

1) Plasmid DNA coding TOM22 is modified to add histidine tag at C terminus.
2) TOM22-HIS is transformed into Neurospora Crassa cells.
3) Mitochondria is isolated
4) Detergent is used to separate TOM complex.
5) Ni-NTA chromatography is used to separate the TOM22-HIS from the TOM complex.

Question 14

What is the histidine tag made of?

Answer 14

Histidine tag is composed of 6 - 10 histidine molecules.

Question 15

What are the steps of Ni-NTA chromatography?

Answer 15

1) Binding: Nickel site binds to the histidine tags separating TOM22 from the rest of the mixture.
2) Washing:
3) Elution: Column is saturated with imidazole which competes with histidine and separates histidine from the the nickel (Nitrilo

Question 16

How many pores does TOM40 contain?

Answer 16

Variable number of pores in TOM40 complex. This depends on the precursor interacting with it.

Question 17

How is Blue-Native PAGE

Answer 17

Mild detergent used to separate TOM complex from mitochondrial membrane.

Protein is incubated with comassie blue G-250 dye. This dye binds to hydrophobic protein and gives it a negative charge.

Protein is then added to electrophoresis gel (acrilamyde)

Question 18

What is an in vitro assay?

Answer 18

In vitro import assay is the use of isolated mitochondria to import protein directly into it.

Question 19

How is an in vitro assay performed?

Answer 19

1) In vitro transcription is performed to generate RNA of protein of interest.
2) Radioactively labelled methionine + all other amino acids are added and ribosomes are added to produce protein of interest.
3) Protein is imported into isolated mitochondria

Question 20

What are the 2 conformations of TIM23?

Answer 20

2 conformations of TIM23:

TIM23motor

TIM23sort

Question 21

What are the components common to both TIM23 complexes?

Answer 21

TIM23

TIM17

TIM50

Question 22

What are the components unique to the TIM23 motor complex?

Answer 22

The PAM proteins

PAM16

PAM18

MtHsp70

TIM44

Question 23

What are the components unique to the TIM23sort complex?

Answer 23

TIM21

Question 24

What are the features of the N-terminal presequence?

Answer 24

20-25 amino acids

Rich in +vely charged amino acids (R,K)

Hydroxylated amino acid

Amphiphillic helix

Question 25

What kind of protein is the MtHsp70?

Answer 25

A chaperone

Question 26

What is the function of TIM23motor and TIM23sort?

Answer 26

TIM23motor facilitates import into matrix.

TIM23 facilitates import (N-terminal + stop-trnsfer signal) inner membrane or intermembrane space.

Question 27

How is a protein imported into the mitochondrial matrix?

Answer 27

1) Protein interacts with TOM20 followed by TOM22 on the outer membrane of the mitochondria.
2) Precursor goes through TOM40 pore.
3) Precursor interacts with TIM50 receptor of the TIM23motor complex. This is dependent on the potential difference (remember chemosomotic gradient from ETC).
4) ATP binding of the mtHsp70 results in pulling of protein towards the matrix.
* Relatively negative charge in the matrix pulls proteins into it when interacting with TIM23motor.