Nuclear encoded proteins that target mitochondria

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Nuclear encoded proteins that target mitochondria

• Characterisation of a eucaryote nuclear genome -
  Rhizopus oryzae
• BIN6002 David To

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Introduction to Eukaryotes

• Cells are much larger and complex than
  prokaryotes
• Characterised as having membrane bound nuclei
• Uni-cellular and multi-cellular
• Span kingdoms of Protists, Plants, Fungi, Animals
• Most contain cellular organelles including
  mitochondria
• Those that lack mitochondria have hydrogenosomes
  or mitosomes
• Plants also have plastids chloroplasts (green
  plants), rhodoplasts (red algae), cyanelles
  (glaucophytes).

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Eukaryote vs. Prokaryote
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Mitochondrion Origin

• Believed to have evolved from an endosymbiotic
  a-proteobacterium several billion years ago
• Closest living relatives of mitochondria seem to
  be within the Rickettsiae.
• Why? The primitive host cell provided nutrients
  for the mitochondrion, and the mitochondrion
  provide the cell a way to extract energy from
  oxygen

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Mitochondrion Origin
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About the mitochondrion

• Grow and divide in a similar way to cells
• Primary function is the generation of ATP and
  aerobic respiration
• Two membranes, outer and inner
• Contain their own genome which greatly vary in
  size throughout the eukaryotes 5kb (protist) -
  2.4 Mb (plant)
• Contain 5 to 100 genes

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About the mitochondrion

• Loss of most functional genes others
  transferred to the nuclear genome
• The mitochondrion didnt require genes for most
  cell mechanisms as the host cell already had them
• very different selective pressure
• Nuclear encoded pre-proteins are targeted to the
  mitochondria, translocated through outer and
  sometimes inner membranes
• These proteins usually tend to be hydrophobic
• Generally, a target sequence is contained within
  N-terminal segments of the pre-protein
• Perhaps 2-10 of the nuclear encoded genes are
  targeted to the mitochondria

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Properties of Targeting signals

• N-terminal extensions (presequences) to the
  protein
• Form an amphipathic helix
• Extensions often around 20-60 amino acids,
  positively charged (mostly)
• Also called matrix-targeting sequences/ signals
• Sometimes internal to the protein

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Protein Import into MitochondriaGeneral pathway

• Pre-proteins are synthesised in the cytosol
• Translated from the nuclear genome
• In an unfolded state
• Import can start during translation

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Protein Import into MitochondriaGeneral pathway

• Chaperone proteins (eg. hsp70) guide unfolded
  pre-proteins to the mitochondria.
• Chaperones prevent proteins folding up while
  passing through the outer membrane

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Protein Import into MitochondriaGeneral pathway

• 3. Targeting sequences on pre-proteins bind with
  receptors on the outer membrane, allow the
  pre-protein through the trans-membrane pore
• Chaperone proteins peel off pre-protein as it
  enters the mitochondria
• Unfolded state allows the protein to fit through
  the pore of the mitochondrial outer/inner membrane

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Protein Import into MitochondriaGeneral pathway

• 3. Continued
• Binding complex TOM (translocase outer
  membrane) complex
• Proteins Tom70, 37, 22, 20 and 40 make up the
  binding site

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Protein Import into MitochondriaGeneral pathway

• 4. In the matrix, internal chaperone proteins
  grab the pre-protein
• Binding complex TIM (translocase inner
  membrane) complex Tim 23, 17, 44
• Internal chaperones also prevent protein from
  folding up (until required)

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Protein Import into MitochondriaGeneral pathway

• Once in the matrix, peptidase cleaves the
  targeting sequence
• The pre-protein is now trapped in the
  mitochondria
• Exception! Some mitochondrion targeted proteins
  do not have targeting signals, so no signal is
  cleaved
• Exception! Some mitochondrion targeted proteins
  are cleaved midway, trapping them between the
  outer/inner layers.

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Protein Import into MitochondriaGeneral pathway

• Pre-protein is folded and/or if there is
  secondary targeting sequence, is redirected
  within the mitochondria
• Proteins can be sent to different compartments
  within the mitochondrion via secondary targeting
  sequences.
• In experiments, by removing the secondary
  targeting sequence, proteins destined for other
  places in the mitochondria remain in the matrix

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Protein Import into MitochondriaExceptions

• In yeast, it has been observed that ribosomes
  gather on the surface of the mitochondria and
  deliver the pre-protein directly
• Some pre-proteins are stopped in between the
  inner and outer membrane

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Detection of proteins targeting mitochondria (in
vivo)

• Molecular genetics - introduction of mutations in
  suspect signal sequences
• Blocking uptake of proteins through the pore, but
  not the binding of proteins (DNP)
• Yeast cells are able to grow aerobically (making
  ATP in mitochondria), or anaerobically, using
  only glycolysis to make ATP changes are usually
  not fatal slow growth
• Protein sequencing of cellular organelles

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Detection of proteins targeting mitochondria (in
silico)

• Numerous number of programs available
• Some are rule based, others are neural network
  based
• Most attempt to use the N-terminal sequence
  information to determine proteins destined for
  mitochondria, chloroplasts, secretory pathways
• The results are not particularly reliable

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Detection of proteins targeting mitochondria (in
silico)

• MitoProt II (1996)
• Rule based mitochondrial, plastid
• Uses aminoacid compositions suffers on accuracy
• TargetP (2000)
• Two layer neural network based plastid,
  mitochondrial
• iPsort/Psort (2002)
• Rule based plastid, mitochondrial
• MITOPRED (2004)
• Pfam domains - genome level

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Detection of proteins targeting mitochondria (in
silico)

• Predotar (2004)
• Neural network based
• Genome level
• Training
• More than 90 of the test sequences are correctly
  predicted (cut-off 0.5)
• Real
• Only 35-50 mitochondrial proteins accurately
  predicted
• 65 for plastid proteins

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Detection of proteins targeting mitochondria (in
silico)
   
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Conclusion

• Focus on mitochondria, but the general theme of
  protein import into cellular organelles
• Pre-proteins targeting mitochondria typically
  have N-Terminal targeting sequences
• Current in-vivo techniques have been mainly
  performed on S. cerevisiae due to their ability
  of anaerobic respiration
• Current in-silco techniques are have high false
  positives rates and mid-range accuracy rates

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References

• Protein Targeting and Organelle Biogenesis
  http//www.lclark.edu/reiness/cellbio/lectures/le
  ct13.htm
• Protein import into mitochondria Neupert,
  Walter. Ann. Rev. Biochemistry 1997,  66863-917
• Minireview - Targeting of proteins to
  mitochondria
• Trevor Lithgow, FEBS Letters 476 (2000) 22-26
• Predicting subcellular localization of proteins
  based on their N-terminal amino acid
  sequence.Olof Emanuelsson, Henrik Nielsen, Søren
  Brunak and Gunnar von Heijne.J. Mol. Biol., 300
  1005-1016, 2000.
• MITOPRED a web server for the prediction of
  mitochondrial proteins
• Chittibabu Guda et al. Nucleic Acids Research,
  2004, Vol. 32
• Predicting Subcellular Localization of Proteins
  Based on their N-terminal Amino Acid Sequence
• Olof Emanuelsson et al. J. Mol. Biol. (2000)
  300, 1005-1016
• AMPDB the Arabidopsis Mitochondrial Protein
  Databases
• Joshua L. Heazlewood and A. Harvey Millar
  Nucleic Acids Research, 2005, Vol. 33
• Mitochondrial Genome Evolution
• B. Franz Lang et al.

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Questions?