Inactivation of Yme1p, a mitochondrially-localized ATP-dependent metallo-protease in the yeast Saccharomyces cerevisiae, causes a high rate of DNA escape from mitochondria to the nucleus as well as pleiotropic functional and morphological mitochondrial defects. The evidence presented here suggests that the abnormal mitochondria of a yme1 strain are degraded by the vacuole. First, electron microscopy of Yme1p-deficient strains revealed mitochondria physically associated with the vacuole via electron dense structures. Second, disruption of vacuolar function affected the frequency of mitochondrial DNA escape from yme1 and wild-type strains. Both PEP4 or PRC1 gene disruptions resulted in a lower frequency of mitochondrial DNA escape. Third, an in vivo assay that monitors vacuole-dependent turnover of the mitochondrial compartment demonstrated an increased rate of mitochondrial turnover in yme1 yeast when compared to the rate found in wild-type yeast. In this assay, vacuolar alkaline phosphatase, encoded by PHO8, was targeted to mitochondria in a strain bearing disruption to the genomic PHO8 locus. Maturation of the mitochondrially localized alkaline phosphatase pro-enzyme requires proteinase A, which is localized in the vacuole. Therefore, alkaline phosphatase activity reflects vacuole-dependent turnover of mitochondria. This assay reveals that mitochondria of a yme1 strain are taken up by the vacuole more frequently than mitochondria of an isogenic wild-type strain when these yeast are cultured in medium necessitating respiratory growth. Degradation of abnormal mitochondria is one pathway by which mitochondrial DNA escapes and migrates to the nucleus.

REFERENCES

Baba
M.
,
Takeshige
K.
,
Baba
N.
,
Ohsumi
Y.
(
1994
).
Ultrastructural analysis of the autophagic process in yeast: detection of autophagasomes and their characterization.
J. Cell Biol
124
,
903
913
Bereiter-Hahn
J.
(
1976
).
Dimethylaminostyrylmethylpyridiniumiodide (DASPMI) as a fluorescent probe for mitochondria in situ.
Biochim. Biophys. Acta
423
,
1
14
Bereiter-Hahn
J.
,
Voth
M.
(
1994
).
Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria.
Microscopy Res. Technique
27
,
198
219
Boriack
R. L.
,
Cortinas
E.
,
Bennett
M. J.
(
1995
).
Mitochondrial damage results in a reversible increase in lysosomal storage material in lymphoblasts from patients with juvenile neuronal ceroid-lipofuscinosis (Batten Disease).
Am. J. Med. Genet
57
,
301
303
Byers
B.
,
Goetsch
L.
(
1990
).
Preparation of yeast cells for thin-section electron microscopy.
Meth. Enzymol
194
,
602
608
Campbell
C. L.
,
Tanaka
N.
,
White
K. H.
,
Thorsness
P. E.
(
1994
).
Mitochondrial morphological and functional defects in yeast caused by yme1 are suppressed by mutation of a 26S protease subunit homologue.
Mol. Biol. Cell
5
,
899
905
Chiang
H. L.
,
Schekman
R.
,
Hamamoto
S.
(
1996
).
Selective uptake of cytosolic, peroxisomal, and plasma membrane proteins into the yeast lysosome for degradation.
J. Biol. Chem
271
,
9934
9941
Corral
M.
,
Baffet
G.
,
Kitzis
A.
,
Paris
B.
,
Tichonicky
L.
,
Kruh
J.
,
Guguen-Guillouzo
C.
,
Defer
N.
(
1989
).
DNA sequences homologous to mitochondrial genes in nuclei from normal rat tissues and from rat hepatoma cells.
Biochem. Biophys. Res. Commun
162
,
258
264
Costa
V.
,
Amorim
M. A.
,
Reis
E.
,
Quintanilha
A.
,
Moradas-Ferreira
P.
(
1997
).
Mitochondrial superoxide dismutase is essential for ethanol tolerance of Saccharomyces cerevisiae in the post-diauxic phase [In Process Citation].
Microbiology
143
,
1649
1656
Dunn
W. A.
Jr.
(
1990
).
Studies on the mechanisms of autophagy: formation of the autophagic vacuole.
J. Cell Biol
110
,
1923
1933
Dunn
W. A.
Jr.
(
1990
).
Studies on the mechanisms of autophagy: maturation of the autophagic vacuole.
J. Cell Biol
110
,
1935
1945
Egner
R.
,
Thumm
M.
,
Straub
M.
,
Simeon
A.
,
Schuller
H. J.
,
Wolf
D. H.
(
1993
).
Tracing intracellular proteolytic pathways. Proteolysis of fatty acid synthase and other cytoplasmic proteins in the yeast Saccharomyces cerevisiae.
J. Biol. Chem
268
,
27269
27276
Egner
R.
,
Mahe
Y.
,
Pandjaitan
R.
,
Kuchler
K.
(
1995
).
Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae.
Mol. Cell Biol
15
,
5879
5887
Egner
R.
,
Kuchler
K.
(
1996
).
The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole.
FEBS Lett
378
,
177
181
Hadler
H. I.
(
1989
).
Comment: mitochondrial genes and cancer.
FEBS Lett
256
,
230
232
Hatfill
S. J.
,
La Cock
C. J. R.
,
Laubscher
R.
,
Downing
T. G.
,
Kirby
R.
(
1993
).
A role for mitochondrial DNA in the pathogenesis of radiation-induced myleodysplasia and secondary leukemia.
Leukemia Res
17
,
907
913
Jones
E. W.
,
Zubenko
G. S.
,
Parker
R. R.
(
1982
).
PEP4 gene function is required for expression of several vacuolar hydrolases in Saccharomyces cerevisiae.
Genetics
102
,
665
677
Jones
E. W.
(
1991
).
Tackling the protease problem in Saccharomyces cerevisiae.
Meth. Enzymol
194
,
428
453
Kaneko
Y.
,
Toh-e
A.
,
Oshima
Y.
(
1982
).
Identification of the genetic locus for the structural gene and a new regulatory gene for the synthesis of repressible alkaline phosphatase in Saccharomyces cerevisiae.
Mol. Cell Biol
2
,
127
137
Kaneko
Y.
,
Tamai
Y.
,
Toh-e
A.
,
Oshima
Y.
(
1985
).
Transcriptional and post-transcriptional control of PHO8 expression by PHO regulatory genes in Saccharomyces cerevisiae.
Mol. Cell. Biol
5
,
248
252
Kaneko
Y.
,
Toh-e
A.
,
Banno
I.
,
Oshima
Y.
(
1989
).
Molecular characterization of a specific p -nitrophenylphosphatase gene, PHO13, and its mapping by chromosome fragmentation in Saccharomyces cerevisiae.
Mol. Gen. Genet
220
,
133
139
Klionsky
D. J.
,
Emr
S. D.
(
1989
).
Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase.
EMBO J
8
,
2241
2250
Klionsky
D. J.
,
Emr
S. D.
(
1990
).
A new class of lysosomal/vacuolar protein sorting signals.
J. Biol. Chem
265
,
5349
5352
Kluck
R. M.
,
Bossy-Wetzel
E.
,
Green
D. R.
,
Newmeyer
D. D.
(
1997
).
The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.
Science
275
,
1132
1136
Knecht
E.
,
Martinez-Ramon
A.
,
Grisolia
S.
(
1988
).
Autophagy of mitochondria in rat liver assessed by immunogold procedures.
J. Histochem. Cytochem
27
,
1433
1140
Leonard
K.
,
Herrmann
J. M.
,
Stuart
R. A.
,
Mannhaupt
G.
,
Neupert
W.
,
Langer
T.
(
1996
).
AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria.
EMBO J
15
,
4218
4229
Liang
B. C.
(
1996
).
Evidence for association of mitochondrial DNA sequence amplification and nuclear localization in human low-grade gliomas.
Mutat. Res
354
,
27
33
Liu
X.
,
Kim
C. N.
,
Yang
J.
,
Jemmerson
R.
,
Wang
X.
(
1996
).
Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c.
Cell
86
,
147
157
Mechler
B.
,
Hirsch
H. H.
,
Muller
H.
,
Wolf
D. H.
(
1988
).
Biogenesis of the yeast lysosome (vacuole): biosynthesis and maturation of proteinase yscB.
EMBO J
7
,
1705
1710
Moradas-Ferreira
P.
,
Costa
V.
,
Piper
P.
,
Mager
W.
(
1996
).
The molecular defenses against reactive oxygen species in yeast.
Mol. Microbiol
19
,
651
658
Nakai
T.
,
Yasuhara
T.
,
Fujiki
T.
,
Ohashi
A.
(
1995
).
Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria.
Mol. Cell. Biol
15
,
4441
4452
Nakashima
R. A.
,
Paggi
M. G.
,
Pedersen
P. L.
(
1984
).
Contributions of glycolysis and oxidative phosphorylation to adenosine 5-triphosphate production in AS-30D hepatoma cells.
Cancer Res
44
,
5702
5706
Noda
T.
,
Matsuura
A.
,
Wada
Y.
,
Ohsumi
Y.
(
1995
).
Novel system for monitoring autophagy in the yeast Saccharomyces cerevisiae.
Biochem. Biophys. Res. Commun
210
,
126
132
Palmer
D. N.
,
Fearnley
I. M.
,
Walker
J. E.
,
Hall
N. A.
,
Lake
B. D.
,
Wolfe
L. S.
,
Haltia
M.
,
Martinus
R. D.
,
Jolly
R. D.
(
1992
).
Mitochondrial ATP synthase subunit c storage in the ceroid-lipofuscinoses (Batten disease).
Am. J. Med. Genet
42
,
561
567
Pearce
D. A.
,
Sherman
F.
(
1995
).
Degradation of cytochrome oxidasesubunits in mutants of yeast lacking cytochrome c and suppressionof the degradation by mutation of yme1.
J. Biol. Chem
270
,
20879
20882
Pinkham
J. L.
,
Dudley
A. M.
,
Mason
T. L.
(
1994
).
T7 RNA polymerase-dependent expression of COXII in yeast mitochondria.
Mol. Cell Biol
14
,
4643
4652
Riballo
E.
,
Herweijer
M.
,
Wolf
D. H.
,
Lagunas
R.
(
1995
).
Catabolite inactivation of the yeast maltose transporter occurs in the vacuole after internalization by endocytosis.
J. Bacteriol
177
,
5622
5627
Richter
C.
(
1988
).
Do mitochondrial DNA fragments promote cancer and aging?.
FEBS Lett
241
,
1
5
Roberts
C. J.
,
Raymond
C. K.
,
Yamashiro
C. T.
,
Stevens
T. H.
(
1991
).
Methods for studying the yeast vacuole.
Meth. Enzymol
194
,
644
661
Robinson
K. S.
,
Lai
K.
,
Cannon
T. A.
,
McGraw
P.
(
1996
).
Inositol transport in Saccharomyces cerevisiae is regulated by transcriptional and degradative endocytic mechanisms during the growth cycle that are distinct from inositol-induced regulation.
Mol. Biol. Cell
7
,
81
89
Shay
J. W.
,
Werbin
H.
(
1992
).
New evidence for the insertion of mitochondrial DNA into the human genome: significance for cancer and aging.
Mutation Res
275
,
227
235
Sikorski
R. S.
,
Hieter
P.
(
1989
).
A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
Genetics
122
,
19
27
Sorensen
S. O.
,
van den Hazel
H. B.
,
Kielland-Brandt
M. C.
,
Winther
J. R.
(
1994
).
pH-dependent processing of yeast procarboxypeptidase Y by proteinase A in vivo and in vitro.
Eur. J. Biochem
220
,
19
27
Takeshige
K.
,
Baba
M.
,
Tsuboi
S.
,
Noda
T.
,
Ohsumi
Y.
(
1992
).
Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction.
J. Cell Biol
119
,
301
311
Thorsness
P. E.
,
Fox
T. D.
(
1990
).
Escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae.
Nature
346
,
376
379
Thorsness
P. E.
,
Fox
T. D.
(
1993
).
Nuclear mutations in Saccharomyces cerevisiae that affect the escape of DNA from mitochondria to the nucleus.
Genetics
134
,
21
28
Thorsness
P. E.
,
White
K. H.
,
Fox
T. D.
(
1993
).
Inactivation of YME1, a gene coding a member of the SEC18, PAS1, CDC48 family of putative ATPases, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae.
Mol. Cell. Biol
13
,
5418
5426
Thorsness
P. E.
,
Weber
E.
(
1996
).
Escape and migration of nucleic acids between chloroplasts, mitochondria, and the nucleus.
Int. Rev. Cytol
165
,
207
234
Thumm
M.
,
Egner
R.
,
Koch
B.
,
Schlumpberger
M.
,
Straub
M.
,
Veenhuis
M.
,
Wolf
D. H.
(
1994
).
Isolation of autophagocytosis mutants of Saccharomyces cerevisiae.
FEBS Lett
349
,
275
280
Toh-e
A.
,
Nakamura
H.
,
Oshima
Y.
(
1976
).
A gene controlling the synthesis of non specific alkaline phosphatase in Saccharomyces cerevisiae.
Biochim. Biophys. Acta
428
,
182
192
Tung
B. S.
,
Unger
E. R.
,
Levin
B.
,
Brasitus
T. A.
,
Getz
G. S.
(
1991
).
Use of unsaturated fatty acid auxotrph of Saccharomyces cerevisiae to modify the lipid composition and function of mitochondrial membranes.
J. Lipid Res
32
,
1025
1038
Tuttle
D. L.
,
Lewin
A. S.
,
Dunn
W. A.
Jr.
(
1993
).
Selective autophagy of peroxisomes in methylotrophic yeasts.
Eur. J. Cell Biol
60
,
283
290
Veenhuis
M.
,
Douma
A.
,
Harder
W.
,
Osumi
M.
(
1983
).
Degradation and turnover of peroxisomes in the yeast Hansenula polymorpha induced by selective inactivation of peroxisomal enzymes.
Arch. Microbiol
134
,
193
203
Walenga
R. W.
,
Lands
W. E.
(
1975
).
Effectiveness of various unsaturated fatty acids in supporting growth and respiration in Saccharomyces cerevisie.
J. Biol. Chem
250
,
9121
9129
Weber
E. R.
,
Rooks
R. S.
,
Shafer
K. S.
,
Chase
J. W.
,
Thorsness
P. E.
(
1995
).
Mutations in the mitochondrial ATP synthase gamma subunit suppress a slow-growth phenotype of yme1 yeast lacking mitochondrial DNA.
Genetics
140
,
435
442
Weber
E. R.
,
Hanekamp
T.
,
Thorsness
P. E.
(
1996
).
Biochemical and functional analysis of the YME1 gene product, an ATP and zinc-dependent mitochondrial protease from S. cerevisiae.
Mol. Biol. Cell
7
,
307
317
Yaffe
M. P.
(
1991
).
Analysis of mitochondrial function and assembly.
Meth. Enzymol
194
,
627
643
Yang
J.
,
Liu
X.
,
Bhalla
K.
,
Kim
C. N.
,
Ibrado
A. M.
,
Cai
J.
,
Peng
T.-I.
,
Jones
D. P.
,
Wang
X.
(
1997
).
Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science
275
,
1129
1132
This content is only available via PDF.