Evolution of Mitochondria and anaerobic lifestyle Martin Kolisko Mitochondria •Often Considered “The Powerhouse of the Cell” • •Contains its own genome – “semi-autonomous organelle” • •Double membrane bound • •Cristae – formed by the folding of the inner membrane • •One of signature characteristics of Eukaryotic cell • • Mitochondria – functions •Oxidative phosphorylation è Pyruvate – Acetyl-CoA – Proton Gradient – ATP synthesis • •Also called cellular respiration Mitochondria – more functions…. •Fe-S cluster assembly •Fatty acid metabolism •Amino acid metabolism •Protein import •Energy generation Mitochondria – more functions…. •Fe-S cluster assembly •Fatty acid metabolism •Amino acid metabolism •Protein import •Energy generation Mitochondria •Often Considered “The Powerhouse of the Cell” • •Contains its own genome – “semi-autonomous organelle” • •Double membrane bound • •Cristae – formed by the folding of the inner membrane • •One of signature characteristics of Eukaryotic cell • • Mitochondria – evolutionary origins Eukaryotic cell α-Proteobacterium Endosymbiosis Unknown host: Bacterium? Primitive Eukaryote? Mitochondrion Endosymbiosis •Internalization of one single cell organism by another single-cell organism • •The internalized organisms is eventually transformed into an organelle (e.g. mitochondrion/ chloroplast) • •Organellar genome is slowly reduced and genes are transferred to the host nucleus EVIDENCE for endosymbiotic origin of mitochondria? •Circular genome – bacterial-like • •Different mitochondrial genome reduction. Different organisms possess different number of genes and vary in sizes: ogenes: 5 genes in Plasmodium vs. 94 genes in Reclinomonas osize: ~5Kb in Plasmodium vs. ~100Kb in Jakoba • •Translational (mRNA into protein) machinery similar to bacteria • • •Some Bacterial-like biochemical pathways and functions • • •Phylogenetic evidence Mitochondria – evolutionary origins Eukaryotic cell α-Proteobacterium Endosymbiosis Unknown host: Bacterium? Primitive Eukaryote? Mitochondrion Gene transfer – This means that there are genes in the eukaryotic genomes that originated from the mitochondria. Making phylogenies based on these genes may help understand the origin of mitochondria α-proteobacterial origin Mol Biol Evol, Volume 34, Issue 4, April 2017, Pages 943–956, https://doi.org/10.1093/molbev/msw298 The content of this slide may be subject to copyright: please see the slide notes for details. Oxford University Press Cristae origins Fig. 1. The function of MICOS is probably conserved between mitochondria and alphaproteobacteria. (A) In the mitochondrion of Saccharomyces cerevisiae, MICOS forms CSs and CJs to maintain and stabilize cristae (Harner et al. 2011; Hoppins et al. 2011; von der Malsburg et al. 2011; Körner et al. 2012; Ott et al. 2012; Zerbes et al. 2012). MICOS is composed of the Mic60–Mic19 subcomplex which establishes CSs with the MOM and marks the sites of CJ formation, and the Mic12–Mic10–Mic26–Mic28 subcomplex which differentiates and bends the MIM at CJs (Pfanner et al. 2014; Friedman et al. 2015; Muñoz-Gómez, Slamovits, Dacks, Baier, et al. 2015; Zerbes et al. 2016). The central MICOS subunit, Mic60, contacts the outer membrane through its interactions with the TOM and SAM complexes (additional interactions have also been reported with the MOM proteins porin and Ugo1), and further interacts with Mia40 to aid in the oxidative import of mitochondrial proteins (von der Malsburg et al. 2011; Körner et al. 2012; Ott et al. 2012; Zerbes et al. 2012). (B) In alphaproteobacteria, alphaMic60 is presumably involved in the formation of ICMJs and CSs to stabilize bioenergetic ICMs. alphaMic60 likely uses its conserved mitofilin domain to interact with the BAM complex, the bacterial homologue of SAM. alphaMICOS might therefore mark the sites of ICM invagination and keep ICMs anchored to the alphaproteobacterial envelope at CSs. Alternatively, alphaMic60 might bring together protein translocases (e.g., SecYEG and BAM) for proper envelope biogenesis (the so called Bayer’s junctions) at sites of murein hypotrophy. The compartmentalization of both mitochondrial cristae and alphaproteobacterial ICMs is achieved by narrow tubules (i.e., CJS and ICMJs) likely made and stabilized by Mic60. Mitochondrial cristae and alphaproteobacterial ICMs are therefore functionally analogous. They both constitute specialized sub-compartments that optimize the efficiency of energy transduction by concentrating bioenergetic metabolism. Thick arrows indicate physical interactions between protein partners. Dashed arrows indicate hypothesized physical interactions between protein partners based on the known function of their homologues. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com Targeting of proteins into Mitochondria Targeting of proteins into the Mitochondria •Mitochondrial genome codes for only a fraction of proteins that function in mitochondrion (1 – 2%). • • •Most of the mitochondrial proteins are imported into the mitochondrion. • • •Targeting of the proteins into the mitochondrion is therefore crucial for the function. Targeting peptide •Proteins are mostly imported to mitochondrion through targeting peptide • Targeting peptide: • usually 10-80 amino acids long • • rich in positively charged residues • • forms amphipathic helix - all charged residues on one site – this is recognized by the translocating proteins • Targeting of proteins into the Mitochondria TIM23 17 HSP70 TIM22 SAM complex Cytosol Intramembrane space Mitochodrial matrix Targeting peptide CPN60 CPN10 MPP HSP70 SAM complex Cytosol Intramembrane space Mitochodrial matrix Inner membrane protein with integral targeting peptide Targeting of proteins into the Mitochondria TOM TIM23 TIM22 Mitochondria – functions HSP70 SAM complex Cytosol Intramembrane space Mitochondrial matrix TOM Targeting of proteins into the Mitochondria TIM23 TIM22 HSP70 SAM complex Cytosol Intramembrane space Mitochondrial matrix Either a stop signal or signal for re-translocation from matrix to inner membrane space Targeting of proteins into the Mitochondria TOM TIM23 TIM22 HSP70 SAM complex Cytosol Intramembrane space Mitochondrial matrix Either a stop signal or signal for re-translocation from matrix to inner membrane space Targeting of proteins into the Mitochondria TOM TIM23 TIM22 Micos Mitochondrila Cristae Organization: MIC proteins, that ficiliate flding of the inner membrane. Fig. 1. The function of MICOS is probably conserved between mitochondria and alphaproteobacteria. (A) In the mitochondrion of Saccharomyces cerevisiae, MICOS forms CSs and CJs to maintain and stabilize cristae (Harner et al. 2011; Hoppins et al. 2011; von der Malsburg et al. 2011; Körner et al. 2012; Ott et al. 2012; Zerbes et al. 2012). MICOS is composed of the Mic60–Mic19 subcomplex which establishes CSs with the MOM and marks the sites of CJ formation, and the Mic12–Mic10–Mic26–Mic28 subcomplex which differentiates and bends the MIM at CJs (Pfanner et al. 2014; Friedman et al. 2015; Muñoz-Gómez, Slamovits, Dacks, Baier, et al. 2015; Zerbes et al. 2016). The central MICOS subunit, Mic60, contacts the outer membrane through its interactions with the TOM and SAM complexes (additional interactions have also been reported with the MOM proteins porin and Ugo1), and further interacts with Mia40 to aid in the oxidative import of mitochondrial proteins (von der Malsburg et al. 2011; Körner et al. 2012; Ott et al. 2012; Zerbes et al. 2012). (B) In alphaproteobacteria, alphaMic60 is presumably involved in the formation of ICMJs and CSs to stabilize bioenergetic ICMs. alphaMic60 likely uses its conserved mitofilin domain to interact with the BAM complex, the bacterial homologue of SAM. alphaMICOS might therefore mark the sites of ICM invagination and keep ICMs anchored to the alphaproteobacterial envelope at CSs. Alternatively, alphaMic60 might bring together protein translocases (e.g., SecYEG and BAM) for proper envelope biogenesis (the so called Bayer’s junctions) at sites of murein hypotrophy. The compartmentalization of both mitochondrial cristae and alphaproteobacterial ICMs is achieved by narrow tubules (i.e., CJS and ICMJs) likely made and stabilized by Mic60. Mitochondrial cristae and alphaproteobacterial ICMs are therefore functionally analogous. They both constitute specialized sub-compartments that optimize the efficiency of energy transduction by concentrating bioenergetic metabolism. Thick arrows indicate physical interactions between protein partners. Dashed arrows indicate hypothesized physical interactions between protein partners based on the known function of their homologues. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com Targeting of proteins into the Mitochondria Quick repeat: Targeting peptide – rich in charged residues TOM complex transports across outer membrane – cytosolic HSP70 are used to deliver the protein to the TOM complex TIM complex transports across inner membrane – mtHSP70 facilitates the transport Tim22 complex – transports inner membrane proteins Tim23 complex – transports into the matrix Mitochondrion – Iron-Sulfur cluster assembly Iron-Sulfur clusters are part of Iron-Sulfur cluster proteins There are vital part of … For example: Ferredoxins aconitase Several Fe-S assembly systems Fe-S cluster assembly system Cytosolic Mitochondrial Plastid Prokaryotes Cytosolic Iron Sulfur Cluster Assembly (CIA) + Iron sulfur cluster (ISC) + + Sulfur Mobilization (SUF) + + Nitrogen fixation (NIF) + IS Mitochondrion – Iron-Sulfur cluster assembly Atm1 X - factor Mitochondrial mature proteins CIA machinery – cytosolic Fe-S cluster assembly Cytosolic mature proteins Fe IscU IscU IscS Cys Cystein desulfurase Scaffold protein Quick rep. •Mitochondria originated through endosymbiosis • • •Most of mitochondrial genes were transferred to nucleus and are imported to mitochondria post-translationaly • •Proteins are imported into mitochondria through TOM and TIM complexes • • •Iron-Sulfur assembly is an obligatory function of mitochondria and is done through bacterial type ISC system Anaerobic lifestyle Anoxic mud Ocean Bacterial mat Gut Deep sea marshes Anaerobic life-style •Few terms: oFacultative anaerobes: tolerate oxygen o oStrict anaerobes: oxygen is toxic o oMicroaerophiles: live in an environment with low-level of oxygen What happens to mitochondria? Anaerobic mitochondria § Electron transport/oxidative phosphorylation § use alternate electron acceptor, not O2 •However many organisms seemed not to contain any mitochondrion • •In past they were considered primarily amitochondriate • •However,… Amitochondriate organisms • •Organisms that do not possess mitochondria • • • • • •Primarily amitochondirate – originated before the acquisition of mitochondria Amitochondriate organisms euktree2011nostem.tif Entamoeba Giardia How are they generating energy? Trichomonas vaginalis TV_hydrogenom •Parasite of men – STD trichomoniasis •Posses Hydrogenosomes: •Double membrane bound organelle •no DNA •no cristae Hydrogenosomes pyruvate ASCT STK Hydrogenosomes pyruvate ASCT STK Hydrogenosomes pyruvate ASCT STK PFO Fe-Hyd Hydrogenosomes pyruvate ASCT STK Amitochondriate organisms euktree2011nostem.tif Entamoeba Giardia Amitochondriate organisms Entamoeba Giardia Intestinal parasite Beaver fever No organelle Intestinal parasite dysentery No organelle In cytosol pyruvate ADP acetate ethanol Alcohol dehydrogenase E ATP Acetyl-CoA Synthetase In cytosol pyruvate ADP acetate ethanol Alcohol dehydrogenase E ATP Acetyl-CoA Synthetase ? In cytosol pyruvate ADP acetate ethanol Alcohol dehydrogenase E ATP Acetyl-CoA Synthetase ? Amitochondriate organisms At this point of the lecture we still think they are truly amitochondriate… Do we? euktree2011nostem.tif Entamoeba Giardia Genes of mitochondrial origin Genes of mitochondrial origin Germot A et al. PNAS 1996;93:14614-14617 IS Atm1 X - factor Mitochondrial mature proteins CIA machinery – cytosolic Fe-S cluster assembly Cytosolic mature proteins Fe IscU IscU IscS Cys Cystein desulfurase Scaffold protein Hydrogenosome = mitochondrion Hydrogenosome = mitochondrion Sutak R et al. PNAS 2004;101:10368-10373 Other evidence: •Other proteins of mitochondrial origin targeted to hydrogenosome • •Similar targeting machinery euktree2011nostem.tif Entamoeba Giardia How about Giardia and Entamoeba Giardia intestinalis 1.CPN60 gene discovered in Giardia genome 2. 2.Subsequently Isc system (Fe-S cluster assembly system) was discovered and localized in Giardia This organelle was called Mitosome Giardia mitosomes •Shown to have common targeting system with Hydrogenosomes and Mitochondria • •TOM complex proteins have been identified • •However, TIM pore complex proteins have not yet been identified • •The only known function is Fe-S cluster assembly • •ATP is being imported into mitosome rather than made Entamoeba mitosomes CPN60 gene of mitochondrial origin Iron-Sulfur cluster assembly •Does not have Ics system but Nif system instead • •Does not localize into the organelle • Entamoeba mitosomes CPN60 gene of mitochondrial origin •Does not have Ics system but Suf system instead •Also functions in cyst formation – again crucial function To summarize… Three basic categories of organelles: Mitochondrion – genome, electron transport chain, Import machinery, Fe-S cluster synthesis Hydrogenosome – no genome, PFO/Hydrogenase – genrates energy, Import machinery, Fe-S cluster synthesis Mitosomes – tiny, no genome, does not generate energy, highly reduced import machinery, Fe-S cluster synthesis (usually) 5 minutes break…. To make it more complicated euk_tree2 •Infects gastro-intestinal tract of humans and animals. •9 species of Blastocystis that infect humans. •Strict anaerobe •Stramenopile Blasto3PEP2004 NucleusandOrganelle Blastocystis •Possess PFO and Fe-Hydrogenase è Therefore it is Hydrogenosome • •But also has genome • •And partial TCA cycle, and classical mitochondrial pyruvate dehydrogenase • •So it is Hydrogenosome/Mitochondria? • •There is several organisms whose organelle are bluring the bouindaries • •Extremely dynamic system Blastocystis organelle Classical mitochondria Hydrogenosomes § No oxidative phosphorylation (usually) § Anaerobic ATP generation producing H2 gas Mitosomes § No electron transport, oxidative phosphorylation § Some mitochondrial-derived proteins § Protein import apparatus and Fe-S cluster biogenesis § Unknown functions Anaerobic mitochondria § Electron transport/oxidative phosphorylation § use alternate electron acceptor, not O2 loss of genome What happens to mitochondria? To make it more complicated euk_tree2 Brevimastigomonas motovehiculus Classical mitochondria Hydrogenosomes § No oxidative phosphorylation (usually) § Anaerobic ATP generation producing H2 gas Mitosomes § No electron transport, oxidative phosphorylation § Some mitochondrial-derived proteins § Protein import apparatus and Fe-S cluster biogenesis § Unknown functions Anaerobic mitochondria § Electron transport/oxidative phosphorylation § use alternate electron acceptor, not O2 loss of genome What happens to mitochondria? Metopid Ciliates euk_tree2 Metopid Ciliates Metopid Ciliates Classical mitochondria Hydrogenosomes § No oxidative phosphorylation (usually) § Anaerobic ATP generation producing H2 gas Mitosomes § No electron transport, oxidative phosphorylation § Some mitochondrial-derived proteins § Protein import apparatus and Fe-S cluster biogenesis § Unknown functions Anaerobic mitochondria § Electron transport/oxidative phosphorylation § use alternate electron acceptor, not O2 loss of genome What happens to mitochondria? Metamonada_backbone.pdf Metamonada Trimastix PCT Trimastix pyriformis Monocercomonoides sp. Tritrichomonas foetus Pentatrichomonas hominis Trichomonas vaginalis Carpediemonas membranifera Kipferlia bialata Ergobibamus cyprinoides CLO NY0171 Chilomastix cuspidata Chilomastix caulleri Dysnectes brevis Giardia intestinalis Spironucleus barkhanus Spironucleus salmonicida Spironucleus vortens Trepomonas PC1 Trichomonads Carpediemonas-like organisms Diplomonads 67 Trimastix + Oxymonads Metamonada_backbone.pdf MROs of metamonada Trimastix PCT Trimastix pyriformis Monocercomonoides sp. Tritrichomonas foetus Pentatrichomonas hominis Trichomonas vaginalis Carpediemonas membranifera Kipferlia bialata Ergobibamus cyprinoides CLO NY0171 Chilomastix cuspidata Chilomastix caulleri Dysnectes brevis Giardia intestinalis Spironucleus barkhanus Spironucleus salmonicida Spironucleus vortens Trepomonas PC1 68 ATP generation Pyruvate Acetyl-CoA Acetate ADP ATP Pyruvate Acetyl-CoA Acetate ADP ATP PFO ASCT + SCS Trichomonas Pyruvate Acetyl-CoA Acetate ADP ATP PFO ACS Giardia 2 enzymes: PFO PFL 2 enzyme systems: ASCT_SCS ACS ATP generation Pyruvate Acetyl-CoA Acetate ADP ATP Pyruvate Acetyl-CoA Acetate ADP ATP PFO ASCT + SCS Trichomonas Pyruvate Acetyl-CoA Acetate ADP ATP PFO ACS Giardia 2 enzymes: PFO PFL 2 enzyme systems: ASCT_SCS ACS Glycine Cleavage system •Important part of amino acid metabolism • •Synthesize or degrades glycine • •In eukaryotes present in mitochondria • •4 component complex: •T_protein •P_protein •H_protein •L_protein 72 Glycine cleavage 72 Trimastix Trichomonads Carpediemonas- like organisms Diplomonads Trichomonads Diplomonads 73 Fornicata_Ancestor.pdf Metamonada Trichomonads Diplomonads 75 Giardia Spironucleus salmonicida Trichomonas Dysnectes brevis Other CLOs Gain of Acetyl-CoA synthetase -> Cytosolic ATP generation Loss of SCS/ASCT -> Organellar ATP generation Loss of Glycine Cleavage System Loss of Hydrogen Production, only ISC assembly and associated pathways remain Transfer of ACS into hydrogenosome -> regain of ATP production Reduction Step By Step Classical mitochondria Hydrogenosomes § No oxidative phosphorylation (usually) § Anaerobic ATP generation producing H2 gas Mitosomes § No electron transport, oxidative phosphorylation § Some mitochondrial-derived proteins § Protein import apparatus and Fe-S cluster biogenesis § Unknown functions Anaerobic mitochondria § Electron transport/oxidative phosphorylation § use alternate electron acceptor, not O2 loss of genome What happens to mitochondria? Monocercomonoides euk_tree2 Metamonada_backbone.pdf Monocercomonoides Trimastix PCT Trimastix pyriformis Monocercomonoides sp. Tritrichomonas foetus Pentatrichomonas hominis Trichomonas vaginalis Carpediemonas membranifera Kipferlia bialata Ergobibamus cyprinoides CLO NY0171 Chilomastix cuspidata Chilomastix caulleri Dysnectes brevis Giardia intestinalis Spironucleus barkhanus Spironucleus salmonicida Spironucleus vortens Trepomonas PC1 78 Metamonada_backbone.pdf Monocercomonoides Trimastix PCT Trimastix pyriformis Monocercomonoides sp. Tritrichomonas foetus Pentatrichomonas hominis Trichomonas vaginalis Carpediemonas membranifera Kipferlia bialata Ergobibamus cyprinoides CLO NY0171 Chilomastix cuspidata Chilomastix caulleri Dysnectes brevis Giardia intestinalis Spironucleus barkhanus Spironucleus salmonicida Spironucleus vortens Trepomonas PC1 79 Monocercomonoides What happens to mitochondria?