Scheffler Immo E. Mitochondria

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EVOLUTIONARY ORIGIN OF MITOCHONDRIA 11

The arrival of oxygen on earth was not an unmixed blessing. Apart from

the fact that uncontrolled respiration is combustion ( “ ﬁ re ” ) with all of its

destructive aspects, even a highly controlled reaction such as respiration can

generate “ reactive oxygen species ” (ROS) such as superoxide, the hydroxyl

radical, and hydrogen peroxide, whose potential for destruction of biological

macromolecules may provide a basis of an explanation for an age - related

deterioration of the mitochondrial genomes and perhaps even for senescence

and death (13, 14) . According to F. Jacob, “ the most important inventions of

evolution are sex and death. ” Did death from free radical bombardments

precede programmed cell death, or apoptosis, as it is now called? Is the mecha-

nism of apoptosis absolutely dependent on mitochondrial functions (see

Chapter 7 )?

It is more fruitful to pursue the molecular fossil record for a proto - mito-

chondrion. Phylogenetic data support an origin of mitochondria from the

alpha - proteobacterial order Rickettsiales (15 – 18) . All mitochondrial genomes

encode two ribosomal RNAs, and most encode 20 or more tRNAs (a notable

exception may be the protozoans such as trypanosomes). As discussed in more

detail elsewhere in this book, mammalian mtDNA also encodes 13 peptides

for complexes of the OXPHOS system, and additional mitochondrial genes

are found in other organisms. From the point of view of evolution, it is particu-

larly illuminating to consider the four genes for complex II: They are all

nuclear in mammals, but diverse organisms have been found recently in which

the mtDNA still carries one or more of the same genes. The iron protein, Ip,

and the ﬂ avoprotein, Fp, of complex II constitute the enzyme succinate dehy-

drogenase (SDH) of the citric acid cycle. SDH is attached to the inner mito-

chondrial membrane via two integral membrane proteins, or anchor proteins,

II - 3

and C

II - 4

. Ip and Fp are remarkably well conserved in evolution at the

protein sequence level and even at the nucleotide sequence level from pro-

karyotes to eukaryotes. This has made it possible to identify and clone the

corresponding genes from many organisms, starting with bacteria (19) . As a

ﬁ rst approximation and from a biochemist ’ s simplest perspective, the complex

in Escherichia coli looks very much like the complex in mammalian mitochon-

dria. In bacteria such as E. coli the four genes form an operon and are coor-

dinately expressed. In mammals the four genes (SDHA, SDHB, SDHC, and

SDHD) are dispersed over various chromosomes. The SDHB (Ip) and SDHC

II - 3

) genes are found on the mitochondrial genome in the red algae Chondrus

crispus (20, 21) , and three of the four genes (SDHB, SDHC, and SDHD) are

found on the mitochondrial genome of another red alga, Porphyra purpurea ,

and on a phylogenetically distant heterotrophic zooﬂ agellate, Reclinomas

americana (22) . Apparently, the transfer of these genes from the mtDNA to

the nuclear genome has not (yet) occurred, and these organisms represent

examples of “ intermediates ” in the evolution of a maximally reduced mito-

chondrial genome. In full agreement with the same train of thought, Chondrus

crispus mtDNA still retains four genes for ribosomal proteins, while certain

12 EVOLUTIONARY ORIGIN OF MITOCHONDRIA

plant mtDNAs encode even more ribosomal proteins. In vertebrates all mito-

chondrial ribosomal protein genes have been translocated to the nucleus.

Studies such as these have provided supporting arguments in favor of a mono-

phyletic origin of mitochondria (22) . The explosion of information on genomic

sequences from many organisms has provided additional examples (6, 23 – 27) .

The diversity of genes still found in mitochondrial genomes can be explained

by a random gene loss over time from a common ancestral genome.

When the complete sequence of the mitochondrial genome of the freshwa-

ter protozoon Reclinomonas americana was completed, some rather surprising

ﬁ ndings strengthened the hypothesized link between mitochondria and eubac-

teria - like endosymbionts ( “ the mitochondrion that time forgot ” ) (28, 29) . With

69,034 basepairs the genome is not exceptional in size, but the existence of at

least 97 genes on this genome was unexpected, since many of the other mito-

chondrial genomes now known have far fewer genes, even when their size was

an order of magnitude greater (see Chapter 4 ). The Reclinomonas mtDNA

encodes 23 components of the electron transport chain and oxidative phos-

phorylation apparatus (compared to 13 in mammals), 18 mitoribosomal pro-

teins (none in mammals, some in plants), 27 distinct tRNAs, 3 ribosomal RNAs,

including a 5S RNA not found in mammals, an RNA component of ribonucle-

ase P (used in RNA processing (see Chapter 4 ), and four genes of a multisub-

unit RNA polymerase resembling a eubacterial - type polymerase. The latter

ﬁ nding is particularly intriguing and unique so far. A preliminary interpreta-

tion is that a eubacterial multisubunit RNA polymerase may have been lost

during evolution from all other mitochondria (analyzed so far) and replaced

by a single - subunit enzyme, encoded by the nuclear genome, with similarities

to the bacteriophage T3/T7 enzyme (see Chapter 4 ). Since then a large number

of genes encoding mitochondrial enzymes for transcription and DNA replica-

tion have been identiﬁ ed and analyzed in fungi, animals, and plants, leading

Shutt and Gray (30) to hypothesize that another early event in the evolution

of the eukaryotic cell (and almost coincident with the establishment of endo-

symbiosis) was the acquisistion of several replication genes from an ancestor

of T - odd bacteriopages. Furthermore, these authors propose that a phage RNA

polymerase initially functioning in primer formation for DNA replication

evolved into the present day mitochondrial RNA polymerase.

Ribosomal RNA sequence comparisons have served as a predominant

source of data in the construction of phylogenetic trees and evolutionary

relationships, and rRNAs have been referred to as the “ ultimate molecular

chronometer ” (31). These ancient, ubiquitous, and functionally identical mol-

ecules and their genes have helped to reevaluate taxonomic subdivisions

between existing life forms on earth. It is not the intent here to enter the

debate about how many kingdoms there should be (the eukaryotes, Animalia,

Plantae, Fungi, Protista, and the prokaryotes, Eubacteria and Archaebacteria),

but to emphasize a major distinction between archaebacteria, eubacteria, and

eukaryotes. Various analyses have shown a closer relationship of archaebacte-

ria to eukaryotes, as well as many similarities in genome organization and gene

EVOLUTIONARY ORIGIN OF MITOCHONDRIA 13

expression between plastid (chloroplast) and eubacterial genomes, in support

of the endosymbiotic origin of plastids. A case for a eubacterial origin of mito-

chondria was more difﬁ cult to make on the basis of the “ molecular biology as

practiced in mitochondria ” (3) , but rRNA sequence analyses strongly favor

the same conclusion. Various aspects such as information content and organi-

zation of the genome, transcription, and translation will be covered in separate

chapters when evolutionary comparisons will be instructive, but not central to

the discussion.

Mitochondria were found to have their own machinery for protein synthe-

sis. A full treatment of this subject is reserved for another chapter. However,

a frequently quoted example in favor of the endosymbiont hypothesis can be

mentioned here. Cytoplasmic protein synthesis employs larger ribosomal sub-

units with more protein subunits compared to mitochondrial ribosomal sub-

units, which in turn resemble bacterial ribosomes (32, 33) . Cycloheximide is a

speciﬁ c inhibitor of cytoplasmic protein synthesis, whereas chloramphenicol

inhibits the bacterial and mitochondrial translation machinery (34) . The dis-

tinction based on the differential sensitivity to these two inhibitors has been

the basis for many elegant experiments elucidating the speciﬁ c mitochondrial

translation products. On the other hand, a closer look at these similarities in

ribosomal properties in bacteria and mitochondria made it apparent that they

were rather superﬁ cial, hiding the uniqueness and variability of mitochondrial

ribosomes (for a detailed discussion see Gray and Doolittle (2) ).

Phylogenetic trees based on rRNA comparisons (for the small as well as

for the large subunit) have not only proved convincing in relating plant mito-

chondria to eubacteria, but have also helped to identify the living eubacteria

that are the closest relative of mitochondria: Members of the α - subdivision of

purple bacteria had been suspected initially on biochemical grounds, and

rRNA sequence comparisons strengthen this conclusion considerably. In con-

trast, similar comparisons with animal, ciliate, and fungal mitochondrial rRNA

sequences are less convincing, because of signiﬁ cant primary sequence diver-

gences. Nevertheless, the eubacterial ancestry of mitochondria is not seriously

challenged by such data. Left open was the question of the monophyletic

origin of mitochondria — that is, whether the symbiosis between proto - eukary-

otes and a eubacterial species was a singular event in evolution, or occurred

more than once. In particular, did plant mitochondria arise independently of

animal mitochondria, for example? The absence of a common root for current

rRNA trees had been interpreted by some authors in support of a biphyletic

or multiphyletic origin (see reference 3 for a summary of the evidence and

interpretation), but the same authors also raised the possibility of artifactual

trees generated by the choice of algorithms, the selection of sequences, and

other methodological subtleties. The most recent studies on complex II genes

in the red algae, Porphyra purpurea , and the zooﬂ agellate, Reclinomonas amer-

icana, have favored a monophyletic lineage for mitochondria (22) .

Has the limit been reached in the reduction of the mitochondrial genome

(35) ? It is generally agreed that the remaining peptides encoded by mtDNA

14 EVOLUTIONARY ORIGIN OF MITOCHONDRIA

are extremely hydrophobic peptides that would not be readily imported by

the mitochondrial translocation machinery. On the other hand, two hydropho-

bic anchor peptides of complex II are encoded by nuclear genes in most

organisms examined so far. Theoretical arguments have been forwarded pre-

dicting that the mitochondrial genome need ultimately retain only two genes

encoding peptides in addition to the ribosomal RNAs and tRNAs (36 – 38) . The

two genes singled out are the apo - cytochrome b and the COX1 genes encoding

peptides with more than three or four hydrophobic transmembrane segments.

Claros et al. (37) deﬁ ne a property referred to as mesohydrophobicity (average

hydrophobicity over an extended stretch of a polypeptide chain) to make

quantitative predictions. The hypothesis was also tested by constructing a

series of chimeric genes expressed in the nucleus yielding cytoplasmically

synthesized peptides. These peptides had a mitochondrial targeting sequence,

a reporter enzyme, and one or more of the eight transmembrane segments of

apocytochrome b. A complete interpretation of these results, however, also

requires a more detailed understanding of how such integral membrane pro-

teins with multiple transmembrane segments are localized in the inner mito-

chondrial membrane, an understanding that is still expanding today (see

Chapter 4 ).

Some recent discoveries provide convincing evidence that the complete loss

of the mitochondrial genome has in fact occurred in evolution, raising several

new questions (35) . Such mitochondria would not be expected to be able to

support respiration. What would be their function if oxidative phosphorylation

was no longer observed? And how would such an organelle still be recognized

as a mitochondrion? An emerging consensus is that several phylogenetically

distinct lineages of eukaryotes may have undergone a reductive evolution in

the course of adapting to anaerobic or micro - aerobic environments (8, 39 – 44) .

In other words, the original mitochondrial endosymbiont has evolved inde-

pendently in diverse species inhabiting such environments, resulting in a

collection of morphologically, genetically, and functionally heterogeneous

organelle variants that include anaerobic and aerobic mitochondria, hydro-

genosomes, and mitosomes (45) . The best - studied example is the parasitic

ﬂ agellate protist Trichomonas vaginalis , an air tolerating anaerobe which has

an energy producing organelle referred to as a hydrogenosome. Hydrogeno-

somes have been identiﬁ ed in a wide variety of organisms (rumen - dwelling

ciliates, fungi, free living ciliates), and by several criteria such hydrogenosomes

are believed to be highly modiﬁ ed mitochondria (16, 41, 42, 46) . They generally

lack DNA, are devoid of cytochromes and most citric acid cycle enzymes, and

produce hydrogen by the use of enzymes usually found in anaerobes (pyru-

vate : ferredoxin oxidoreductase, hydrogenase). They make ATP by substrate -

level phosphorylations involving succinyl CoA synthetase, a Krebs cycle

enzyme. However, they have a double membrane, they import proteins post -

translationally, and they divide by ﬁ ssion. Further evidence for the relationship

of hydrogenosomes to mitochondria comes from the characterization of

Trichomonas nuclear genes of the heat - shock proteins Hsp10, Hsp60, and

Hsp70 (47) . These proteins are found in the hydrogenosome, and by phyloge-

netic analysis these Hsps have been found to be among the “ most reliable

tracers of the eubacterial ancestry of both the mitochondrion and the chloro-

plast ” (35) . Thus, the evidence supports a common origin for hydrogenosomes

and mitochondria, rather than the hypothesis of an independent endosymbi-

otic event involving an anearobic eubacterium yielding a hydrogenosome (48) .

A hydrogenosome with a genome has been identiﬁ ed in the anaerobic ciliate

Nyctotherus ovalis , which thrives in the hindgut of cockroaches (49) . More

recently it was shown that it is a rudimentary genome encoding components

of a mitochondrial electron transport chain (39) . The authors interpret the

Nycotherus hydrogenosome as a “ true missing link ” between mitochondria

and hydrogenosomes. As discussed by Gray (50) , the hydrogenosome ’ s

past is still muddied by recent ﬁ ndings in the genome - less hydrogenosome

of Trichomonas vaginalis . Two groups arrived at conﬂ icting evolutionary

positions, based on the sequence analysis of two nuclear genes encoding

T. vaginalis homologues of the mitochondrial complex I (51, 52) .

Such evidence may strengthen arguments over which genes were originally

present in the proto - mitochondria. However, at this time the more challenging

questions to ask are questions about the evolution of more individual differ-

ences between organisms: the size of the mitochondrial genome, the organiza-

tion of the remaining genes, and some rather unusual mechanisms in gene

expression. Plants have mitochondrial genomes that are one to two orders of

magnitude larger than metazoan mitochondrial genomes, without a propor-

tionate increase in the number of genes. What is this “ junk DNA ” doing and

where did it come from? Mitochondrial plasmids and transposon - like parasitic

DNA have invaded even mitochondrial genomes. Understanding the evolu-

tion of the mind - boggling RNA editing process in trypanosomes (see Chapter

4 ) is a major challenge by itself. Mitochondrial DNA sequences continue to

to be altered by mutations, providing an ever - richer database for evolutionary

studies — not about the evolution of mitochondria per se, but about the much

more recent evolution of closely related species such as the primates, including

humans (see Chapter 8 ).

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Mitochondria, Second Edition, by Immo E. Schefﬂ er

STRUCTURE AND MORPHOLOGY.

INTEGRATION INTO THE CELL

Summary

A detailed morphological description of mitochondria became possible only with

the perfection of the electron microscope, when their double membranes and the

existence of cristae were established. The shape and size of mitochondria is quite

variable, and most signiﬁ cantly, there is an amazing variation in the number and

structure of the cristae. While it is clear that cristae serve to increase the surface area

of the inner membrane, the shape of cristae as revealed by cross sections viewed in

the electron microscope can still be a puzzle. More reﬁ ned three - dimensional images

of outer and inner mitochondrial membranes and cristae have recently been achieved

by electron tomography, but the full signiﬁ cance of novel ﬁ ndings (cristae junctions)

remains to be generalized and even fully explained.

A very challenging and exciting new area of research is concerned with the

dynamics of the mitochondrial morphology and the distribution of mitochondria

within specialized cells. They have to divide by ﬁ ssion; in many organisms they

also constantly fuse with each other, and their interaction with the cytoskeleton

assures their distribution in dividing and differentiating cells, as well as their optimal

localization within cells. The elucidation of components of the fusion and ﬁ ssion

apparatus has been a frontier where major progress has been achieved in the past

decade.

3.1 STRUCTURE AND MORPHOLOGY

When the nineteenth - century morphologists with their light microscopes dis-

covered grains (chondria) and ﬁ laments (mito), they could not be sure that

they were all looking at the same functionally distinct structure in different

cells, and undoubtedly they were not. Nevertheless, variability in number and

shape must have been apparent. A more systematic approach became possible

when selective staining methods were used (fuchsin, Altman, 1890; crystal

violet, Benda, 1898; and Janus green, Michaelis, 1898). The capacity of mito-

chondria to reduce Janus green, an indicator of oxidation – reduction reactions,

should have been a major clue, which was evidently missed at the time or,

rather, not interpreted fully.

Major progress in the characterization and understanding of the morphol-

ogy and ultrastructure of mitochondria was not possible until the techniques

for electron microscopy and specimen preparation were perfected, but the use

of the light microscope in the study of mitochondria was revived in the past

few decades by two technical innovations. First, mitochondria could be viewed

with the ﬂ uorescence microscope in permeabilized cells after staining with

speciﬁ c antibodies and secondary antibodies conjugated to ﬂ uorescent dyes.

Another very important discovery was the observation that certain rhodamine

derivatives and other lipophilic “ vital dyes ” would stain mitochondria, because

the dye became concentrated in these organelles by an uptake mechanism

driven by the membrane potential (1) . The biophysical basis of this phenom-

enon will be addressed in a later chapter. Rhodamine staining can be per-

formed with live cells; and in combination with time - lapse photography, and

more recently image intensiﬁ cation and videotaping, it has opened up a window

on the dynamic behavior of mitochondria (changes in size and shape), their

distribution in the cytosol, and their redistribution following certain perturba-

tions of the cytoskeleton, for example.

While the resolution of the light/ﬂ uorescence microscope is limited, it does

offer a view of the entire cell and the global distribution of mitochondria

within it (see Section 3.2 for further discussion). With the increased under-

standing of protein targeting sequences, it has become possible to target

recombinant reporter proteins such as the green ﬂ uorescent protein (GFP) to

the mitochondrial matrix, or even the intermembrane space (see, for example,

references 2 and 3 ). Such “ labeled ” mitochondria can be studied in relation to

other subcellular structures identiﬁ ed by probes with a second chromophore.

Mitochondria have no ﬁ xed size, but typically in hepatocytes and ﬁ broblasts

they have a sausage - like shape with dimensions of 3 – 4 μ m (length) and ∼ 1 μ m

(diameter); as a ﬁ rst approximation they do in fact resemble small bacteria in

size and shape. The number of mitochondria per cell is a parameter that

appears to vary signiﬁ cantly from cell type to cell type, and this will become

an important consideration when mitochondrial biogenesis is considered in

the context of cell differentiation and specialization (see also below). Esti-

mates from serial sections of cells yield values in the range of a few hundred

STRUCTURE AND MORPHOLOGY 19

20 STRUCTURE AND MORPHOLOGY. INTEGRATION INTO THE CELL

to a few thousand per cell — for example, ∼ 800 per hepatocyte. Specialized

vertebrate cells show signiﬁ cant variations. The human oocyte is estimated to

contain > 100,000 mitochondria, while spermatozoa have relatively few (but

these may be exceptionally large). A somewhat simpliﬁ ed view of mitochon-

dria in some sperm cells is that of a small number of highly elongated mito-

chondria wrapped around the axoneme of the sperm tail. In lower eukaryotes

the number can also be substantial, for example, 500,000 mitochondria have

been found in the giant amoebae Chaos chaos . The actual number is most

likely not the most relevant parameter to consider. Of far more signiﬁ cance

may be the mitochondrial volume as a fraction of the cellular volume, or even

the total surface area of the mitochondrial cristae per cell (see below). Clearly,

details in the morphology depend on the organism. Another interesting number

from a geneticist ’ s point of view is the number of mitochondrial genomes per

cell. This subject will be taken up in a later chapter, but it can be mentioned

here that there are multiple mtDNAs per mitochondrion and therefore thou-

sands of mtDNAs per cell.

Morphological studies of trypanosomes drew attention to a structure at the

base of the ﬂ agellum referred to as kinetoplast. Over 70 years ago the kineto-

plast was found to be stainable with Janus green (oxidation – reduction) and,

shortly thereafter, to give a positive Feulgen reaction (nucleic acid). Later,

electron microscopic examination revealed cristae similar to those found in

mitochondria, and the kinetoplast was recognized to be a highly specialized

mitochondrion. The ﬁ rst deﬁ nite report of DNA in this organelle appeared

in 1960 (4) , but the kinetoplast was considered to be so specialized and

exceptional that the ﬁ nding was not immediately generalized to all

mitochondria.

A new chapter in the study of the morphology of mitochondria began with

the perfection of the electron microscope as a tool for biological studies. The

design of the microscope was a physics and engineering problem. The prepara-

tion of a sufﬁ ciently thin specimen, a slice of a single cell many times thinner

than the diameter of the cell, was a challenge that required a more empirical

approach. In sum, the tissue had to be ﬁ xed, dehydrated, embedded “ in plastic, ”

stained, and sectioned to give a specimen that could hold together in a high

vacuum, provide contrxast in the ﬁ nal image based on differential diffraction

of the electron beam (uptake of electron - dense stains such as uranyl acetate

and osmium tetroxide by different constituents of the cell), and still reﬂ ect

biological reality — that is, meaningful images of subcellular structures. The

number of skeptics was rapidly diminished by the spectacular images pro-

duced by the pioneers at the Rockefeller Institute. A new view of the morphol-

ogy of individual mitochondria emerged from the studies of Palade and his

colleagues, and of Sjostrand, who not only conﬁ rmed that mitochondria are

membrane - bound organelles, but recognized that there were two membranes:

an outer membrane and an inner membrane which was convoluted and folded

into cristae mitochondriales (Palade) (Figures 3.1 and 3.2 ) . It should also be

reemphasized that the development of buffers for cell fractionation studies