Scheffler Immo E. Mitochondria
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REFERENCES
1 . Klausner , R. D. , Rouault , T. A. , and Harford , J. B. ( 1993 ) Cell 72 , 19 – 28 .
2 . Philpott , C. C. , Klausner , R. D. , and Rouault , T. A. ( 1994 ) Proc. Natl. Acad. Sci. USA
91 , 7321 – 7325 .
3 . Hentze , M. W. and K ü hn , L. C. ( 1996 ) Proc. Natl. Acad. Sci. USA 93 , 8175 – 8182 .
4 . Scheffl er , I. E. ( 1986 ) Biochemical genetics of respiration - defi cient mutants of
animal cells . In Morgan , M. J. , editor. Carbohydrate Metabolism in Cultured Cells ,
Plenum Press , London , pp. 77 – 109 .
5 . Broderick , K. E. , Potluri , P. , Zhuang , S. , Scheffl er , I. E. , Sharma , V. S. , Pilz , R. B. , and
Boss , G. R. ( 2006 ) Exp. Biol. Med . 231 , 641 – 649 .
6 . Lanks , K. W. and Li , P. - W. ( 1988 ) J. Cell Physiol . 135 , 151 – 155 .
7 . Zitomer , R. S. and Lowry , C. V. ( 1992 ) Microbiol. Rev . 56 , 1 – 11 .
8 . Daum , G. ( 1985 ) Biochim. Biophys. Acta 822 , 1 – 42 .
9 . Hackenbrock , C. R. , Chazotte , B. , and Gupte , S. S. ( 1986 ) J. Bioenerg. Biomembr . 18 ,
331 – 368 .
10. Chazotte , B. and Hackenbrock
, C. R. ( 1991 ) J. Biol. Chem . 266 , 5973 – 5979 .
11. Schlame , M. , Rua , D. , and Greenberg , M. L. ( 2000 ) Prog. Lipid Res . 39 , 257 – 288 .
12. Hostetler , K. Y. ( 1982 ) Polyglycerophospholipids: Phosphatidylglycerol, diphospha-
tidylglycerol and bis(monoacylglycero)phosphate . In Hawthorne , J. N. and Ansell ,
G. B. , editors. Phospholipids , Elsevier Biomedical Press , Amsterdam , pp.
215 – 261 .
13. Schlame , M. , Brody , S. , and Hostetler , K. Y. ( 1993 ) Eur. J. Biochem . 212 , 727 – 735 .
14. Dowhan , W. ( 1997 ) Annu. Rev. Biochem . 66 , 199 – 232 .
15. Schlame , M. and Greenberg , M. L. ( 1997 ) Biochim. Biophys. Acta Lipids Lipid
Metab . 1348 , 201 – 206 .
16. Schlame , M. and Hostetler , K. Y. ( 1997 ) Biochim. Biophys. Acta Lipids Lipid Metab .
1348 , 207 – 213 .
17. Schlame , M. and Haldar , D. ( 1993 ) J. Biol. Chem . 268 , 74 – 79 .
18. Hauff , K. D. and Hatch , G. M. ( 2006 ) Prog. Lipid Res . 45 , 91 – 101 .
19. Xu , Y. , Malhotra , A. , Ren , M. , and Schlame , M. ( 2006 ) J. Biol. Chem . 281 ,
39217 – 39224 .
20. Li , G. , Chen , S. , Thompson
, M. N. , and Greenberg , M. L. ( 2007 ) Biochim. Biophys.
Acta 1771 , 432 – 441 .
21. Xu , Y. , Condell , M. , Plesken , H. , Edelman - Novemsky , I. , Ma , J. , Ren , M. , and Schlame ,
M. ( 2006 ) Proc. Natl. Acad. Sci USA 103 , 11584 – 11588 .
22. Ohtsuka , T. , Nishijima , M. , and Akamatsu , Y. ( 1993 ) J. Biol. Chem . 268 ,
22908 – 22913 .
23. Bereiter - Hahn , J. and Voth , M. ( 1994 ) Microsc. Res. Tech . 27 , 198 – 219 .
24. Kent , C. ( 1995 ) Annu. Rev. Biochem . 64 , 315 – 343 .
25. Zinser , E. , Sperka - Gottlieb , C. D. , Fasch , E. V. , Kohlwein , S. D. , Paltauf , F. , and Daum ,
G. ( 1991 ) J. Bacteriol . 173 , 2026 – 2034 .
26. Stewart , L. C. and Yaffe , M. P. ( 1991 ) J. Cell. Biol . 115 , 1249 – 1257 .
27. Tzagoloff , A. and Dieckmann , C. L. ( 1990 ) Microbiol. Rev . 54 , 211 – 225 .
REFERENCES 341
342 METABOLIC PATHWAYS INSIDE MITOCHONDRIA
28. Baba , S. W. , Belogrudov , G. I. , Lee , J. C. , Lee , P. T. , Strahan , J. , Shepherd , J. N. , and
Clarke , C. F. ( 2004 ) J. Biol. Chem . 279 , 10052 – 10059 .
29. Barkovich , R. J. , Shtanko , A. , Shepherd , J. A. , Lee , P. T. , Myles , D. C. , Tzagoloff ,
A. , and Clarke , C. F. ( 1997 ) J. Biol. Chem . 272 , 9182 – 9188 .
30. Barros , M. H. , Johnson , A. , Gin , P. , Marbois , B. N. , Clarke , C. F. , and Tzagoloff , A. A.
( 2005 ) J. Biol. Chem . 280 , 42627 – 42635 .
31. Do , T. Q. , Hsu , A. Y. , Jonassen , T. , Lee , P. T. , and Clarke , C. F. ( 2001 ) J. Biol.
Chem . 276 , 18161 – 18168 .
32. Gin , P. , Hsu , A. Y. , Rothman , S. C. , Jonassen , T. , Lee , P. T. , Tzagoloff , A. , and Clarke ,
C. F. ( 2003 ) J. Biol. Chem . 278 , 25308 – 25316 .
33. Olsen , R. E. and Rudney , H. ( 1983 ) Vitamins and Hormones
40
, 1 – 43 .
34. Grunler , J. , Ericsson , J. , and Dallner , G. ( 1994 ) Biochem. Biophys. Acta 1212 ,
259 – 277 .
35. Tielens , A. G. M. ( 1994 ) Parasitol. Today 10 , 346 – 352 .
36. Van Hellemond , J. J. , Luijten , M. , Flesch , F. M. , Gaasenbeek , C. P. H. , and Tielens ,
A. G. M. ( 1996 ) Mol. Biochem. Parasitol . 82 , 217 – 226 .
37. Van Hellemond , J. J. , Klockiewicz , M. , Gaasenbeek , C. P. H. , Roos , M. H. , and
Tielens , A. G. M. ( 1995 ) J. Biol. Chem . 270 , 31065 – 31070 .
38. Tielens , A. G. M. and Van Hellemond , J. J. ( 1998 ) Parasitol. Today 14 , 265 – 271 .
39. Beinert , H. , Holm , R. H. , Munck , E. , and M ü nck , E. ( 1997 ) Science 277 , 653 – 659 .
40. Johnson , D. C. , Dean , D. R. , Smith , A. D. , and Johnson , M. K. ( 2005 ) Annu. Rev.
Biochem . 74 , 247 – 281 .
41. Lill , R. and Muhlenhoff , U. ( 2005 ) Trends Biochem. Sci . 30 , 133 – 141 .
42. Kispal , G. ,
Sipos , K. ,
Lange , H. , Fekete , Z. , Bedekovics , T. , Janaky , T. , Bassler , J. ,
Aguilar Netz , D. J. , Balk , J. , Rotte , C. , and Lill , R. ( 2005 ) EMBO J . 24 , 589 – 598 .
43. Yarunin , A. , Panse , V. G. , Petfalski , E. , Dez , C. , Tollervey , D. , and Hurt , E. ( 2005 )
EMBO J . 24 , 580 – 588 .
44. Alper , G. and Narayanan , V. ( 2003 ) Pediatr. Neurol . 28 , 335 – 341 .
45. Van Der Giezen , M. and Tovar , J. ( 2005 ) EMBO Rep . 6 , 525 – 530 .
46. van der Giezen M. , Tovar , J. , and Clark , C. G. ( 2005 ) Int. Rev. Cytol . 244 , 175 –
225 .
47. Tielens , A. G. M. , Rotte , C. , Van Hellemond , J. J. , and Martin , W. ( 2002 ) Trends
Biochem. Sci . 27 , 564 – 572 .
48. Gunter , K. K. and Gunter , T. E. ( 1994 ) J. Bioenerg. Biomembr . 26 , 471 – 485 .
49. Hansford , R. G. ( 1994 ) J. Bioenerg. Biomembr .
26 , 495 – 508 .
50. Hatefi
, Y. and Yamaguchi , M. ( 1996 ) FASEB J . 10 , 444 – 452 .
51. Rostovtseva , T. K. , Tan , W. , and Colombini , M. ( 2005 ) J. Bioenerg. Biomembr . 37 ,
129 – 142 .
52. Lemasters , J. J. and Holmuhamedov , E. ( 2006 ) Biochim. Biophys. Acta 1762 ,
181 – 190 .
53. Klingenberg , M. , Winkler , E. , and Huang , S. ( 1995 ) Methods Enzymol . 260 ,
369 – 389 .
54. Wohlrab , H. ( 2005 ) Biochim. Biophys. Acta 1709 , 157 – 168 .
55. Pebay - Peyroula , E. and Brandolin , G. ( 2004 ) Curr. Opin. Struct. Biol . 14 , 420 – 425 .
56. Pebay - Peyroula , E. , Dahout - Gonzales , C. , Kahn , R. , Trezeguet , V. , Lauquin , G. J. M. ,
and Brandolin , G. ( 2003 ) Nature 426 , 39 – 44 .
57. Nury , H. , Dahout - Gonzalez , C. , Trezeguet , V. , Lauquin , G. J. M. , Brandolin , G. , and
Pebay - Peyroula , E. ( 2006 ) Annu. Rev. Biochem . 75 , 713 – 741 .
58. Ciapaite , J. , Bakker , S. J. , Diamant , M. , van Eikenhorst , G. , Heine , R. J. , Westerhoff , H.
V. , and Krab , K. ( 2006 ) FEBS J . 273 , 5288 – 52302 .
59. Dahout - Gonzalez , C. , Nury , H. , Trezeguet , V. , Lauquin , G. J. , Pebay - Peyroula , E. , and
Brandolin , G. ( 2006 ) Physiology (Bethesda) 21 , 242 – 249 .
60. Kunji , E. R. ( 2004 ) FEBS Lett . 564 , 239 – 244 .
61. Garlid , K. D. ( 1994 ) J. Bioenerg. Biomembr . 26 , 537 – 542 .
62. Brierley , G. P. , Baysal , K. , and Jung , D. W. ( 1994 ) J. Bioenerg. Biomembr . 26 ,
519 – 526 .
63.
Ardehali , H. and
O ’ Rourke , B. ( 2005 ) J. Mol. Cell Cardiol . 39 , 7 – 16 .
64. O ’ Rourke , B. , Cortassa , S. , and Aon , M. A. ( 2005 ) Physiology (Bethesda.) 20 ,
303 – 315 .
65. Yarov - Yarovoy , V. , Paucek , P. , Jaburek , M. , and Garlid , K. D. ( 1997 ) Biochim.
Biophys. Acta 1321 , 128 – 136 .
66. Paucek , P. , Yarov - Yarovoy , V. , Sun , X. , and Garlid , K. D. ( 1996 ) J. Biol. Chem . 271 ,
32084 – 32088 .
67. Garlid , K. D. ( 1996 ) Biochim. Biophys. Acta 1275 , 123 – 126 .
68. Szewczyk , A. , Skalska , J. , Glab , M. , Kulawiak , B. , Malinska , D. , Koszela - Piotrowska ,
I. , and Kunz , W. S. ( 2006 ) Biochim. Biophys. Acta 1757 , 715 – 720 .
69. Costa , A. D. , Quinlan , C. L. , Andrukhiv , A. , West , I. C. , Jaburek , M. , and Garlid , K.
D. ( 2006 ) Am. J. Physiol. Heart Circ. Physiol . 290 , H406 – H415 .
70. Jaburek , M. , Costa , A. D. , Burton , J. R. , Costa
, C. L. , and Garlid
, K. D. ( 2006 ) Circ.
Res . 99 , 878 – 883 .
71. Jung , D. W. and Brierley , G. P. ( 1994 ) J. Bioenerg. Biomembr . 26 , 527 – 536 .
72. Jung , D. W. , Panzeter , E. , Baysal , K. , and Brierley , G. P. ( 1997 ) Biochim. Biophys.
Acta 1320 , 310 – 320 .
73. Leo , S. , Bianchi , K. , Brini , M. , and Rizzuto , R. ( 2005 ) FEBS J . 272 , 4013 – 4022 .
74. DeLuca , H. F. and Engstrom , G. W. ( 1961 ) Proc. Natl. Acad. Sci. USA 47 ,
1744 – 1750 .
75. Putney , J. W. Jr , . and Thomas , A. P. ( 2006 ) Curr. Biol . 16 , R812 – R815 .
76. Jung , D. W. , Baysal , K. , and Brierley , G. P. ( 1995 ) J. Biol. Chem . 270 , 672 – 678 .
77. Rizzuto , R. and Pozzan , T. ( 2006 ) Physiol. Rev . 86 , 369 – 408 .
77a. Tsien , R. Y. and Poenie , M. ( 1986 ) Trends in Biochem. Sci . 11 , 450 – 455 .
78. Rizzuto , R. , Simpson , A. W. M. , Brini , M. , and Pozzan , T. ( 1992 )
Nature
358 ,
325 – 327 .
79. Sheu , S. S. and Jou , M. J. ( 1994 ) J. Bioenerg. Biomembr . 26 , 487 – 493 .
80. Bianchi , K. , Rimessi , A. , Prandini , A. , Szabadkai , G. , and Rizzuto , R. ( 2004 ) Biochim.
Biophys. Acta 1742 , 119 – 131 .
81. Rizzuto , R. , Pinton , P. , Carrington , W. , Fay , F. S. , Fogarty , K. E. , Lifshitz , L. M. , Tuft ,
R. A. , and Pozzan , T. ( 1998 ) Science 280 , 1763 – 1766 .
82. Rutter , G. A. and Rizzuto , R. ( 2000 ) Trends Biochem. Sci . 25 , 215 – 221 .
REFERENCES 343
344 METABOLIC PATHWAYS INSIDE MITOCHONDRIA
83. Rapizzi , E. , Pinton , P. , Szabadkai , G. , Wieckowski , M. R. , Vandecasteele , G. , Baird ,
G. , Tuft , R. A. , Fogarty , K. E. , and Rizzuto , R. ( 2002 ) J. Cell Biol . 159 , 613 – 624 .
84. Szabo , I. , Bathori , G. , Wolff , D. , Starc , T. , Cola , C. , and Zoratti , M. ( 1995 ) Biochim.
Biophys. Acta 1235 , 115 – 125 .
85. Bernardi , P. and Petronilli , V. ( 1996 ) J. Bioenerg. Biomembr . 28 , 131 – 138 .
86. Jung , D. W. , Bradshaw , P. C. , and Pfeiffer , D. R. ( 1997 ) J. Biol. Chem . 272 ,
21104 – 21112 .
87. Kroemer , G. , Zamzami , N. , and Susin , S. A. ( 1997 ) Immunol. Today 18 , 44 – 51 .
88. Scorrano , L. , Petronilli , V. , and Bernardi , P. ( 1997 ) J. Biol. Chem . 272 ,
12295 – 12299 .
89. Halestrap , A. P. , Woodfi eld , K. Y. , and Connern , C. P. ( 1997 ) J. Biol. Chem . 272 ,
3346 – 3354 .
90. Connern
, C. P. and Halestrap , A. P. ( 1996 ) Biochemistry 35 , 8172 – 8180 .
91. Connern , C. P. and Halestrap , A. P. ( 1994 ) Biochem. J . 302 , 321 – 324 .
92. Kowaltowski , A. J. , Castilho , R. F. , and Vercesi , A. E. ( 2001 ) FEBS Lett . 495 ,
12 – 15 .
93. Halestrap , A. P. , Griffi ths , E. J. , and Connern , C. P. ( 1993 ) Biochem. Soc. Trans . 21 ,
353 – 358 .
345
7
Mitochondria, Second Edition, by Immo E. Scheffl er
Copyright © 2008 by John Wiley & Sons, Inc.
MITOCHONDRIAL MUTATIONS
AND DISEASE
Summary
When mitochondrial DNA was discovered, it could be anticipated that there could
be mutations, and an explanation was provided for “ cytoplasmic mutations ” and
non - mendelian inheritance fi rst observed in Saccharomyces cerevisiae . Since yeast
could proliferate under anaerobic conditions, mtDNA mutations affecting respira-
tion and oxidative phosphorylation could be isolated and characterized. In humans
the detection of such mutations seemed inconceivable for two reasons: (1) Respira-
tion - defi cient humans would be expected to be dead, and (2) with > 1000 mtDNAs
per cell it was diffi cult to imagine how a mutation in one of these genomes could ever
be expressed as a phenotype. In the late 1980s, human pedigrees with mtDNA muta-
tions and a phenotype were discovered, and the term “ mitochondrial disease ” was
introduced. Soon it was recognized that such diseases were more common than
expected; symptoms ranged from mild to severe, and combinations of symptoms
made it a challenge to classify these diseases into distinct categories. However, neu-
ropathies, myopathies, or a combination of these are typical. Initially, mitochondrial
diseases were characterized by a partial defect in oxidative phosphorylation (an
energy defi ciency) due to mtDNA mutations, but it is immediately obvious that
nuclear mutations could also lead to defective electron transport and ATP synthesis.
Such mutations should follow mendelian genetics in a pedigree. Severe mutations
are lethal (in early embryogenesis/development), and affected patients must still
have partial function of the affected enzyme/complex. With > 1000 proteins in mito-
chondria, one could in principle have thousands of “ mitochondrial diseases. ” Here
346 MITOCHONDRIAL MUTATIONS AND DISEASE
the term will be applied in a limited sense to mutations that affect oxidative phos-
phorylation and energy metabolism. Included in this category are diseases that are
the result of problems in mtDNA replication and maintenance. Numerous other
defects in mitochondrial proteins are the cause of “ inborn errors of metabolism ” or
“ metabolic diseases ” that will not be discussed in this chapter.
There has also been an increasing awareness over the last two decades that mito-
chondria and mitochondrial defects are implicated in age - dependent neurological
degeneration (Alzheimer ’ s disease, Parkinson ’ s disease, Friedreich ’ s ataxia, and
others). The current state of knowledge will be summarized. Finally, the phenome-
non of aging (senescence) has attracted attention for a long time and for obvious
reasons. There are clearly multiple facets of this problem and the underlying causes.
Mitochondria are thought to play a prominent role as a major source of reactive
oxygen species (ROS); an accumulation of mutations in mtDNA and damage to
mitochondrial proteins due to ROS provides a rationale for the diminishing capacity
of mitochondria and for the accumulation of cellular debris that can overwhelm the
cellular garbage disposal machinery (proteasome).
7.1 GENERAL INTRODUCTION
Human genetic diseases due to defective mitochondrial enzymes can be
grouped broadly into two major classes: (1) Nuclear mutations causing defects
in enzymes affecting specifi c metabolic pathways have been studied exten-
sively for more than three decades. Many inborn errors of metabolism are due
to nuclear mutations and hence enzyme defi ciencies in metabolic reactions
taking place in the mitochondrial matrix. As an example, one can mention
ornithine transcarbamylase defi ciency, one of several defects causing disorders
in the urea cycle. A discussion of such metabolic diseases is not intended here.
(2) The present chapter will emphasize the mutations affecting the capacity of
cells for respiration and the rate of oxidative phosphorylation. Severe defects
in energy metabolism would be expected to be lethal, but leaky mutations
resulting in partial defi ciencies are now recognized as the major cause of a
broad spectrum of neurological and other diseases.
Of greatest interest for the present discussion are mutations in mitochon-
drial DNA. The large number of mtDNAs per cell requires the consideration
of conditions such as homoplasmy and heteroplasmy, defi ned as the presence
of at least two populations of mtDNA molecules, one being normal (wild type)
and the other being mutated. The fraction of mutated mtDNAs can range from
barely detectable to 100%, and hence the expression of the defect can range
between undetectable and severe. Consequently, the symptoms and their
severity vary widely. The mechanisms giving rise to such mixtures of genomes
are intriguing and still relatively obscure. Distinctions also arise from the pos-
sibility of mutations in mitochondrial (structural) genes encoding, for example,
(a) a subunit of cytochrome oxidase and (b) mutations in a mitochondrial
tRNA gene, affecting mitochondrial protein synthesis and hence the biogene-
sis of multiple complexes.
Previous chapters have made frequent reference to nuclear and mitochon-
drial mutations that have had global effects on mitochondrial biogenesis.
Mutations affecting DNA replication and transcription, RNA processing, and
translation of mitochondrial mRNAs, as well as specifi cally nuclear mutations
that cause defects in the protein import machinery of mitochondria, were dis-
cussed in the context of the mechanisms that operate in mitochondrial prolif-
eration, fusion and fi ssion, and the assembly of the mitochondrial electron
transport chain. The mutations were of interest because they illuminated the
role of specifi c gene products in the biogenesis of mitochondria. Obviously,
such mutations also affect the capacity for respiration. In this chapter the focus
will initially be on mutations in the mtDNA affecting the capacity for oxidative
phosphorylation, and hence one of the major functions of the mitochondria
as the powerhouse of the cell. The existence of mtDNA mutations was fi rst
recognized in cell culture with lower eukaryotes such as yeast, and to a very
limited extent with mammalian cells in culture. During the past decade a new
fi eld of “ mitochondrial medicine ” has been created from the discovery that a
complex and variable set of symptoms in humans could be caused by partial
defi ciencies in oxidative phosphorylation due to mitochondrial mutations. It
should be stated at the outset that nuclear mutations can also have a direct
effect on oxidative phosphorylation, since both genomes contribute to the
complexes of the mitochondrial electron transport chain. While the well -
known rules of Mendelian inheritance will apply to such nuclear mutations,
mitochondrial mutations passed along the maternal lineage pose a much more
signifi cant challenge to the genetic counselor. The severity of the resulting
phenotype will depend on the degree of heteroplasmy that can vary widely
among siblings, as will be discussed below.
Included in this chapter are discussions of several diseases such as
Alzheimer ’ s disease, Parkinson ’ s disease, Huntington ’ s disease, and amyo-
trophic lateral sclerosis (ALS), where partially defective mitochondria have
been implicated, but the question is still unresolved as to whether the mito-
chondrial defect is a primary cause or a secondary consequence of a more
fundamental defect within specifi c (neuronal) tissues. An intriguing aspect of
these diseases is their delayed onset, similar to the delayed onset of mitochon-
drial diseases resulting from mtDNA mutations. Investigations of programmed
cell death, apoptosis, have linked the pathway to mitochondrial components,
and they have established a signifi cant role of mitochondria in determining
the fate of a cell. Research into mechanisms responsible for normal aging
(senescence) in multicellular organisms has led to the formulation of very
plausible and stimulating hypotheses, proposing an accumulation of mitochon-
drial mutations and a gradual deterioration of OXPHOS capacity as a funda-
mental cause of aging.
Finally, this chapter includes a summary and discussion of the salient fea-
tures of fungal senescence and male sterility in plants, because mitochondria
GENERAL INTRODUCTION 347
348 MITOCHONDRIAL MUTATIONS AND DISEASE
and mitochondrial genomes (and mitochondrial plasmids) are strong genetic
determinants in the expression of these phenomena.
7.2 IN CELL CULTURE
7.2.1 Mitochondrial Mutations in Microorganisms
For very familiar practical and theoretical reasons the budding yeast, Saccha-
romyces cerevisiae , has become one of the most widely used model systems in
which to study eukaryotic molecular genetics, cell biology, and even cell physi-
ology. Growth requirements are minimal, and growth will occur under a wide
variety of conditions. The yeast cell has all or most of the typical subcellular
organelles and compartments of a eukaryotic cell. Its genome is relatively
small, and the complete sequences of all of its genome has been available for
some time. Large numbers of cells can be easily cultured in suspension or on
plates. Mutant selections on a relatively large scale are possible, and clones
are readily isolated. A system of mating and sexual reproduction has made the
standard genetic methodology applicable for mapping and complementation
analysis. Most readers will not need to be reminded of the enormous advan-
tages of yeast as a model system, and it may at times be more interesting to
ask, What biological problems cannot be tackled with the help of yeast? Even
prions appear to be found in yeast (1) . The complex I of the ETC is not present
in the common yeasts, S. cerevisiae and S. pombe , but the yeast Yarrowia lipo-
lytica promises to fi ll this gap (2, 3) .
Studies on plant variants early in this century had noted non - Mendelian
(maternal) types of inheritance, a phenomenon that became known as cyto-
plasmic inheritance (4, 5) . Mutations in chloroplast DNA were eventually
demonstrated to be responsible for these original observations. The fi eld
received a major boost when cytoplasmic inheritance was discovered in yeast
(6) , and a large variety of yeast mutants classifi ed as pet mutants, mit mutants,
ρ
−
or ρ
0
mutants has been useful in making pioneering discoveries on mito-
chondrial inheritance, mitochondrial functions, oxidative phosphorylation, and
so on. A detailed description of all of these would constitute a book all by
itself (7) . Only some general principles and insights will be discussed in the
following section and illustrated with some specifi c examples.
A property of yeast cells is that they can grow under a variety of different
conditions. Such conditions are likely to be encountered in the wild as well,
and the ability of yeast to adapt to different defi ned conditions in the labora-
tory continues to amaze and to provide systems for the study of gene regula-
tion in many laboratories. Of primary signifi cance for the present discussion
is the ability to adapt to the carbon source in the medium and to the avail-
ability of oxygen. The expression of multiple genes is adjusted to prevailing
conditions. When a fermentable carbon source and oxygen are present, many
genes involved in transport, metabolism of other sugars, and, most signifi cantly,
the biosynthesis of the entire electron transport chain and ATPsynthase are
repressed. This phenomenon, referred to as “ glucose repression, ” has been the
subject of numerous reviews (8 – 12) . In a previous chapter on nuclear mito-
chondrial interactions, many aspects of this phenomenon relevant to mito-
chondrial biogenesis have already been presented.
Here it is simply relevant to emphasize that wild - type yeast cells can grow
on media either rich in glucose (a fermentable carbon source) or defi cient in
glucose (in the presence of nonfermentable carbon sources such as acetate,
glycerol, or ethanol). In combination with replica plating, it is therefore
straightforward to isolate mutant clones that can grow on glucose but are
unable to grow in the presence of glycerol alone. Among the possible mutants,
respiration - defi cient mutants (res - ) form a large group; and because they tend
to form small (petite) colonies under defi ned conditions of glucose depriva-
tion, they are routinely referred to as pet mutants. Collectively, they have been
expertly described and reviewed by Dujon (7) and Tzagoloff and Dieckmann
(13) . Mutations giving rise to this phenotype can be subdivided into nuclear
and mitochondrial mutations. Nuclear mutations are local and generally affect
single, specifi c genes whose function has been exemplifi ed in numerous
instances in earlier chapters of this book. The mitochondrial mutations can
again be subdivided into point mutations affecting single functions (referred
to as mit
−
) and large deletions of mtDNA (termed cytoplasmic petites or rho
−
or ρ
−
) (14) . Mutants completely lacking mtDNA are termed rho
0
( ρ
0
) strains,
and they can be obtained by prolonged culture of yeast cells in glucose and
sublethal concentrations of ethidium bromide. Since the deletions in rho
−
mutants include tRNA genes most of the time, they are incapable of mito-
chondrial protein synthesis and thus lack the entire electron transport chain.
When mit
−
mutants are crossed with rho
−
mutants, marker rescue by recom-
bination can occur, if the mit mutation is in a region of the genome still present
as a wild - type sequence in the rho
−
mitochondria. Respiration - competent
diploid cells arise at high frequency by this mechanism, which involves recom-
bination rather than complementation. A recombination event is necessary
to generate wild - type diploid cells, because it is found that “ merodiploid ” or
heteroplasmic mitochondria are not maintained under routine culture condi-
tions. Instead, rapid mitotic segregation of mitochondrial genomes takes place
resulting in a loss of potentially complementing sequences (14) .
A particularly useful new method in yeast mitochondrial genetics has been
the development of the transformation of mitochondria by high - velocity bom-
bardment of yeast cells with tungsten microprojectiles to which cloned DNA
sequences are adsorbed. By this method, DNA constructs made in vitro can
be introduced into mitochondria of rho
0
strains to form “ synthetic rho
−
”
strains. After mating with rho
+
strains, the DNA constructs can recombine
with the mtDNA and deliberate mutations can be introduced into mtDNA.
Details of the procedure can be found in the original publications and authori-
tative reviews (14, 15) . Using this approach, Fox and colleagues have been
able to perform site - directed mutagenesis to study sequences at the 5 ′ end of
IN CELL CULTURE 349
350 MITOCHONDRIAL MUTATIONS AND DISEASE
mitochondrial mRNAs encoding subunits of complex IV that are required
for effi cient translation and insertion of the proteins into the inner
membrane (16) .
7.2.2 Mitochondrial Mutations in Mammalian Cells in Culture
Thirty years ago it would probably have been considered impossible to obtain
mammalian cell mutants in tissue culture with severe defects in respiration.
Attempts to grow mammalian cells under anaerobic conditions had consis-
tently failed, and since normally such cells would not have experienced severe
anaerobiosis, it was believed that oxidative phosphorylation was an essential
activity in the energy metabolism of such cells. There had been a great deal of
speculation about an altered energy metabolism in cancer versus normal cells
since the days when O. Warburg had proposed that tumor cells had partially
reverted to a more “ primitive ” type of energy metabolism in which glycolysis
played a larger role (17) , but no specifi c enzyme or activity had been shown
to be affected in cancer cells, which would have explained the elevated secre-
tion of lactate by cells grown under normal aerobic conditions. An informative,
entertaining and highly personal review of the literature on this subject up to
about 1975 has been written by Racker (18) . The Warburg effect in cancer cells
has recently had a notable revival (see, for example, references 19 – 21 ).
Serendipity played a large role in the isolation and characterization of the
fi rst respiration - defi cient Chinese hamster fi broblast mutant cells in tissue
culture (22 – 24) . The original mutant cell line was eventually found to have a
defect in complex I of the mitochondrial electron transport chain, making it
unable to oxidize NADH in the mitochondria. The elevated levels of NADH,
in turn, inhibited the Krebs cycle enzyme α - ketoglutarate dehydrogenase
by a simple feedback mechanism (24) . The mutation was clearly established
to be a nuclear mutation, and a complementing gene was mapped on the X -
chromosome of mammals (25) .
A complete understanding of the phenotype of this fi rst mutant allowed
the Scheffl er laboratory to devise a selection scheme for the isolation of addi-
tional mutants of Chinese hamster fi broblasts in tissue culture with severe
defects in respiration (26) . Complementation tests were carried out by pair-
wise fusions, and the mutants were sorted into seven complementation groups
(27) . Similar mutants were subsequently isolated in two other laboratories (28,
29) in the course of their search for Chinese hamster cell mutants in tissue
culture which would reproduce the phenotype of human cells from galactose-
mic patients. It was expected that defects in galactokinase or other enzymes
in the Leloir pathway would make cells unable to grow in galactose. Clearly,
respiration - defi cient mutants of the type described above would resemble
galactokinase mutants. Reactions of the Leloir pathway are too slow to sustain
the high rate of glycolysis required by such mutants. Reviews summarizing the
earlier efforts in the various laboratories have been written by Scheffl er (30)
and Whitfi eld (28) .