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Mitochondria Flashcards

1
Q

Where do mitochondrial proteins come from and why?

A

From the cytosol, because it doesn’t make many of its own anymore due to reduced size of mitochondrial genome - which means more genes need to be imported

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2
Q

Why has the size of the mitochondrial genome been reduced?

A
  • some genes expendable in the internal environment of the host (e.g. no need for cell wall now its inside a host)
  • nuclear genes replaced function of some organellar genes or genes moved to nucleus
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3
Q

How many mitochondria are there per cell?

A

over 1000

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4
Q

What are important features of mitochondrial signal peptides?

A
  • Rich in positively charged residues (Arg/Lys)
  • Often Arginine residues are close to the processing site (generally at position –2 or –3)
  • Rich in hydrophobic residues
  • Form an a-helical structure
  • But there is no primary sequence homology
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5
Q

What is another name for the mitochondrial signal peptide?

A

CoxIV Presequence

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6
Q

What is the structure of the mitochondrial signal peptide?

A

It forms an amphipathic helix – the charged amino acids end up on one side and hydrophobic amino acids on the other.

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7
Q

Is amphipathicity important in the mitochondrial signal peptide?

A

Yes
The first methionine was removed so that it becomes charged right in the middle of the hydrophobic regions of the helix which destroyed amphipathicity. This stops the protein from targeting to the mitochondria and it ends up at the cytosol instead.

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8
Q

What is MPP?

A

matrix protein peptidase, the equivalent to signal peptidase

It results in cleaved matrix proteins having an Arg at -2 or -3

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9
Q

Why is amphipathicity important in the mitochondrial signal peptide?

A

It allows proteins to bind to receptors in the outer membrane.

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10
Q

How the necessary length of nascent chains investigated?

A

Assessed the import of import of cytochrome b2-DHFR fusion proteins. Different lengths of b2 with fixed length of DHFR.
Assessed in the presence of methotrexate (stabilises folded form of DHFR and prevents it engaging with transport machinery.
Only if the region between folded DHFR and processing site (Cyb2) is long enough can the protein emerge into the matrix and is cleaved by MPP

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11
Q

How long must the nascent chain be in order to emerge into the matrix?

A

Minimum length about 50 residues: more or less the width of 2 membranes, indicating that translocation occurs at contact sites between inner and outer membrane

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12
Q

What are contact sites?

A

Regions where the inner and outer membranes are very close together. It can be seen via electron microscope.
They allow proteins to cross both membranes with minimal possible effort. It is a concerted effort to cross both membranes at the same time.

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13
Q

When are matrix proteins imported?

A

Post-translationally

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14
Q

What is the mechanism of matrix protein import?

A
  1. Precursor protein is unfolded by cytosolic chaperones (e.g. Hsc70) so the signal sequence to engage with transport machinery.
  2. Presequence engages with the Tom20/22 import receptor on the OM
  3. It is then transferred to the Tom40 general import pore (GIP)
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15
Q

What are the receptors of the TOM complex?

A

Tom20 = 1st receptor
Tom22 = 2nd receptor
Binding of these docks the protein at the pore.
Tom5 = 3rd receptor - transfers protein into GIP

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16
Q

What are the components of the General Import Pore?

A

Tom40 = 2x forms the channel
Tom6 = required for assembly of Tom22-40
Tom 7 = supports dissociation of Tom complex to allow release of preproteins.

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17
Q

What drives translocation across the TIM complex?

A

Proton gradient

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18
Q

What are the important components of the TIM complex?

A 
Tim50 = receptor for preproteins after release from TOM complex, it guides protein to import channel
TIM23 = N terminal domain is a receptor, C terminal domain is the channel
19
Q

What is the PAM complex?

A

Presequence Translocase-Associated Motor – it generates the energy to pull the protein through the complex.

20
Q

What does the PAM consist of?

A
  • TIM44: Anchors mtHsp70 to the membrane
  • mtHsp70: Chaperone; ATP-dependent motor protein
  • Mge1: Nucleotide exchange factor; promotes mtHsp70 reaction cycle
  • PAM18: co-chaperone: stimulates ATPase activity of mtHsp70
  • PAM 16: Involved in recruitment of PAM18 to TIM complex
21
Q

What is the mechanism of threading proteins through into the matrix?

A

Chaperones hydrolyse ATP to create energy
There are two mechanisms:
A. Power-stroke model
B. Brownian-ratchet model

22
Q

What is the Power-stroke model?

A

Pulling
One chaperone binds to the protein and pulls it out of the pore into the mitochondria via conformational change.
Another chaperone does the same so its like two hands pulling.

23
Q

What is the Brownian-ratchet model?

A

Trapping
Protein in the pore is oscillating in the pore. Chaperones don’t actively pull but prevent the protein from sliding back out.

24
Q

Which model of proteins threading through the complexes is correct?

A

It is still a debated topic. It may be that both are correct, and that the mechanism used depends on the substrate. Loosely folded proteins can use a “Brownian ratchet”, whereas more tightly folded proteins may require active pulling via the power-stroke.

25
Q

What are the different places that proteins can be transported in the mitochondria?

A

Other than into the matrix:
Membrane proteins can go to the inner or outer membrane
Proteins can also go to the intermembrane space by passing though TOM but avoiding passing through TIM

26
Q

How does the structure of inner membrane proteins differ from outer membrane proteins?

A

Inner membrane proteins are alpha helical

Outer membrane proteins have beta-sheets also called beta-barrel proteins.

27
Q

Which membrane in the mitochondria has the most amount of proteins and why?

A

The inner membrane because it has the electron transport machinery like:
components of respiratory complexes, ATP synthase and carriers e.g. ATP/ADP carriers

28
Q

What are the two main routes that have been proposed for proteins to get into the inner membrane?

A
  1. Stop-transfer mechanism

2. Conservative sorting

29
Q

How is an inner membrane protein distinguished from a matrix protein?

A

It has a signal peptide and a TM domain so that the N terminus ends up in the matrix and the C terminus in the intermembrane space

30
Q

What recognises inner membrane proteins?

A

Oxa1

t is a membrane protein in the inner membrane with bacterial origin.

31
Q

What is the conservative sorting mechanism?

A

Oxa1 in the inner membrane recognises transmembrane sequences of inner membrane proteins in the matrix and inserts it into the membrane just like mitochondrially encoded proteins.

32
Q

What determines the route of an inner membrane protein?

A

Whether or not the protein has a bacterial homolog:
Those WITHOUT use the stop-transfer mechanism (e.g. TIM proteins)
Those WITH bacterial homologues use the conservative sorting mechanism (e.g. subunit 9 of F1F0 ATPase; Oxa1)
Also sequences and charges flanking the hydrophobic domain probably play an important role

33
Q

What determines whether or not a protein has a bacterial homolog?

A

Whether a protein evolved pre or post symbiotic events

34
Q

How are inner membrane carrier proteins imported?

A

via the TIM22 pathway:

they are recognised by Tom70 and then they go through one of three mechanisms to pass the membrane.

35
Q

What are the 5 stages of import of carrier proteins?

A
  1. Cytoplasmic chaperones target carrier proteins to the OM
  2. TOM70 subunits bind to carrier protein: release from TOM70 is ATP dependent
  3. Proteins cross GIP via loop formation translocation and TIM9/10 assist in targeting to the IM by binding to TIM12
  4. Insertion into the IM occurs via the TIM22/TIM18/TIM54 complex; this step requires the membrane potential
  5. The carrier is released via lateral diffusion and assembles
36
Q

What are Tim22’s 4 stages of action?

A
  1. Proteins of AAC family (carrier proteins) bind to the TIM22 complex, independent of membrane potential
  2. It drives docking of protein into the complex
    • Matrix exposed loops of are ++ charged; this step may be electrophoretic
  3. ∆ψ and internal signal peptide activate channel gating. One pore is kept closed; other is rapidly gated to promote insertion of additional transmembrane segments
  4. Protein is released laterally into the membrane
37
Q

What do Tim9/10 do?

A

probably have chaperone-like activities that prevent aggregation of hydrophobic precursor proteins; essential for viability

38
Q

How are proteins imported to the intermembrane space?

A

Using intermembrane space-targeting sequences which resemble Sec-type signal peptides.
The signal peptide is processed by Imp1, a type I SPase (similar to e.g. Lep (E. coli), Sec11 (yeast ER), and TPP (thylakoid)

39
Q

What are the two pathways with proteins targeted to the intermembrane space can follow?

A
  1. TIM23-dependent pathway - which is the same as route A for IM proteins, but with extra proteolytic cleavage
  2. TIM23-independent pathway - proteins engage with TOM40, then are ‘trapped’ in the intermembrane space by S-S bonds, and it is mediated by MIA redox relay
40
Q

How are outer membrane proteins targeted to the outer membrane?

A

Simple OM proteins just have one TMD so only require the TOM complex.
Those with complex topology like beta-barrel proteins require TOM, TIM9/10 and the SAM complex

41
Q

What is the SAM complex?

A

Sorting and Assembly Machinery

42
Q

What are the 4 components of the SAM complex?

A

SAM50: Central component of SAM complex, β-barrel topology, channel
SAM35: Binding of β signal, partner of Sam50
SAM37: Subunit of SAM complex, promotes release of precursors
Mdm10: specifically required for assembly of TOM complex

43
Q

Give examples of proteins that are encoded by the human mitochondrial genomes

A

NADH hydrogenase
Cytochrome C oxidase
ATP synthase complex
as well as tRNA components and rRNA

44
Q

What is TargetP?

A

It is a software/program that predicts the subcellular location of eukaryotic proteins

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