Copyright  2000 by the Genetics Society of America
Expression of the Saccharomyces cerevisiae Gene YME1 in the Petite-Negative
Yeast Schizosaccharomyces pombe Converts It to Petite-Positive
Douglas J. Kominsky and Peter E. Thorsness
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071
Manuscript received September 11, 1998
Accepted for publication September 27, 1999
ABSTRACT
Organisms that can grow without mitochondrial DNA are referred to as “petite-positive” and those that
are inviable in the absence of mitochondrial DNA are termed “petite-negative.” The petite-positive yeast
Saccharomyces cerevisiae can be converted to a petite-negative yeast by inactivation of Yme1p, an ATP- and
metal-dependent protease associated with the inner mitochondrial membrane. Suppression of this yme1
phenotype can occur by virtue of dominant mutations in the a- and g-subunits of mitochondrial ATP
synthase. These mutations are similar or identical to those occurring in the same subunits of the same
enzyme that converts the petite-negative yeast Kluyveromyces lactis to petite-positive. Expression of YME1
in the petite-negative yeast Schizosaccharomyces pombe converts this yeast to petite-positive. No sequence
closely related to YME1 was found by DNA-blot hybridization to S. pombe or K. lactis genomic DNA, and
no antigenically related proteins were found in mitochondrial extracts of S. pombe probed with antisera
directed against Yme1p. Mutations that block the formation of the F1 component of mitochondrial ATP
synthase are also petite-negative. Thus, the F1 complex has an essential activity in cells lacking mitochondrial
DNA and Yme1p can mediate that activity, even in heterologous systems.
M
ITOCHONDRIAL biogenesis requires the coordinated expression of genes encoded by mitochondrial and nuclear genomes, as well as the regulated
assembly of a number of multicomponent protein complexes. Recent work in the yeast Saccharomyces cerevisiae
has revealed the importance of a related set of mitochondrial proteases in the assembly of energy transduction complexes. Located in the inner mitochondrial
membrane is a hetero-oligomer, composed of the homologous proteins Yta10p and Yta12p, that is necessary
for the assembly of cytochrome oxidase and ATP synthase (Paul and Tzagoloff 1995; Arlt et al. 1996;
Guélin et al. 1996). The yeast homolog of the Escherichia
coli lon protease has also been implicated in the assembly of inner membrane protein complexes (Rep et al.
1996). These proteases have an ATP requirement, and
mutational analysis has led to a proposal that these proteins function largely as assembly factors with editing
capability. Misfolded and damaged proteins or proteins
present in excess that cannot be assembled into a
higher-order complex are degraded by these proteases.
Yme1p forms another inner mitochondrial membrane
complex with protease activity (Leonhard et al. 1996).
Yme1p determines the stability of cytochrome oxidase
subunit II that is present in excess or is not assembled
into the multicomponent cytochrome oxidase complex
(Nakai et al. 1995; Pearce and Sherman 1995; Weber
Corresponding author: Peter Thorsness, Department of Molecular
Biology, University of Wyoming, Laramie, WY 82071-3944.
E-mail: thorsnes@uwyo.edu
Genetics 154: 147–154 ( January 2000)
et al. 1996). While Yme1p is envisioned to have a role
as an assembly/editing factor similar to Yta10p/Yta12p
and mitochondrial Lon, this has not yet been demonstrated experimentally. YME1 was originally identified
in a screen for mutations that increase the rate at which
mitochondrial DNA (mtDNA) is transferred to the nucleus (Thorsness and Fox 1993). yme1 strains are also
heat sensitive for growth on nonfermentable carbon
sources (pet-ts) and cold-sensitive for growth on richglucose media and have altered mitochondrial morphology (Thorsness et al. 1993; Campbell et al. 1994).
S. cerevisiae strains in which YME1 has been inactivated
are petite-negative; that is, they are incapable of growth
in the absence of mtDNA (Thorsness et al. 1993). Wildtype S. cerevisiae is a petite-positive strain, capable of
growing on fermentable carbon sources in the absence
of mtDNA. As a consequence of dysfunctional mitochondrial structure and function, yme1 strains have an
increased rate of mitochondrial compartment turnover
by the vacuole (Campbell and Thorsness 1998).
Clearly, Yme1p is involved in a number of important
mitochondrial processes that must extend beyond the
turnover of unassembled Cox2p.
In an effort to understand the physiological role of
Yme1p in mitochondrial biogenesis and function, a
number of suppressors of yme1 phenotypes have been
characterized. A bypass suppressor of the yme1 null allele
that suppresses all yme1 phenotypes was identified as
YNT1/RPT3, a gene that encodes a regulatory subunit
of the 26S protease (Campbell et al. 1994). Inactivation
of YNT20, which encodes a putative 39-59 exonuclease
148
D. J. Kominsky and P. E. Thorsness
located in mitochondria, suppresses the high rate of
mtDNA escape in yme1 strains and also suppresses the
synthetic nonrespiring phenotype of yme1 yme2 strains
(Hanekamp and Thorsness 1998). Specific dominant
mutations in the gene encoding the g-subunit of the
mitochondrial ATP synthase, ATP3, suppress the petitenegative phenotype of yme1 yeast (Weber et al. 1995).
For none of these suppressors has it been possible to
demonstrate a substrate/product relationship between
the identified gene products and the Yme1p protease.
In the work presented here, a number of additional
mutations that result in the suppression of various yme1
phenotypes are described, and another suppressor of
the petite-negative phenotype of yme1 yeast is fully described. Additionally, the heterologous expression of
Yme1p in the petite-negative yeast Schizosaccharomyces
pombe was used to define the genetic basis of yeast strains
that require mtDNA for viability.
MATERIALS AND METHODS
Strains: The E. coli strains used for preparation and manipulation of DNA were DH5a [F2 end, hsdR17 (rk2 mk1), supE44,
thi-1, l recA, gyrA96, relA1, D(argF-lacZYA) U169, φ80,
lacZDM15] and XL1 Blue [recA1, endA1, gyrA96, thi-1, rsdR17,
supE44, relA1, lac (F 9 proAB, lacI qZDM15, Tn10 (tet r))]. The E.
coli strains ES1301 [lacZ53, mutS201::Tn5, thyA36, rha-5, metB1,
deoC, IN(rrnD-rrnE)] and JM109 [endA1, recA1, gyrA96, thi,
hsdR17(rk2 mk1), relA1, supE44, l-, D(lac-proAB), (F 9, traD36,
proA1B1, lacIqZDM15)] were used for in vitro mutagenesis
and were obtained from Promega (Madison, WI). The genotypes of S. cerevisiae strains used in this study are listed in Table
1. Standard genetic techniques were used to construct and
analyze the various yeast strains (Sherman et al. 1986; see
below). The S. pombe strain PNY10 was a gift of Dr. Paul Nurse.
The Kluveromyces lactis strain Y11401 was a gift of Dr. Claudia
Abeijon. The genotypes of both strains are listed in Table 1.
Media: E. coli strains containing plasmids were grown in
Luria-Bertani (LB) medium (10 g bactotryptone, 10 g NaCl,
5 g yeast extract per liter; Maniatis et al. 1982) supplemented
with 125 mg/ml of ampicillin. Yeast strains were grown in
complete glucose medium (YPD) containing 2% glucose, 2%
Bacto-peptone, 1% yeast extract; complete ethanol-glycerol
medium (YPEG) containing 3% glycerol, 3% ethanol, 2%
Bacto-peptone, 1% yeast extract; or minimal glucose medium
(SD) containing 2% glucose, 6.7 g/liter yeast nitrogen base
without amino acids (Difco, Detroit) and supplemented with
the appropriate nutrients. Nutrients were uracil at 40 mg/
liter, adenine at 40 mg/liter, tryptophan at 40 mg/liter, lysine
at 60 mg/liter, and leucine at 100 mg/liter. For agar plates,
Bacto-agar was added at 20 g/liter. Where indicated, ethidium
bromide was added at 25 mg/ml (Weber et al. 1995).
yme1D1::URA3 suppressor isolation and analysis: Bypass suppressors of yme1-D1::URA3 were isolated by screening for spontaneous revertants of the pet-ts phenotype of PTY52. Overnight
cultures grown in YPD at 308 were plated on YPEG plates at
z2000 cells/plate and incubated for several days at 378. Isolated revertants able to respire at high temperatures were
colony purified, rescreened on YPEG at 378, analyzed for suppression of other yme1-D1::URA3 phenotypes, and screened
for recessive collateral phenotypes by scoring for growth on
different carbon sources at different temperatures. Each suppressed strain was mated to every other suppressed strain.
Diploids were analyzed to identify complementation groups.
Tetrad analysis of matings between the suppressed strains and
a wild-type strain provided confirmation that any collateral
phenotype segregated with the suppressing mutation. Dominant suppressors were isolated in the same manner as the
recessive suppressors except that the screen was performed
using the diploid strain PTY52 3 PTY60. Isolation of suppressors of the yme1 petite-negative phenotype was performed as
described (Weber et al. 1995).
TABLE 1
Yeast strains
Genotypea
Strain
PTY44
PTY52
PTY62
PTY78
PTY93
DKY1
DKY21
DKY22
DKY25
DKY26
DKY27
DKY30
NTY1
W303DATP12
PNY10b
Y11401c
a
MATa ura3-52 lys2 leu2-3,112 trp1-D1
TRP1]
MATa ura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 [rho1, TRP1]
MATa ura3-52 lys2 leu2-3,112 trp1-D1 yme1-1 [rho1, TRP1]
MATa ura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ATP1-75 [rho0]
MATa ura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ATP1-75 [rho1, TRP1]
MATa ura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 YNT9-1 [rho1, TRP1]
MATa ura3-52 ade2 leu2-3,112 trp1-D1 yme1-D1::URA3
MATa ura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ynt4-1 [rho1, TRP1]
MATa ura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ynt5-1 [rho1, TRP1]
MATa ura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ynt6-1 [rho1, TRP1]
MATa ura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ynt7-1 [rho1, TRP1]
MATa ura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ynt8-1 [rho1, TRP1]
MATa ura3-52 lys2 leu2-3,112 trp1-D1 atp1-D1::LEU2 [rho1, TRP1]
MATa ura3-52 lys2 leu2-3,112 trp1-D1 ynt1-1 [rho1, TRP1]
MATa ade2-1 his3-11,15 leu2-3,112 ura3-1 trp1-1 atp12::LEU2 [rho1]
h2 leu1-32
MATa ura3
The mitochondrial genotype is bracketed.
S. pombe strain.
c
K. lactis strain.
b
[rho1,
Source
Thorsness and Fox (1993)
Thorsness et al. (1993)
Thorsness and Fox (1993)
Weber et al. (1995)
Weber et al. (1995)
This study
This study
This study
This study
This study
This study
This study
Campbell et al. (1994)
Bowman et al. (1991)
Paul Nurse
Claudia Abeijon
Basis for Petite-Positive Yeast
Nucleic acid techniques and DNA sequencing: All manipulations of DNA were performed using standard techniques
(Maniatis et al. 1982). Restriction enzymes and DNA modification enzymes were purchased from New England Biolabs
(Beverly, MA). Double-stranded DNA templates were prepared for sequencing by boiling lysis (Maniatis et al. 1982)
and were sequenced by the nucleotide chain termination
method (Sanger et al. 1977) using the Sequenase Version 2.0
DNA sequencing kit (United States Biochemical, Cleveland).
DNA-blot hybridization analysis was performed using standard
techniques (Maniatis et al. 1982). Blots were washed in 23
SSC with 0.5% SDS for 2 hr at 508. Random primed probes
corresponding to the entire open reading frames (ORFs) of
YME1 and ATP1 from S. cerevisiae were prepared using the
NEBlot kit from New England Biolabs.
Isolation of ATP1-75: Briefly, genomic DNA was prepared
as described (Rose and Broach 1991) from the yeast strain
PTY78 bearing the ATP1-75 mutation. Genomic DNA was partially digested with Sau3A; 6–10-kb DNA fragments were isolated and cloned into the yeast CEN-vector pRS316 (Sikorski
and Hieter 1989). Library DNA was prepared by large-scale
alkaline lysis (Maniatis et al. 1982) and was used to transform
PTY62 using the alkali cation treatment method (Ito et al.
1983). Approximately 2 3 105 transformants were inoculated
into 10 ml of selective SD and incubated with agitation at 308
for 2 hr. Aliquots (1 ml) of the SD culture were inoculated
into each of 10 tubes containing 10 ml selective SD media
plus ethidium bromide (25 mg/ml) and grown to saturation
at 308 (8 days). Ten microliters of each culture was inoculated
into 10 ml of fresh SD plus ethidium bromide media and
grown to saturation at 308 (12 days). Dilutions of each culture
were plated on YPD media. Colonies were replica plated to
media containing 5-fluoroorotic acid (5-FOA; Boeke et al.
1984) to identify cells unable to grow without plasmid DNA.
Thirty-two colonies unable to grow on 5-FOA were recovered
from the YPD plates and the phenotype was rechecked. Plasmid DNA was prepared from four isolates. Three clones contained identical inserts of z2.4 kb. One of these, pDK1, was
sequenced and found to contain ATP1. This clone was sent
to Macromolecular Resources (Fort Collins, CO) for sequencing of the entire ATP1-75 allele.
In vitro mutagenesis of plasmid DNA: In vitro mutagenesis
of plasmid DNA was performed using the Altered Sites in vitro
mutagenesis system from Promega. Oligonucleotides were obtained from Integrated DNA Technology. The ATP1-75 allele
was cloned into pALTER-Ex1 as follows. pDK1 was digested
with KpnI and the ends were made blunt with mung bean
nuclease. After digestion with EcoRI, the 2.2-kb ATP1-75 fragment was isolated and inserted into pALTER-Ex1 that had
been digested with EcoRI and StuI. The resulting construct,
pDK3, was the starting substrate for site-specific mutagenesis.
The mutant ATP1 allele was restored to wild type using the
oligonucleotide 59-GGCTTTGAACTTGGAGCC-39, generating the plasmid pDK5. The sequencing oligonucleotide 59GCAGTCGGTGATGGTATTGC-39 was used to verify that the
wild-type ATP1 sequence was restored in pDK5. This plasmid
was then used as a substrate to recreate the ATP1-75 allele
using the oligonucleotide 59-GGCTTTGATCTTGGAGCC-39.
The sequence of the resulting plasmid, pDK6, was again verified using the sequencing oligonucleotide described above.
The 2.2-kb EcoRI-HpaI inserts in pDK5 and pDK6 were transferred to the yeast CEN vector pRS316, creating the plasmids
pDK9 and pDK10, respectively. These constructs, along with
pDK1, were transformed into PTY62 to verify that the ATP175 mutation was able to suppress the petite-negative yme1
phenotype.
Creation of an ATP1 null allele: The ATP1 null mutant was
created using the plasmid pG50/ST6, a gift from Dr. Sharon
149
Ackerman. The gene disruption was made by removing an
internal 200-bp BglII fragment of the ATP1 gene and replacing
it with a 3-kb BglII fragment containing the LEU2 gene. The
chromosomal disruption was made by digesting pG50/ST6
partially with KpnI and then digesting completely with HindIII.
The 4-kb KpnI-HindIII fragment containing the atp1::LEU2
construct was gel purified and used to transform PTY44. The
resulting strain, DKY30, was then tested for the ability to respire and whether it was petite-positive or petite-negative. Creation of the null mutant was verified by performing PCR with
the oligonucleotides 59-TAAAGGTCCTATTGACGCTGC-39
and 59-TATTGTAGAGGAGCGGCTTCA-39.
S. pombe expression clones: The S. pombe expression constructs were made using the plasmid pART1, a gift from Dr.
Paul Nurse. To isolate the YME1 coding sequence, the plasmid
pYME1-NdeI was digested with NdeI and the ends were made
blunt by filling in the site with Klenow. After digestion with
SacI, the 2-kb YME1 fragment was isolated and ligated into
pART1 that had been digested with SacI and SmaI, generating
pART1-YME1. The plasmid pyme1Dm was constructed by digesting pART1-YME1 with MluI. This site was then filled in
using Klenow and the plasmid was religated. Transformation
of S. pombe was performed by treatment of cells with alkali
cations (Ito et al. 1983).
Preparation of cellular extracts and detection of Yme1p
and Atp1p: Protein extracts were prepared as described (Pillus and Solomon 1986). Mitochondrial fractions were isolated by differential centrifugation (Daum et al. 1982). Protein
fractions were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antisera directed
against Yme1p or Atp1p as described previously (Hanekamp
and Thorsness 1996).
RESULTS
Isolation and characterization of bypass suppressors
of yme1: To identify gene products that interact with
Yme1p, suppressors of a yme1-D1::URA3 null allele were
isolated. The yme1 null allele was used for this analysis
because suppressors of yme1-1, a missense mutation,
were all intragenic. Mutants were isolated either as dominant or recessive suppressors of the yme1 pet-ts phenotype or as suppressors of the yme1 petite-negative phenotype. These mutants were then screened for their ability
to suppress other yme1 phenotypes, as well as for inherent collateral phenotypes. Nine strains were identified
with mutations that suppress various yme1 phenotypes
(Figure 1). Complementation analysis indicates that
these suppressors represent mutations at distinct genetic loci. These mutations can be placed in classes
based on the yme1 phenotypes suppressed and collateral
phenotypes linked to the suppressing mutation. Class
1 contains the recessive suppressor ynt1, which suppresses all of the yme1 phenotypes and has a recessive
collateral phenotype of cold-sensitive growth on the
nonfermentable carbon sources ethanol and glycerol
(Campbell et al. 1994). Class 2 includes the recessive
mutants ynt4 and ynt5. These mutations suppress the
yme1 pet-ts, the high rate of mtDNA escape, and coldsensitive growth on rich-glucose phenotypes. These suppressing mutations display no collateral phenotypes.
The third class of suppressors includes the recessive
150
D. J. Kominsky and P. E. Thorsness
Figure 1.—Suppression of yme1 phenotypes. Top, the indicated yeast strains were scored for mtDNA escape by patching
on a YPEG plate, incubating at 308 for 2 days, and then replica
plating to synthetic glucose lacking tryptophan. The plate was
then scored after 4 days of incubation at 308. Strains that have
an increased rate of mtDNA escape have an increase in the
relative number of Trp1 papillae (Thorsness et al. 1993).
Middle, the indicated strains were patched to YPD, incubated
for 1 day and then replica plated to YPD and YPEG and
incubated at the temperatures shown for 1–7 days, depending
on temperature and media. Bottom, the indicated strains were
streaked on a synthetic glucose plate containing 25 mg/ml
ethidium bromide and incubated for 5 days at 308. Strains:
yme-1D (PTY52), yme1D ynt1 (NTY1), yme1D ynt4 (DKY21),
yme1D ynt5 (DKY22), yme1D ynt6 (DKY25), yme1D ynt7
(DKY26), yme1D ynt8 (DKY27), yme1D ATP1-75 (PTY93), yme1D
YNT9-1 (DKY1), WT (PTY44).
mutants ynt7 and ynt8, which suppress the pet-ts and
cold-sensitive growth on rich-glucose media phenotypes
of yme1 strains. Additionally, these mutations have a
recessive collateral phenotype of temperature-sensitive
growth on rich-glucose media. The fourth class of recessive yme1-D1::URA3 suppressors includes the mutant
ynt6, which suppresses only the pet-ts phenotype. This
mutation has a recessive collateral phenotype of pet-ts
growth in a YME1 background. Also shown in Figure 1
is the mutation YNT9-1, which is presumably a dominant
suppressor of the yme1 pet-ts phenotype since it was isolated in a yme1-D1::URA3 homozygous diploid. However,
further characterization of this mutation has proven
difficult because this strain grows poorly on glucose
media and does not sporulate. The final class of suppressors includes the dominant mutations ATP3-1 and ATP175, previously referred to as YNT3-1 (Weber et al. 1995).
These mutants suppress only the petite-negative phenotype of yme1. Cells grown in the presence of ethidium
bromide rapidly lose all DNA from their mitochondria
(Slonimski et al. 1968; Fox et al. 1991) and are termed
rho0. Unlike wild-type yeast, which readily grows in the
presence of ethidium bromide, yme1 rho0 yeast are very
slow growing. To better understand the basis for this
petite-negative phenotype of yme1 strains, we pursued
analysis of the ATP1-75 mutation.
Isolation of ATP1-75, a dominant suppressor of the
yme1 rho0 slow-growth phenotype: Previous work in our
laboratory demonstrated that mutations in the ATPsynthase g-subunit suppress the petite-negative phenotype of yme1 yeast (Weber et al. 1995). Additionally,
work in the petite-negative yeast K. lactis has shown that
mutations in the ATP-synthase a-, b-, and g-subunits
convert this yeast to petite-positive (Chen and ClarkWalker 1995, 1996). To assess the similarities of petite
negative suppressors in yme1 S. cerevisiae and K. lactis,
we cloned the ATP1-75 mutation. A genomic DNA library was constructed from a yme1-D1::URA3 ATP1-75
strain. This genomic library was transformed into a
yme1-1 strain, and transformed cells were screened for
the ability to grow upon mtDNA loss. Several clones were
isolated from this screen. Figure 2 shows the growth of
a rho0 yme1-1 mutant after introduction of one of these
plasmids, pDK1. DNA sequence analysis demonstrated
that the plasmid insert contained the complete open
reading frame of ATP1, the structural gene for the
a-subunit of mitochondrial ATP synthase. Sequence
analysis of the entire ORF identified a single nucleotide
change that resulted in the conversion of the strictly
conserved asparagine residue at position 102 to isoleucine. To demonstrate that the amino acid change at
position 102 in ATP1-75 was responsible for suppression
of the petite-negative phenotype, site-directed mutagenesis was performed. First, the residue was changed to
the wild-type asparagine, followed by reversion of the
asparagine residue back to the mutant isoleucine. As
shown in Figure 2, a plasmid bearing the reverted wildtype ATP1 allele is unable to rescue the petite-negative
phenotype, while both the original ATP1-75 allele and
the reverted ATP1-75 allele restore growth on ethidium
bromide.
A requirement of functional mitochondrial ATP-synthase F1 component in rho0 yeast: It has been reported
that null mutations in the ATP-synthase g-subunit
(Weber et al. 1995) and the d-subunit (Giraud and
Velours 1997) cause a petite-negative phenotype in S.
Basis for Petite-Positive Yeast
Figure 2.—Complementation of the yme1 petite-negative
phenotype by cloned ATP1-75. Wild-type yeast (PTY44) or a
strain bearing the yme1-1 mutation (PTY62) was transformed
with the indicated plasmids and then streaked on a synthetic
glucose plate (A) and incubated for 3 days at 308, or streaked
on a synthetic glucose plate containing 25 mg/ml ethidium
bromide (B) and incubated for 5 days at 308. The indicated
plasmids are the vector (pRS316), the cloned ATP1-75 allele
(pATP1-75), the cloned ATP1 allele reverted to wild type via
site directed mutagenesis (pATP1r), and the ATP1 wild-type
clone reverted back to the ATP1-75 allele (pATP1-75r).
cerevisiae. These observations have been taken as evidence for a requirement of assembled and functional F1
subunits in mitochondria that lack DNA. To determine
whether an ATP1 null mutation would confer a similar
phenotype, an ATP1-D1::LEU2 strain, DKY30, was constructed and streaked on media containing ethidium
bromide. Additionally, another strain that had an
ATP12 null mutation was also cultured on media containing ethidium bromide. Atp12p is involved in the
assembly of ATP-synthase F1 subunits (Bowman et al.
1991). As shown in Figure 3, both null mutant strains
exhibit a petite-negative phenotype, demonstrating the
requirement for an assembled and functional F1 portion
of the ATP-synthase complex, even in the absence of a
functional F0 component.
An absence of closely related YME1 genes in S. pombe
151
Figure 3.—Growth of rho0 yeast bearing yme1, atp1, or atp12
null alleles. Yeast cells were streaked on a synthetic glucose
plate (A) and incubated for 3 days at 308 and on a synthetic
glucose plate containing 25 mg/ml ethidium bromide (B) and
incubated for 5 days at 308. The indicated yeast strains are
wild type (PTY44), yme1D (PTY52), atp1D (DKY30), and atp12D
(W303DATP12).
and K. lactis: The results presented above suggest that
the activity of Yme1p in S. cerevisiae is essential to maintain the petite-positive phenotype of this yeast. Because
of the striking similarities between a yme1 mutant and
the petite-negative yeasts S. pombe and K. lactis, we wondered if this growth characteristic was due to the absence
of a Yme1p-like activity. To address this issue DNA-blot
hybridization analysis was performed to determine
whether the genomes S. pombe and K. lactis encode a
YME1 homolog. Total DNA prepared from the strains
PTY44, PTY52, PNY10, and Y11401 was digested with
EcoRI or SspI, blotted, and probed with a randomprimed probe prepared from a PCR product corresponding to the YME1 ORF under low stringency conditions. Figure 4 shows that a strong signal was detected
for the S. cerevisiae DNA (Figure 4, lanes 5, 6, 9, and
10), corresponding to YME1. An additional weak signal
was detected in the SspI-digested S. cerevisiae DNA at
z6.5 kb (Figure 4, lanes 9 and 10). These fragments
correspond to the AFG3 gene (data not shown). Digestion of the AFG3 locus with EcoRI produces a fragment
of z15 kb; thus the AFG3 signal in lanes 5 and 6 was
152
D. J. Kominsky and P. E. Thorsness
Figure 4.—Detection of DNA sequences homologous to
YME1. DNA-blot hybridization was performed on genomic
DNA prepared from S. cerevisiae, S. pombe, and K. lactis. Designations for the DNA sources are as follows: Sc (lanes 1, 5, 9, 13,
16), wild-type S. cerevisiae (PTY44); Sc9 (lanes 2, 6, 10), yme1D1::URA3 S. cerevisiae (PTY52); Sp (lanes 3, 7, 11, 15, 18), S.
pombe (PNY10); Kl (lanes 4, 8, 12, 14, 17), K. lactis (Y11401).
Restriction enzyme digestions were as indicated. Lanes 1–12
were probed with S. cerevisiae YME1 sequences and lanes 13–18
with S. cerevisiae ATP1 sequences. Hybridization signals were
detected with a Bio-Rad (Richmond, CA) phosphorimager.
masked by the intense YME1 signal at the same position.
The AFG3 gene is the nearest homolog to YME1 in the
S. cerevisiae genome, sharing an internal coding region
that is 61% identical. Weak signals were also detected
in the S. pombe DNA (Figure 4, lanes 7 and 11), but not
in K. lactis (lanes 8 and 12). The relative strength of
the hybridization signal generated with the S. pombe
DNA indicates that the YME1 probe recognizes a DNA
sequence in S. pombe that is no more homologous to
the YME1 sequence than is the AFG3 gene. As a control,
the highly conserved ATP1 genes in S. pombe and K.
lactis were easily detected when S. cerevisiae ATP1 sequences were used to probe genomic DNA (Figure 4,
lanes 13–18).
To identify a closely related YME1 gene product in
S. pombe, whole cell extracts were prepared from a S.
pombe culture, proteins were separated on polyacrylamide gels and blotted to a nitrocellulose filter, and
the filter was probed with polyclonal antisera directed
against Yme1p. No cross-reaction with the Yme1p antisera was observed (Figure 6, lane 4). In contrast, antisera
directed against the conserved S. cerevisiae Atp1p gene
product detected S. pombe Atp1p (Figure 6, lanes 4
and 5).
Heterologous expression of YME1 in S. pombe: To
determine if Yme1p activity was able to convert a petitenegative yeast to petite-positive, YME1 was expressed in
S. pombe. The YME1 gene was cloned into the S. pombe
expression vector pART1. As an additional control, the
pART1-YME1 construct was digested with MluI, filled
in, and religated. This plasmid, pyme1Dm, has a frameshift in the YME1 ORF that creates a nonfunctional gene
product. These two plasmids, along with the vector, were
transformed into a S. cerevisiae yme1 mutant and the S.
pombe strain PNY10. As shown in Figure 5, expression
of YME1 in both the S. cerevisiae yme1 and S. pombe strains
Figure 5.—Heterologous expression of YME1 in S. pombe.
S. cerevisiae and S. pombe were transformed with the indicated
plasmids and streaked on a synthetic glucose plate (A) and
incubated for 5 days at 308 or streaked on a synthetic glucose
plate containing 25 mg/ml ethidium bromide (B) and incubated for 7 days at 308. The indicated strains are as follows:
S. c. wild type, S. cerevisiae wild-type strain PTY44; S. c. yme1D,
S. cerevisiae yme1 strain PTY52; S. p. wild-type, S. pombe strain
PNY10. The indicated plasmids are as follows: pART1, S. pombe
expression vector; pYME1, the vector plasmid pART1 bearing
wild-type YME1; pyme1Dm, the vector plasmid pART1 bearing
an inactivated yme1 allele.
allows growth in the absence of mtDNA, while the
pART1 vector and the inactivated yme1 allele do not.
Thus, Yme1p provides an activity that is essential for
viability of two yeast when they lack mtDNA. Western
blot analysis using whole cell extracts from these strains
detected Yme1p in mitochondria of S. pombe transformed with pART1-YME1 (Figure 6).
DISCUSSION
To identify possible Yme1p substrates and to elucidate
the role of Yme1p in yeast mitochondria, we undertook
a suppressor analysis of a yme1 null mutant. Several
Basis for Petite-Positive Yeast
Figure 6.—Detection of Yme1p in S. cerevisiae and S. pombe.
Mitochondrial fractions prepared from the indicated strains
bearing the indicated plasmids were subjected to SDS-PAGE,
transferred to nitrocellulose filters, and probed with antisera
directed against Yme1p and Atp1p. Lane 1, PTY44/pART1;
lane 2, PTY52/pART1; lane 3, PTY52/pYME1; lane 4, PNY10/
pART1; lane 5, PNY10/pYME1. Whole cell extracts and postmitochondrial supernatants prepared from PTY52/pART1,
PNY10/pART1, or PNY10/pYME1 do not produce a crossreacting signal with Yme1p antisera.
genes were identified that, when mutated, lead to suppression of various yme1 phenotypes (Figure 1). Because
mutation of different genes suppress different spectrums of yme1 phenotypes, we anticipate multiple roles
for Yme1p in mitochondrial biogenesis and function.
Previous work in our laboratory identified mutations
in the ATP3 gene that suppress the petite-negative phenotype of yme1 strains (Weber et al. 1995). Here we
identify a mutation in the ATP1 gene that suppresses
the same phenotype. The F1 complex of the mitochondrial ATP synthase is a multi-subunit structure with a
subunit stoichiometry of a3b3gdε. Deletion of ATP1
leads to the inability to use nonfermentable carbon
sources (Takeda et al. 1986) and causes a petite-negative
phenotype (Figure 3). Mutational inactivation of the
g- and d-F1 subunits (Weber et al. 1995; Giraud and
Velours 1997) and of Atp12p, a protein involved in F1
subunit assembly (Bowman et al. 1991), also cause this
phenotype (Figure 3). These results indicate that
Yme1p, as well as the F1 portion of the ATP synthase, are
important for the growth of S. cerevisiae in the absence of
mtDNA. One extensively characterized mutation that
causes S. cerevisiae to become petite-negative is op1, a
mutation in the major ADP/ATP translocator encoded
by AAC2 (Kovacova et al. 1968; Lawson and Douglas
1988; Kolarov et al. 1990). Both op1 and a null mutation
of ACC2 render cells unable to grow on nonfermentable
carbon sources (Kovacova et al. 1968; Lawson et al.
1990). The petite-negative nature of op1 strains has been
taken as evidence for the absolute requirement for ATP
in the matrix of mitochondria.
The yeasts K. lactis and S. pombe are petite-negative.
Work in K. lactis has shown that specific mutations in
the ATP-synthase a-, b-, and g-subunits convert this yeast
to a petite-positive organism (Chen and Clark-Walker
1995, 1996). Based on the similarities between these
petite-negative strains and an S. cerevisiae yme1 mutant,
153
we surmised that these yeasts may be petite-negative
because they lack a Yme1p-like activity. DNA-blot analysis indicated that neither K. lactis nor S. pombe encode
a close YME1 homolog (Figure 4), nor is there an antigenically related Yme1p homolog in S. pombe (Figure
6). Additionally, heterologous expression of YME1 in
S. pombe converted this petite-negative yeast to petitepositive. Thus, Yme1p has an activity that is missing in
S. pombe, and presumably also in K. lactis, that results in
these yeast being petite-negative.
We utilized the bovine F1-ATPase crystal structure
(Abrahams et al. 1994) to identify the relative location
of mutations in the a- and g-subunits of mitochondrial
ATP synthase that convert petite-negative yeast to petitepositive. The amino acid changed in ATP1-75, Asn102,
is located near the interface of the a- and g-subunits in
the region referred to as the “dimple” of the F1 complex,
distal to the stalk connecting F1 to F0 (Abrahams et al.
1994). The two amino acid positions mutated in the K.
lactis a-subunit, Ala333 and Phe443, are also located
along this interface, in direct contact with the g-subunit
(Chen and Clark-Walker 1996). Thus, it is likely that
the interaction of the a- and g-subunits is altered in
these mutant F1 complexes.
The information presented here suggests that Yme1p
must be involved in the regulation of mitochondrial
ATP synthase, although the nature of this regulation is
unknown. The consequences of the absence of Yme1p
in yeast that contain a complete mitochondrial genome
is not deleterious at 308, as these cells can use nonfermentable carbon sources. Upon loss of mtDNA, however, the absence of Yme1p leads to the petite-negative
phenotype. It has previously been shown that Yme1p is
a metallo-protease that is involved in the turnover of
cytochrome oxidase subunit II (Nakai et al. 1995;
Pearce and Sherman 1995; Weber et al. 1996). One
possibility is that Yme1p may also have a direct role in
the turnover of F1 subunits. In mitochondria lacking
mtDNA, which encodes the F0 subunits Atp6p, Atp8p,
and Atp9p, excess F1 subunits might be degraded in a
Yme1p-dependent manner. Without Yme1p, F1 subunits
may accumulate, leading to aberrant complex formation. This scenario seems unlikely since the accumulation of Atp3p has been shown to be independent of
Yme1p (Weber et al. 1995). Alternatively, Yme1p may
affect the F1 complex indirectly by interacting with F0
subunits, at least six of which are encoded in the nucleus. In the absence of the mitochondrially encoded
F0 subunits, the nuclearly encoded subunits may be subject to degradation by Yme1p, allowing the F1 portion
of the ATP synthase to function properly. In the absence
of Yme1p, these F0 subunits may accumulate and interfere with an F1 activity that is essential for cells lacking
mtDNA. The mutations that have been identified in
ATP1 and ATP3 would thus allow the F1 complex to
assume a conformation that is not subject to interference by inhibitory factors that exist in the absence of
Yme1p activity.
154
D. J. Kominsky and P. E. Thorsness
We thank Dr. Sharon Ackerman, Dr. Paul Nurse, and Dr. Claudia
Abeijon for generously providing plasmids and yeast strains. We also
thank the members of the Thorsness laboratory for critical review of
this manuscript. This work was supported by Public Health Service
grant GM-47390.
LITERATURE CITED
Abrahams, J. P., A. G. W. Leslie, R. Lutter and J. Walker, 1994
Structure at 2.8 A resolution of F1-AtPase from bovine heart mitochondria. Nature 370: 621–628.
Arlt, H., R. Tauer, H. Feldmann, W. Neupert and T. Langer, 1996
The YTA10-12 complex, an AAA protease with chaperone-like
activity in the inner membrane of mitochondria. Cell 85: 875–885.
Boeke, J. D., F. Lacroute and G. R. Fink, 1984 A positive selection
for mutants lacking orotidine-59-phosphate decarboxylase activity
in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197:
345–346.
Bowman, S., S. H. Ackerman, D. E. Griffiths and A. Tzagoloff,
1991 Characterization of ATP12, a yeast nuclear gene required
for the assembly of the mitochondrial F1-ATPase. J. Biol. Chem.
266: 7517–7523.
Campbell, C. L., and P. E. Thorsness, 1998 Escape of mitochondrial DNA to the nucleus in yme1 yeast is mediated by vacuolardependent turnover of abnormal mitochondrial compartments.
J. Cell Sci. 111: 2455–2464.
Campbell, C. L., N. Tanaka, K. H. White and P. E. Thorsness, 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.
Chen, X. J., and G. D. Clark-Walker, 1995 Specific mutations in
alpha- and gamma-subunits of F1-ATPase affect mitochondrial
genome integrity in the petite-negative yeast Kluyveromyces lactis.
EMBO J. 14: 3277–3286.
Chen, X. J., and G. D. Clark-Walker, 1996 The mitochondrial
genome integrity gene, MGI1, of Kluyveromyces lactis encodes
the beta-subunit of F1-ATPase. Genetics 144: 1445–1454.
Daum, G., P. C. Böhni and G. Schatz, 1982 Import of proteins
into mitochondria. Energy-dependent uptake of precursors by
isolated mitochondria. J. Biol. Chem. 257: 13028–13035.
Fox, T. D., L. S. Folley, J. J. Mulero, T. W. McMullin, P. E. Thorsness et al., 1991 Analysis and manipulation of yeast mitochondrial genes. Methods Enzymol. 194: 149–165.
Giraud, M. F., and J. Velours, 1997 The absence of the mitochondrial ATP synthase delta subunit promotes a slow growth phenotype of rho2 yeast cells by a lack of assembly of the catalytic
sector F1. Eur. J. Biochem. 245: 813–818.
Guélin, E., M. Rep and L. A. Grivell, 1996 Afg3p, a mitochondrial
ATP-dependent metalloprotease, is involved in the degradation
of mitochondrially-encoded Cox1, Cox3, Cob, Su6, Su8, and Su9
subunits of the inner membrane complexes III, IV and V. FEBS
Lett. 381: 42–46.
Hanekamp, T., and P. E. Thorsness, 1996 Inactivation of YME2/
RNA12, which encodes an integral inner mitochondrial membrane protein, causes increased escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae. Mol. Cell. Biol. 16:
2764–2771.
Hanekamp, T., and P. E. Thorsness, 1998 YNT20, a bypass suppressor of yme1 yme2, encodes a putative 39-5-exonuclease localized in
mitochondria of Saccharomyces cerevisiae. Curr. Genet. 34: 438–448.
Ito, H., Y. Fukuda, K. Murata and A. Kimura, 1983 Transformation
of intact yeast cells treated with alkali cations. J. Bacteriol. 153:
163–168.
Kolarov, J., N. Kolarova and N. Nelson, 1990 A third ADP/ATP
translocator gene in yeast. J. Biol. Chem. 265: 12711–12716.
Kovacova, V., J. Irmlerova and L. Kovac, 1968 Oxidative phosphorylation in yeast, IV: combination of a nuclear mutation affect-
ing oxidative phosphorylation with cytoplasmic mutation to respiratory deficiency. Biochim. Biophys. Acta 162: 157–163.
Lawson, J. E., and M. G. Douglas, 1988 Separate genes encode
functionally equivalent ADP/ATP carrier proteins in Saccharomyces cerevisiae. J. Biol. Chem. 263: 14812–14818.
Lawson, J. E., M. Gawaz, M. Klingenberg and M. G. Douglas, 1990
Structure-function studies of adenine nucleotide transport in
mitochondria. I. Construction and genetic analysis of yeast mutants encoding the ADP/ATP carrier protein of mitochondria.
J. Biol. Chem. 265: 14195–14201.
Leonhard, K., J. M. Herrmann, R. A. Stuart, G. Mannhaupt, W.
Neupert et al., 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.
Maniatis, T., E. F. Fritsch and J. Sambrook, 1982 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
Nakai, T., T. Yasuhara, T. Fujiki and A. Ohashi, 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.
Paul, M. F., and A. Tzagoloff, 1995 Mutations in RCA1 and AFG3
inhibit F1-ATPase assembly in Saccharomyces cerevisiae. FEBS Lett.
373: 66–70.
Pearce, D. A., and F. Sherman, 1995 Degradation of cytochrome
oxidase subunits in mutants of yeast lacking cytochrome c and
suppression of the degradation by mutation of yme1. J. Biol. Chem.
270: 20879–20882.
Pillus, L., and F. Solomon, 1986 Components of microtubular
structures in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA
83: 2468–2472.
Rep, M., J. M. van Dijl, K. Suda, G. Schatz, L. A. Grivell et al.,
1996 Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon. Science 274: 103–106.
Rose, M. D., and J. R. Broach, 1991 Cloning genes by complementation in yeast. Methods Enzymol. 194: 195–230.
Sanger, F., S. Nicklen and A. R. Coulson, 1977 DNA sequencing
with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:
5463–5467.
Sherman, F., G. R. Fink and J. B. Hicks, 1986 Methods in Yeast
Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Sikorski, R. S., and P. Hieter, 1989 A system of shuttle vectors and
yeast host strains designed for efficient manipulation of DNA in
Saccharomyces cerevisiae. Genetics 122: 19–27.
Slonimski, P. P., G. Perrodin and J. H. Croft, 1968 Ethidium
bromide induced mutation of yeast mitochondria: complete
transformation of cells into respiratory deficient non-chromosomal “petites.” Biochem. Biophys. Res. Commun. 30: 232–239.
Takeda, M., W. J. Chen, J. Saltzgaber and M. G. Douglas, 1986
Nuclear genes encoding the yeast mitochondrial ATPase complex. Analysis of ATP1 coding the F1-ATPase alpha-subunit and
its assembly. J. Biol. Chem. 261: 15126–15133.
Thorsness, P. E., and T. D. Fox, 1993 Nuclear mutations in Saccharomyces cerevisiae that affect the escape of DNA from mitochondria
to the nucleus. Genetics 134: 21–28.
Thorsness, P. E., K. H. White and T. D. Fox, 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.
Weber, E. R., R. S. Rooks, K. S. Shafer, J. W. Chase and P. E.
Thorsness, 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., T. Hanekamp and P. E. Thorsness, 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.
Communicating editor: M. Lichten