Nuclear encoded proteins that target mitochondria

1. Nuclear encoded proteins that target mitochondria Characterisation of a eucaryote nuclear genome - Rhizopus oryzae BIN6002 – David To

2. 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).

3. Eukaryote vs. Prokaryote

4. Mitochondrion Origin • Believed to have evolved from anendosymbiotic a-proteobacterium several billion years ago • Closest living relatives ofmitochondria 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

5. Mitochondrion Origin

6. 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

7. About the mitochondrion • Loss of most functional genes – others transferred to the nuclear genome • The mitochondrion didn’t 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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)

14. 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.

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. Plant networks Sensitivity Target Predotar Predotar V0.5 TargetP iPSORT mitochondria 72% 83% 70% 89% plastids 82% 76% 80% 70% ER 93% - 80% 70% Specificity mitochondria 84% 52% 70% 42% plastids 88% 41% 71% 43% ER 96% - 90% 93% Overall accuracy 92% 78% 84% 75% Non-Plant networks Sensitivity Target Predotar Predotar V0.5 TargetP iPSORT mitochondria 80% 80% 84% 94% ER 93% 95% 87% Specificity mitochondria 84% 85% 49% 51% ER 97% 96% 92% Overall accuracy 94% 94% 86% 89% Detection of proteins targeting mitochondria (in silico)

22. 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

23. References • Protein Targeting and Organelle Biogenesishttp://www.lclark.edu/~reiness/cellbio/lectures/lect13.htm • Protein import into mitochondriaNeupert, Walter. Ann. Rev. Biochemistry 1997,  66:863-917 • 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 ProteinDatabases Joshua L. Heazlewood and A. Harvey MillarNucleic Acids Research, 2005, Vol. 33 • Mitochondrial Genome Evolution B. Franz Lang et al.

24. Questions?