Endosymbiosis and the Origin of Eukaryotes: Are mitochondria really just bacterial symbionts?

1. Endosymbiosis and the Origin of Eukaryotes:Are mitochondria really just bacterial symbionts? Timothy G. Standish, Ph. D.

2. Outline • Mitochondria - A very brief overview • Endosymbiosis - Theory and evidence • Archaezoa - Eukaryotes lacking mitochondria • Gene expression - Mitochondrial proteins coded in the nucleus • Mitochondrial genetic codes • Gene transport - Mitochondria to nucleus • Conclusions

3. Mitochondria • Mitochondria are organelles found in most eukaryotic organisms. • The site of Krebs cycle and electron transport energy producing processes during aerobic respiration • Are inherited only from the mother during sexual reproduction in mammals and probably all other vertebrates. • Because of their mode of inheritance genetic material found in mitochondria appears to be useful in determining the maternal lineage of organisms.

4. Outer membrane Inner membrane mtDNA Mitochondria Matrix Inter membrane space

5. Extranuclear DNA • Mitochondria and chloroplasts have their own DNA • This extranuclear DNA exhibits non-Mendelian inheritance • Recombination is known between some mt and ctDNAs • Extranuclear DNA may also be called cytoplasmic DNA • Generally mtDNA and ctDNA is circular and contains genes for multimeric proteins, some portion of which are also coded for in the nucleus • Extranuclear DNA has a rate of mutation that is independent of nuclear DNA • Generally, but not always, all the RNAs needed for transcription and translation are found in mtDNA and ctDNA, but only some of the protein genes

6. mtDNA • Mitochondrial DNA is generally small in animal cells, about 1.65 kb • In other organisms sizes can be more than an order of magnitude larger • Plant mtDNA is highly variable in size and content with the large Arabidopsis mtDNA being 200 kb. • The largest known number of mtDNA protein genes is 97 in the protozoan Riclinomonas mtDNA of 69 kb. • “Most of the genetic information for mitochondrial biogenesis and function resides in the nuclear genome, with import into the organelle of nuclear DNA-specified proteins and in some cases small RNAs.” (Gray et al.,1999)

7. Endosymbiosis

8. Origin of Eukaryotes Two popular theories presupposing naturalism seek to explain the origin of membrane-bound organelles: 1Endosymbiosis to explain the origin of mitochondria and chloroplasts (popularized by Lynn Margulis in 1981) 2Invagination of the plasma membrane to form the endomembrane system

9. Mitochondria Origin of Eukaryotes Two popular theories presupposing naturalism seek to explain the origin of membrane-bound organelles: 1Endosymbiosis to explain the origin of mitochondria and chloroplasts (popularized by Lynn Margulis in 1981) 2Invagination of the plasma membrane to form the endomembrane system

10. Endoplasmic Reticulum Mitochondria Nucleus Golgi Body Chloroplast Origin of Eukaryotes Two popular theories presupposing naturalism seek to explain the origin of membrane-bound organelles: 1Endosymbiosis to explain the origin of mitochondria and chloroplasts (popularized by Lynn Margulis in 1981) 2Invagination of the plasma membrane to form the endomembrane system

11. Endoplasmic Reticulum Mitochondria Nucleus Golgi Body Chloroplast Origin of Eukaryotes Two popular theories presupposing naturalism seek to explain the origin of membrane-bound organelles: 1Endosymbiosis to explain the origin of mitochondria and chloroplasts (popularized by Lynn Margulis in 1981) 2Invagination of the plasma membrane to form the endomembrane system

12. How Mitochondria Resemble Bacteria Most general biology texts list ways in which mitochondria resemble bacteria. Campbell et al. (1999) list the following: • Mitochondria resemble bacteria in size and morphology. • They are bounded by a double membrane: the outer thought to be derived from the engulfing vesicle and the inner from bacterial plasma membrane. • Some enzymes and inner membrane transport systems resemble prokaryotic plasma membrane systems. • Mitochondrial division resembles bacterial binary fission • They contain a small circular loop of genetic material (DNA). Bacterial DNA is also a circular loop. • They produce a small number of proteins using their own ribosomes which look like bacterial ribosomes. • Their ribosomeal RNA resembles eubacterial rRNA.

13. How Mitochondria Don’tResemble Bacteria • Mitochondria are not always the size or morphology of bacteria: • In some Trypanosomes (i.e., Trypanosoma brucei) mitochondria undergo spectacular changes in morphology that do not resemble bacteria during different life cycle stages (Vickermann, 1971) • Variation in morphology is common in protistans, “Considerable variation in shape and size of the organelle can occur.” (Lloyd, 1974 p 1) • Mitochondrial division and distribution of mitochondria to daughter cells is tightly controlled by even the simplest eukaryotic cells

14. How Mitochondria Don’tResemble Bacteria • Circular mtDNA replication via D loops is different from replication of bacterial DNA (Lewin, 1997 p 441). • mtDNA is much smaller than bacterial chromosomes. • Mitochondrial DNA may be linear; examples include: Plasmodium, C. reinhardtii, Ochromonas, Tetrahymena, Jakoba (Gray et al., 1999). • Mitochondrial genes may have introns which eubacterial genes typically lack (these introns are different from nuclear introns so they cannot have come from that source) (Lewin, 1997 p 721, 888). • The genetic code in many mitochondria is slightly different from bacteria (Lewin, 1997).

15. Archaezoa

16. Giardia - A “Missing Link”? • The eukaryotic parasite Giardia has been suggested as a “missing link” between eukaryotes and prokaryotes because it lacks mitochondria (Friend, 1966; Adam, 1991) thus serving as an example of membrane invagination but not endosymbiosis • Giardia also appears to lack smooth endoplasmic reticulum, peroxisomes and nucleoli (Adam, 1991) so these must have either been lost or never evolved

17. A Poor “Missing Link” • As a “missing link” Giardia is not a strong argument due to its parasitic life cycle which lacks an independent replicating stage outside of its vertebrate host • Transmission is via cysts excreted in feces followed by ingestion • As an obligate parasite, to reproduce, Giardia needs other more derived (advanced?) eukaryotes • Some other free-living Archaezoan may be a better candidate

18. Origin of Giardia • Giardia and other eukaryotes lacking mitochondria and plastids (Metamonada, Microsporidia, and Parabasalia ) have been grouped by some as “Archaezoa” (Cavalier-Smith, 1983; Campbell et al., 1999 p 524-6) • This name reflects the belief that these protozoa split from the group which gained mitochondria prior to that event. • The discovery of a mitochondrial heat shock protein (HSP60) in Giardia lamblia (Soltys and Gupta, 1994) has called this interpretation into question. • Other proteins thought to be unique to mitochondria, HSP70 (Germot et al., 1996), chaperonin 60 (HSP60) (Roger et al., 1996; Horner et al., 1996) and HSP10 (Bui et al., 1996) have shown up in Giardia’s fellow Archaezoans

19. Origin of Archaezoa • The authors who reported the presence of mitochondrial genes in amitochondrial eukaryotes all reinterpreted prevailing theory in saying that mitochondria must have been present then lost after they had transferred some of their genetic information to the nucleus. • The hydrogenosome, a structure involved in carbohydrate metabolism found in some Archaezoans (Muller, 1992), is now thought to represent a mitochondria that has lost its genetic information completely and along with that loss, the ability to do the Krebs cycle (Palmer, 1997). • Alternative explanations include transfer of genetic material from other eukaryotes and the denovo production of hydrogenosomes by primitive eukaryotes.

20. Origin of Archaezoa:Mitochondrial Acquisition

21. mtGenes Origin of Archaezoa:Gene Transfer and Loss Lost genetic material

22. Origin of Archaezoa:Option 1 - Mitochondrial Eukaryote Production

23. Hydrogenosome Origin of Archaezoa:Option 2 - Mitochondrial DNA Loss/Hydrogenosome production

24. Origin of Archaezoa:Option 2A - Mitochondria/Hydrogenosome Loss

25. Gene Transport

26. “All in all then, the host nucleus seems to be a tremendous magnet, both for organellar genes and for endosymbiotic nuclear genes.” Palmer, 1997

27. Fusion of Rickettsia with either a nucleus containing Archaezoan or an archaebacterium Rickettsia Host Cell Primitive eukaryote DNA reduction/transfer to nucleus Ancestral eukaryote (assuming a nucleus) Steps in Mitochondrial Acquisition:The Serial Endosymbiosis Theory

28. Fusion of proteobacterium with an archaebacterium Hydrogen producing proteobacterium Hydrogen requiring archaebacterium Ancestral eukaryote With nucleus containing both archaebacterium and proteobacterium genes DNA reduction/transfer nucleus production Steps in Mitochondrial Acquisition:The Hydrogen Hypothesis

29. Microsporidia, and Parabasalia Bacteria Metamonada Eukaryota Bacteria mtDNA loss Hydrogenosome/ mitochondria loss mtDNA loss Gene transfer Origin of Life Phylogeny Cell fusion

30. Timing of Gene Transfer • Because gene transfer occurred in eukaryotes lacking mitochondria, and these are the lowest branching eukaryotes known: • Gene transfer must have happened very early in the history of eukaryotes. • The length of time for at least some gene transfer following acquisition of mitochondria is greatly shortened. • No plausible mechanism for movement of genes from the mitochondria to the nucleus exists although intraspecies transfer of genes is sometimes invoked to explain the origin of other individual nuclear genes.

31. Gene Expression

32. Cytoplasmic Production of Mitochondrial Proteins • Mitochondria produce only a small subset of the proteins used in the Krebs cycle and electron transport. The balance come from the nucleus • As mitochondrial genomes vary spectacularly between different groups of organisms, some of which may be fairly closely related, if all came from a common ancestor, different genes coding for mitochondrial proteins must have been passed between the nucleus and mitochondria multiple times

33. The Unlikely Movement of Genes Between Mitochondria and the Nucleus Movement of genes between the mitochondria and nucleus seems unlikely for at least two reasons: • Mitochondria do not always share the same genetic code with the cell they are in • Mechanisms for transportation of proteins coded in the nucleus into mitochondria seem to preclude easy movement of genes from mitochondria to the nucleus

34. Cytoplasm AAAAAA Nucleus G Export Mitochondrion Chloroplast Protein Production Mitochondria and Chloroplasts

35. Cytoplasm Nucleus Mitochondrion Chloroplast Protein Production Mitochondria and Chloroplasts

36. Outer membrane Inner membrane Protein Production Mitochondria Matrix Inter membrane space

37. Outer membrane Leader sequence binding receptor ATP P +ADP ATP P +ADP MLSLRQSIRFFKPATRTLCSSRYLL Inner membrane Protein Production Mitochondria Inter membrane space Matrix

38. Outer membrane Leader sequence binding receptor Peptidease cleaves off the leader MLSLRQSIRFFKPATRTLCSSRYLL Inner membrane Inter membrane space Matrix Protein Production Mitochondria

39. Outer membrane Leader sequence binding receptor Inner membrane Protein Production Mitochondria MLSLRQSIRFFKPATRTLCSSRYLL Inter membrane space Matrix

40. Outer membrane Leader sequence binding receptor Inner membrane Protein Production Mitochondria Inter membrane space Matrix

41. Outer membrane Leader sequence binding receptor Hsp60 Hsp60 Inner membrane Inter membrane space Chaperones Matrix Protein Production Mitochondria

42. Outer membrane Leader sequence binding receptor Inner membrane Inter membrane space Mature protein Matrix Protein Production Mitochondria

43. M L S L Polar R Q S First 12 residues are sufficient for transport to the mitochondria Non- polar I R F F K P A T R T L Polar Recognized by peptidase? C S S R Y L P Yeast Cytochrome C Oxidase Subunit IV Leader Neutral Non-polar Polar Basic Acidic • This leader does not resemble other eukaryotic leader sequences, or other mtProtein leader sequences. • Probably forms an a helix • This would localize specific classes of amino acids in specific parts of the helix • There are about 3.6 amino acids per turn of the helix with a rise of 0.54 nm per turn MLSLRQSIRFFKPATRTLCSSRYLL

44. Charged leader sequence signals for transport to mitochondria First cut Second cut Uncharged second leader sequence signals for transport across inner membrane into the intermembrane space Yeast Cytochrome C1 Leader • Cytochrome c functions in electron transport and is thus associated with the inner membrane on the intermembrane space side • Cytochrome c1 holds an iron containing heme group and is part of the B-C1 (III) complex • C1 accepts electrons from the Reiske protein and passes them to cytochrome c MFSNLSKRWAQRTLSKTLKGSKSAAGTATSYFE-KLVTAGVAAAGITASTLLYANSLTAGA-------------- Neutral Non-polar Polar Basic Acidic

45. Outer membrane Inner membrane Protein Production Mitochondria Matrix Inter membrane space

46. Outer membrane Leader sequence binding receptor ATP P +ADP ATP P +ADP Peptidease cleaves off the leader Inner membrane Protein Production Mitochondria Inter membrane space Matrix

47. Outer membrane Leader sequence binding receptor Inner membrane Protein Production Mitochondria Inter membrane space Matrix

48. Outer membrane Leader sequence binding receptor Inner membrane Protein Production Mitochondria Inter membrane space Matrix

49. Outer membrane Leader sequence binding receptor Inner membrane Protein Production Mitochondria Inter membrane space Matrix

50. Outer membrane Leader sequence binding receptor Inner membrane Protein Production Mitochondria Inter membrane space Matrix