11. How do we test function of yeast homologues in animals and plants as lethal if mutated or deleated - in both yeast and higher eukaryotes

12. Tim17:23 Membrane location (Yeast)

13. AOX Tim17:23 Import channel on inner membrane Recently shown that channel activity associated with Tim23. Four membrane spanning section has voltage dependant channel activity and the N-terminal region which binds presequences gives channel specificity. Role in Apoptosis or programmed cell death ?

14. H.s TIM17 S.c TIM17At TIM17-2 0 50 100 150 200 250 At TIM17-5 0 40 80 120 160 0 40 80 120 0 40 80 120 160 AT-2 AT-5 S.c H.s AT-2 AT-5 S.c H.s AT-2 AT-5 S.c H.s AT-2 AT-5 S.c H.s AT-2 AT-5 S.c H.s TIM17

15. Arabidopsis Tim17

16. TIM23

17. 1 50 Yeasttim23 MSWLFGDKTP TDDANAAVGG QDTTKPKELS LKQSLGFEPN INNIISGP.G Arabtim23.....................................MA ANNRSDHGSD 51 100 Yeasttim23 GMHVDTARLH PLAGLDKGVE YLDLEEEQLS SLEGSQGLIP SRGWTDDLCY Arabtim23 ENTRLYNPYQ NYEVPINKSQ YLYKLPTSPE FLFTEEALRQ RRSWGENLTF 101 150 Yeasttim23 GTGAVYLLGL GIGGFSGMMQ GLQNIPPNSP GKLQLNTVLN HITKRGPFLG Arabtim23 YTGTAYLGGS VAGASVGVIT GVKSFESGDT TKLKINRILN SSGQTGRTWG 151 200 Yeasttim23 NNAGILALSY NIINSTIDAL RGKHDTAGSI GAGALTGALF KSSKGLKPMG Arabtim23 NRIGIIGLVY AGIESGIVAA TDRDDVWTSV VAGLGTGAVC RAARGVRSAA 201 227 Yeasttim23 YSSAMVA.AA CAVWCS..VK KRLLEK. Arabtim23 VAGALGGLAA GAVVAGKQIV KRYVPI* Arabidopsis Tim23 Outermembrane

18. AOX Characterise Tim17:23 in Plants Q - Given the differences observed does it play the same role in plants via the same mechanisms as elucidated in yeast? The acquisition of mitochondria(or endosymbiosis) has been proposed to be vital for the evolution of multi-cellular organisms. Yeast is an excellant model system but cannot claim to represent diverse multicellular organisms that display developmental and tissue specific functions.

19. AOX Function of AtTim17 and AtTim23 Knock-out mutants of yTim17 and yTim23 are lethal so must use a replacement strategy to see if plant genes can complement yeast genes.

22. EUROPEAN..........SACCHAROMYCES....................CEREVISIAE..............................ARCHIVE FOR........................................FUNCTIONAL ANALYSIS Yeast deletions for 5943 genes Haploid non essential genes Diploid essential genes Deletion strains for mitochondrial import Tim 10, 11, 12, 17, 22, 23, 44, 54 Tom 5, 6, 7, 20, 22, 72, 34, 37, 40, 71, 72 Deletions have kanMX4 inserted into gene of interest

23. tetrad kanMX4 Tim17 kanMX4 Tim17 2:2 genotype segregation Tetrad analysis

26. AOX Function of AtTim17 and AtTim23 Different AtTim17 genes have different activities C-terminal region of AtTim17-2 prevents it activity in yeast which implies a different function in plants AtTim23 cannot complement a yeast mutant implying different functions