Lecture 7 - Mitochondria 1 Flashcards
What are the 2 modes of translocation?
2 modes of translocation; co and post translational
How are mitochondrial and ER proteins imported?
majority of mitochondrial proteins are imported post-translationally and majority of ER proteins are transported co-translationally (SRP)
What are the main principles governing protein translocation?
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
What is the function of the N-terminal presequence? What happens if a stop-transfer signal is added?
N-terminal presequence guides proteins to mitochondrial matrix. With a stop-transfer signal it stops at the inner membrane or the intermembrane space.
Where do beta signals target proteins?
beta signal targets proteins to outer membrane.
Where do MISS signals target proteins?
MISS signal targets proteins to inner membrane
What proteins go through the TOM complex?
All proteins transported into the mitochondria go through the TOM complex
What is the size of the TOM complex?
440 kda
What is the function of TOM20 & TOM70?
They are peripheral receptors that engage with the incoming precursor.
What is the function of TOM22?
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.
What is the function of TOM5, 6, and 7?
Maintain TOM complex dynamics.
What is the function of the TOM40 complex?
Pore though which the proteins traverse the membrane
What are the steps of performing affinity chromatography? (use TOM22 as the example)
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.
What is the histidine tag made of?
Histidine tag is composed of 6 - 10 histidine molecules.
What are the steps of Ni-NTA chromatography?
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
How many pores does TOM40 contain?
Variable number of pores in TOM40 complex. This depends on the precursor interacting with it.
How is Blue-Native PAGE
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)
What is an in vitro assay?
In vitro import assay is the use of isolated mitochondria to import protein directly into it.
How is an in vitro assay performed?
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
What are the 2 conformations of TIM23?
2 conformations of TIM23:
TIM23motor
TIM23sort
What are the components common to both TIM23 complexes?
TIM23
TIM17
TIM50
What are the components unique to the TIM23 motor complex?
The PAM proteins
PAM16
PAM18
MtHsp70
TIM44
What are the components unique to the TIM23sort complex?
TIM21
What are the features of the N-terminal presequence?
20-25 amino acids
Rich in +vely charged amino acids (R,K)
Hydroxylated amino acid
Amphiphillic helix
What kind of protein is the MtHsp70?
A chaperone
What is the function of TIM23motor and TIM23sort?
TIM23motor facilitates import into matrix.
TIM23 facilitates import (N-terminal + stop-trnsfer signal) inner membrane or intermembrane space.
How is a protein imported into the mitochondrial matrix?
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.
