Download A Genetic Link Between an mRNA-Specific Translational

Copyright 0 1990 by the Genetics Society of America
A Genetic Link Between an mRNA-Specific Translational Activator and
the
Translation Systemin Yeast Mitochondria
Pascal Haffier, Thomas W. McMullin and Thomas D. Fox
Section of Genetics and Development, Cornell University, Zthaca,New York 14853-2703
Manuscript received November 30, 1989
Accepted for publicationMarch 24, 1990
ABSTRACT
Translation of the Saccharomyces cerevisiae mitochondrial mRNA encoding cytochrome c oxidase
subunit 111 (~0x111)
specifically requires the products
of at least three nuclear genes,PET122, PET494
and PET54. pet122 mutations that remove 24-67 amino acid residues from the carboxyterminus of
thegeneproductwerefoundtobesuppressed
by unlinkednuclearmutations.Theseunlinked
suppressors fail to suppress both a pet122 missense mutation and a complete pet122 deletion. One of
the suppressor mutations causes a heat-sensitive nonrespiratory growth phenotype in an otherwise
wild-type strain and reduces translation of
all mitochondrial gene products in cells grown at high
temperature. This suppressor maps to a newly identified gene on chromosome XV termed PET123.
The sequence of a DNA fragment carryingPET123 contains one major open reading frame encoding
a basic protein of 318 amino acids. Inactivation
of the chromosomal copy of PET123 by interruption
of this open reading frame causes cellsto become rho- (sustain large deletionsin their mtDNA). This
phenotype is characteristic fornull alleles of genes whose products
are essential for general mitochondrial protein synthesis. T h u s our data strongly suggest that the PET123 protein is a component of
the mitochondrial translation apparatus that interacts directly with the coxIII-mRNA-specific translational activatorPETl 22.
M
ITOCHONDRIAL genes are expressed by an
organellar genetic system whose protein components are (with few exceptions) encoded by nuclear
genes (reviewed in TZAGOLOFF
and MYERS1986; ATTARDI and SCHATZ 1988).T h e majority of these nuclearly encoded proteins are required generally for
the expression of all mitochondrial genes. However
in the yeast Saccharomyces cerevisiae, a number of additional nuclear genes have been identifiedthat exert
positive control over the expression of specijic mitochondrial genes(reviewed in FOX 1986; ATTARDIand
SCHATZ1988). Many of these specific regulators act
at the level of translation and thus must mediate, in
some fashion, an interaction betweenspecific mRNAs
and components of the general mitochondrial translation system.
Translation of the mitochondrially coded mRNA
for cytochrome c oxidase subunit 111 (~0x111)specifically requires theaction ofat least three nucleargenes,
PET122, PET494 and PET54 (CABRALand SCHATZ
1978; MULLER et al. 1984; COSTANZO
and Fox 1986;
COSTANZO, SEAVER
and FOX 1986; COSTANZO
et al.
1988; KLOECKENER-GRUISSEM,
MCEWENand POYTON
1987, 1988). T h e site (or sites) at which these nuclear
genes act to promote
translation has been mapped
genetically to the upstream two-thirds of the cox111
mRNA5’-untranslatedleader
(COSTANZO
and Fox
1988a). Furthermore, these three nuclear genes encode proteins thatare localized in mitochondria (CosC.enrtics 125: 495-503 (July, 1990)
and Fox 1986; OHMEN, KLOECKENER-GRUISand MCEWEN1988; COSTANZO,
SEAVER
and Fox
1989; ourunpublished results). Taken together,these
studies suggest a model in which at least one of the
threenucleargeneproducts
contacts the cox111
mRNA 5’-leader and activates translation by an unknown mechanism. Clearly, this mechanism could involve direct protein-protein interactions between one
or more of the cox111 mRNA-specific activators and
proteincomponents
of thegeneral
mitochondrial
translation machinery.
T o identifyinteractions
involving the PET122,
PET54 and PET494 gene products we are looking for
allele-specific suppressors of mutations that alter these
proteins. Such suppressors can define genes coding
for interacting proteins (HARTMAN andROTH 1973)
and are especially informative when the suppressor
mutations themselves produce a phenotype (JARVIK
and BOTSTEIN1975). In this paper we describe the
isolation and characterization of allele-specific suppressorsof pet122 mutations. T h e suppressorsdescribed here, some of which cause a heat sensitive
nonrespiratory phenotype, are mutations in a newly
identified nuclear gene termed PET123. By examining the phenotypes of theheat sensitive mutation
petl23-1 and of pet123 null mutations, we found that
the PETl23 protein is essential for the translation of
most if not all mitochondrial mRNAs. These findings
strongly suggest that we have genetically identified a
TANZO
SEM
496
P. Haffter, T. W. McMullin and T. D. Fox
TABLE 1
Strains used in this study
Genotype
Stvain
DBY947
‘IF1x9
TMAP2-3
T M K2- 1
WH20
PTH25
PTH37
PTH39
l”IH64
PTH78
PTH211
PTH86
PTH161
T F I 75
GRFlS
405
x55
Source
MATa, ade2-101,
3-52
ura
MATa, ade2-101,
ura3-52
MZ4Ta,
ura3-52,
ade2-101,
pet122-6
MATa, ade2-101,
ura3-52,
pet122-6,
pet123-1
petl23-l
MATa,
ura3-52,
ade2-101,
MATa, ade2-101,
ura3-52,
pet122-6,
petL23-l
MATa, ade2-101,
ura3-52,
pet122-7
MATaIlMATa, ade2-l0l/ade2-101,
ura3-52/ura3-52
MATa, leu2-3,
leu2-I
12,
his3-11,
his3-15,
pet123-I
MATa, ade2-101,
ura3-52,
petl23::URA3
MATa, ade2-101, u r d - 5 2 , pet123-12
(::URAJ)
MATa, leu2-3,
petl23-1
(possibly canR)
MATa, ade2-101,
ura3-52,
pet122-9
MATa, ura3-52,
leu2-3,
leu2-112,
his4-519,
pet122-5
M,4Ta, his3-1 I, his3-15,
leu2-3,
leu2-I
12,
canR
,MATa,leu2-3,
his3,
ade2,
WF5-I
MATa, ade9, l e d - 3 , lys2
link between the coxIII-mRNA-specific translational
activator PET122 a n d a component of the general
mitochondrial translation machinery.
MATERIALS AND METHODS
Yeast strains, media and genetic methods: S. cerevisiae
strains used in this study are listed in Table 1. All strains
were isogenic except PTH64,PTH86,TF175,GRF18,
405, and 855. PTH64 was derived from a cross between
PTH2O and GRF18; PTH86 from a cross between PTH64
and 855. SUF5 was scored as a suppressor of the leu2-3
mutation (CULBERTSON,
UNDERBRINK FINK
and 1980). Nonfermentable medium was YPEG (1% yeast extract, 2% peptone, 3% ethanol, 3% glycerol). Complete medium containing glucose (YPD), other media and genetic methods were
as described by ROSE,WINSTON
and HIETER (1
988), except
that some yeast transformations were carried out by agitation with glass beads (COSTANZO
and FOX 1988b).
DNA manipulations,
sequencing
and
computer
searches: DNA manipulations were carried out as described
by MANIATIS,FRITSCHand SAMBROOK
(1982). DNAsequence determination was performed by the dideoxynucleotide chain terminationmethod (SANGER,
NICKLENand
COULSON
1977). Computer searches were done using the
FASTA-MAIL program (PEARSON
and LIPMAN 1988).
Isolation and manipulation of pet122 mutations: The
pet122-5 mutation was previously isolated in this laboratory
(COSTANZO
and FOX 1988a). [We formerly referred to
PET122 as PET55 (COSTANZO
et al. 1988).] The PET122
gene was isolated by selecting a plasmid that could complement the pet122-5 mutation in TF175 from a genomic bank
in the vector YEP13 (NASMYTHand TATCHELL
1980). The
pet122-6 allele was generated by deletion of the 468-bp
PuuII fragment (Figure 1) as follows: an 1820-bp BamHIXbaI fragment carrying PET122 (OHMEN,KLOECKENERGRUISSEM
and MCEWEN1988) was subcloned from the
original complementing plasmid intothe vector YCp50
(ROSEet al. 1987)to generatepC122. pC122 was then
digested with PvuII and religated. The corresponding
BamHI-XbaI fragment, carrying the deletion, was then sub-
01-
Ref.
NEFFet al. (1983)
This study
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study
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study
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DONAHUE
et al. (1983)
CULBERTSON,UNDERBRINK
and FINK( 1 980)
CULBERTSON, UNDERBRINK
and FINK(1980)
cloned into theintegrating vector YIp5 (STRUHL
et al. 1979).
The pet122-7 allele was generated by deletion of the 1342bp Hind1 fragmentcontaining theentire gene,froma
subclone of PET122 in BLUESCRIPT M13 (Stratagene
Inc.) which was then subcloned on an approximately 1-kb
BamHI-EcoRV fragment into YIp5,resulting inplasmid
pPHY2. Both deletions were then used to replace the
PET122 gene of strain DBY947 as described (BOEKE,LACROUTE
and FINK1984) andverified by gel-blot hybridization analysis (not shown). DNA carrying the petl22-5 mutation was cloned by taking advantage of the fact that
transformation ofyeastwith
gapped plasmids frequently
results in repair of the gap using informationfrom the
chromosomal region corresponding
to
the gap (ORRWEAVER,
SZOSTAK
and ROTHSTEIN 1981).The pet122-9
allele was made by mutagenizing a plasmid carrying PET122
on an approximately 2.3-kb BamHI-EcoRV fragment subcloned into YCp50 (ROSEet al. 1987) in E. coli using Nmethyl-N’-nitro-N-nitrosoguanidine
as described by MILLER
1972). The mutation was mapped upstream of the PvuI site
of PET122 by exchanging PvuI fragments with the original
wild-type derivative and analyzing thephenotype of the
resulting plasmids by transformation intoPTH37.The
pet122-9 allele was then sequenced between the HincII and
PvuI sites and foundto be achange of codon 82 from
CTA to CCA changing leucine to proline. The pet122-9
allele was subcloned into YIp5 and the resulting plasmid
(pPHY41CX82) was used to replace the chromosomal copy
of PET122 in DBY947 as described by BOEKE,LACROUTE
and FINK(1 984).
DNA sequence analysisof PET122 was previously reported by OHMEN,KLOECKENER-GRUISSEM
and MCEWEN
(1 988). Duringanalysis of the wild-type and mutant genes
described here we noted a discrepancy between our results
and this published DNA sequence: we found an additional
T residue following position 996. Figure 1 reportsour
sequence data between positions 950 and 1200. The additional T residue at position 997 generates an alteredreading
frame that predicts a PET122 protein of 254 amino acids.
Two lines of evidence indicate the sequence of Figure 1 is
correct. First, we found that the pet122-5 mutation is due
Yeast Translation in
to a C toT transition at position 1091. In the reading frame
of Figure 1 this substitution generates a UAA codon, accounting for the mutant phenotype. However, in the previously published open reading frame (OHMEN,KLOECKENER-GRUISSEM
and MCEWEN1988) this mutation would
produce a silent third base change. A second result consistent with the sequence of Figure 1, butinconsistent with the
previously published sequence, was obtained by fusing lacZ
to PET122 at position 113 1,which would have maintained
the previously reported PET122 termination codon. However, as predicted by the sequence of Figure l , this chimeric
gene directed the synthesis of a large fusion polypeptide in
Escherichia coli with antigenic cross-reactivity to PET1 22
(our unpublished data).
I n vivo labeling of mitochondrial translation products
were
and analysis of mitochondrial RNA: Yeastcells
grown to saturation in YPD medium at either 30" or 35"
for 24 to 36 hr prior to labeling at the respective temperatures. Radioactive labeling in vivo with ["Slmethionine
in the presence of cycloheximide, preparation of crude
mitochondria and SDS-polyacrylamide gel electrophoresis
were as previously described (DOUGLAS
and BUTOW1976;
MULLERet al. 1984) except that the labeling medium contained 0.5% glucose instead of galactose. Total cellular RNA
JENSEN and HERSKOWwas isolated as described (SPRAGUE,
ITZ 1983) from cells grown at either 30" or 37" and subjected to electrophoresis, blotting and hybridization to the
coxIII-specific probe pZHS3 as previously described (COSTANZO, SEAVER and Fox
1986).
To check that the mutant cells had remained rho+, aliquots of the mutant cultures grown at nonpermissive temperature were diluted and plated on YPD for single colonies.
The colonies were then mated to a Pet+, rho" tester strain
and the ability of the resulting diploids to grow on YPEG
medium was scored. More than 95% of the mutant cells
remained rho+.
Isolation and manipulation of P E T 1 2 3 The PET123
gene was cloned from a yeast genomic bank in the vector
YCp50 (ROSEet al. 1987), selecting for complementation of
the Pet"' allele pet123-1. T w o independent clones were
obtained, oneof which was within the other.A 1.9-kb XhoIBamHI fragment (this BamHI site is not present in the larger
clone and is probably the result of ligating a Sau3AI end
into the BamHI site of YCp50) was subcloned between the
Sal1 and BamHIsitesof
YCp50. The resulting plasmid
pPHY7 complemented pet123-1 and its 1.9-kb insert was
sequenced. Templates were made by subcloning DNA restriction fragments, generated from the sites shown in Figure 3, into the vectors M13mp18 or M13mp19 (MESSING
1983). A smaller SspI-BamHI fragment subcloned into the
NruI and BamHI sites of YCp50 (pPHY30) also complemented thepetl23-1 allele, whereas the DraI fragment
inserted into the NruI site of YCp50 (pPHY35) did not
(Figure 3). From pPHY3O and pPHY35 two derivatives
(pPHY39 and pPHY40) were constructed by swapping PvuI
fragments, using the PvuI site within the insert and another
one in the vector YCp50. pPHY40 is the subclone from the
first DraI site to the BamHI site, pPHY39 contains the
sequence from the Ssp1 site to thesecond DraI site. A HincIIBamHI fragment was subcloned separately into the NruIBamHI sites of YCp50. These three additional subclones
were then also tested for complementation of the pet123-1
allele (Figure 3).
Constructionof null alleles of PET123: The 1.9-kbXhoIBamHI fragment that complements petl23-1 was subcloned
intothe plasmid pUCl8 (YANISCH-PERRON,
VIEIRAand
MESSING1985), yielding the plasmid pPHB5. Into this plasmid, a 1.6-kbXbaI fragment containing the URA3 gene was
Mitochondria
497
........................................
951
GAG
ATA GCA TTG ATG CAG CAA GAT
ile ala l e u met g l n g l n a s p
r PVUII
CAA
GCA
GCT
GCC
CTG TTG
GCG
TTT
GGA
CGACAG CCC CTA GTG
ATA AAG
g l n pro leu Val i l e l y s
q l u q l n ala ala a l a l e u a l a l e u phe g l y a r q
1002
AAC GAA TGG TCA CTA CCG CTA CTA
asn q l u t r p s e r l e u pro l e u l e u l e u
CTG GGT
GCT GTC CTT TGG CAT
ala glyvalleutrphis
GTT CCC
v a l pro
r
T in pet122-5
1053
GGC CCA GCG CAG GCG CGA CGT
GTG CTG GCG GAG TTC CGT CAA
AGT TAT CGC
g l y pro a l a q l n a l a arqarq val l e u a l a glu phe a r g g l n s e r t y r arg
1104
GGG CTG
glyleu
CCG CTG CTG
pro l e u l e u a s p
GAT GCC GAA CTAATAGTG
AAG AGA AGA GGA TTT GAA
a l a g l u l e u val i l e l y s a r ga r g q l y phe glu
1155
ATC AAC ACA TAA ATCTGGGTGGAGCATCGCTGTAACAAGGAACAACG.....GTACGTTTCTG
i l e asn t h r OCH
1420
rPvuII
AATGATCAGCT GTT TAA
v a l OCH
FIGURE1.-Sequence
analysis of thedownstreamregion
of
PET122, containing alleles suppressible by pet123 mutations. T h e
positions of the two PvuII sites used to make the deletion mutation
pet122-6 are shown. As indicated, a Val residue is added at the C
terminus of theresultingprotein.
T h e location ofthe C to T
substitution that generates theUAA codon ofpet122-5is indicated.
This DNA sequence differs from that reported
by OHMEN, KLOECKENER-GRUISSEM
and MCEWEN(1988) by an additional T residue
following base 996 (see MATERIALS AND METHODS).
inserted creating anew plasmid pPHY21. [This 1.6-kb XbaI
fragment was derived by inserting the URA3 gene on an
Nrul-SmaI fragment from the plasmid YCp50 between the
HincII sites of pUC1318 (KAYand MCPHERSON
1987).] A
BamHI-PstI fragment,containingthedisruptedPET123
gene was isolated from pPHY21 and used to transform the
diploid strain PTH39. Haploids carrying the petl23::URAjr
allele (PTH78) were obtained by sporulation and verified
by their inability to complement pet123-1 and by DNA gelblot hybridization.
A large deletion of PET123 replacing the sequence between the two DraI sites (Figure 3)with the URA3 gene was
constructed as follows. A 527-bp KpnI-DraI fragment containing the start of PET123 and upstream sequences and a
304-bp DraI-BamHI fragment containing the carboxyterminus of PET123 and downstream sequences had been
subcloned into M13mp18, giving PHFl6 and PHFl2 respectively. A KpnI-Hind111 fragment from PHFl2 and an
EcoRI-SphI fragmentfrom PHF16 were then ligated between the KpnI and EcoRI sites of pUC18 in the presence
of a 1.6-kb HindII-SphI fragment containingthe URA3 gene,
yielding plasmid pPHY59. The 1.6-kb fragment carrying
the URA3 gene was derived from a subclone of the URA3
gene on an NruI-SmaI fragment from the plasmid YCp50
between the HincII sites of pUCl8 13
(KAYand MCPHERSON
1987). A 2.7-kb EcoRI-BamHI fragmentcontaining the
large pet123 deletion was used totransform the diploid
strain PTH39. Haploid strains with this deletion (PTH21 I),
termed pet123-12, were obtained upon sporulation of the
transformed diploid and verified by their inability to complement pet123-1 and by DNA gel-blot hybridization.
RESULTS
Primary sequence of pet122 mutant alleles: The
pet122 allele for which unlinked suppressors were
isolated was adeletion mutation, termed pet122-6,
that truncated the PET122 protein at its C terminus
by 67 amino acid residues (Figure 1). pet122-6 was
498
P. Haffter, T. W. McMullin and T. D. Fox
generated by replacement of the wild-type gene with
a sequence from which a 468-bp PvuII fragment had
beendeleted (MATERIALS AND METHODS). An additional allele affecting the C terminus was the previously isolated mutation pet122-5 (COSTANZO
and FOX
1988a; COSTANZO
et al. 1988), which was found to be
an ochre (UAA) mutation that truncated the PET122
protein by 24 amino acid residues (Figure 1; MATERIALS ANDMETHODS).
Two other pet122 mutations
used in this study were a complete deletion, termed
pet122-7, and a point
mutation,pet122-9, that changed
residue 82 from Leu to Pro (MATERIALS AND METHODS).
Isolation and characterization of suppressors of
pet122-6: A straincarrying the pet122-6 mutation
(TMAP2-3; Table 1) failed to grow on medium containing nonfermentable carbon sources (Pet- phenotype), as expected. However, TMAP2-3 yielded spontaneousrespiratory-competentrevertants
at afrequency of approximately 10-8,despite the fact that its
PET122 protein lacked 67 C-terminalamino acids
(Figure1). These revertants exhibited a
mitotically
stable Pet+ phenotypeat 30 O but were unable to grow
on nonfermentable carbonsources at 37 O . They grew
normally on glucose at all temperatures.
Eighteen heat sensitive (Pet") revertants of
TMAP2-3 were analyzed further, as outlined below
fortherevertantTMR2-1,todeterminewhether
their ability to respire was due to mitochondrial mutations, nuclear mutations linked to PET122, or unlinked nuclear suppressors. First, TMR2-1 was treated
with ethidium bromideto remove its mtDNA (GOLDRINC et al. 1970). Thisrhoo derivative was then crossed
to an isogenic Pet+, rho+ wild-type strain (TF189),
andthe
resulting diploid was sporulated. Tetrad
analysis of the progeny indicated that TMR2-1 contained an unlinked nuclear suppressor of pet122-6,
and furthermore that this suppressor mutation conferred itsownPet""
phenotype:approximatelyone
sixth of the tetrads (1 8 total) contained 2 Peths: 2 Pet+
(parental ditype), while one sixth contained 2 Peth": 2
Pet- (nonparental ditype) and two thirds contained 1
Pet+: 2 Peth': 1 Pet- (tetratype). Since this suppressor
mutationdefineda
new nucleargene (see below)
required for respiration even in the presence of a
wild-type PET122 gene, we have termed it pet123-I.
Similar crosses to wild type showed thatthe17
additional revertants of TMAP2-3 were also due to
nuclearmutations unlinked to PET122. However,
only one of these additional suppressors conferred an
intrinsic Pet"' phenotype while the other 16 had no
dramatic effect on respiratory growth in an otherwise
wild-type background.
As a first step toward determininghow many genes
could mutateto suppress pet122-6, we determined
whether the 17 additional suppressors were linked to
TABLE 2
Tetrad analysis of genetic linkage between markers in the
PET123 region on chromosomeXV
Ascus type (no.)
PD
Interval
pet12?-his3".'.'
pet123-ade2".'
petl23-ade9'
pet 123-SUF5'
ade9-his3'
SUFS-ade2'
SUF5-his3'
ade2-his3",'
163
183
60
161
73
145
62
76
NPD
5
3
0
0
0
1
4
12
Distance
T?'
210
100
34
31
19
29
117
221
(CM)
32
21
18
8
10
10
39
47
" Cross of G R F l B X PTHPO.
' Cross of PTH64 X 855.
' Cross of PTH86 X 405.
pet123-I. A pet122-6, pet123-I strain (PTH25) was
crossed to each of these revertants and the resulting
diploids were sporulated. Crosses involving 9 of the
17 additional revertants (including that with the second intrinsically heat insensitive suppressor) yielded
tetradsthat all contained 4 PethSspores, indicating
that their suppressor mutations
were tightly linked
and probably allelic to petl23-I. The suppressors in
the other 8 revertants (not described further here)
were unlinked to pet123-1.
The intrinsic Peths phenotype caused by pet123-I
(and that of the second intrinsically PethS suppressor
mentioned above)was recessive to wild type (therefore
the mutant allele is indicated in lowercase). However,
the suppresssion of pet122-6 by the pet123-1 mutation
was dominant: a diploid strain that was heterozygous
for the suppressor(pet123-IIPET123)and carried the
suppressible pet122-6 allele over the complete deletion, petI22-7, grew on
nonfermentable
carbon
sources at 30 (although more slowly than the haploid
TMR2-1) but not 37
at Similar results were obtained
with all of the suppressors linked to pet123-1.
Genetic mapping of PETl23: Analysis of a cross
between strain PTH20andGRF18(Table
l), revealed that petl23-1 was linked to ade2 and his3, two
markers on the right arm of chromosome XV (MORTIMER and SCHILD1985). We performed further tetrad analyses to map pet123 with respect to ade2 and
his3, as well as SUF5 and ade9 (Table 2). pet123 was
located between SUFS and ade9, most tightly linked
to SUFS (8 cM) and more weakly linked to ade9 (18
cM). N o pet mutations have been previously mapped
to this position.
The pet123 suppressorsarespecificfor
pet122
alleles that truncate the C terminus of the PET122
protein: The pet123 mutations could either be allelespecific suppressors that depend on residual function
of the pet122-6 allele, or mutations that completely
bypass PET122 function. T o distinguish between
these alternatives, we first tested the ability of the ten
O
O .
Translation
Mitochondria
in Yeast
suppressor mutationsin pet123 to suppress a complete
deletion of PET122 (the pet122-7 allele). Haploid
strains were constructed that each contained pet122-7
and one of the ten pet123 mutations. None of these
haploids was able to grow on nonfermentable carbon
sources, demonstrating that thepet123 mutations did
not simply bypass the requirement for PET122 function. Similarly, we found that the pet123-I mutation
failed to suppress (bypass) deletions of PET54 and
PET494, the other nuclear genes required specifically
for cox111 translation.
We also found that the pet123 mutations could not
suppress the missense mutation pet122-9 (Leu to Pro
at position 82; MATERIALS AND METHODS). First, diploids were constructed that contained pet122-9 over
the complete deletion pet122-7, and each pet123 mutation over wild-type PET123. These diploids failed
to respire,demonstratingthatnone
of the pet123
mutations were dominant suppressors of pet122-9.
Second, when strains carrying each of seven pet123
alleles (including petl23-I) were crossed to a strain
containing pet122-9 (PTH161) and sporulated, the
resulting tetrads each contained two Pet- spores, demonstrating that the pet123 mutations could not suppress the missense mutation, pet122-9, in haploids.
Interestingly, the pet123 mutations were able to
suppress pet122-5, an ochre mutation that truncates
the PET122 proteinby 24 C-terminal residues (Figure
1): diploids of genotype pet122-5/pet122-7, pet123/
PET123 were able to respire at 30", but not at 37".
Thus, the suppressor mutations in PET123 that were
selected to restore partial function to a PET122 protein lacking 67 C-terminal amino acids (the pet122-6
product), were also able to restore partial function to
a PETl 22protein lacking 24 C-terminal residues.
However as noted above, the pet123 mutations could
neither bypass the PET122 function nor suppress a
missense mutation affecting the N-terminal domain of
the PET122 protein. Taken together, these results
strongly suggest that the products of PET122 and
PET123 interact directly.
The petZ23-1 mutation causes temperature-sensitive mitochondrial protein synthesis: If the PET123
gene product does interact with the coxIII-mRNAspecific translational activator PETl 22, then the Pethr
mutation petl23-1 would be expected to affect mitochondrial translation at the nonpermissive temperature. T o test this prediction, cells were grown at
permissive and non-permissive temperatures,and
their mitochondrial translation products were selectively labeled in vivo in the presence of cycloheximide
(MATERIALS AND METHODS). The mitochondrially
coded proteinswere then detectedby SDS-gel electrophoresis and autoradiography (Figure 2; top panel).
In cells grown at permissive temperature, mitochondrial translation in the petl23-1 mutantstrain was
499
1 2 3 4
4
FIGURE 2.-Thepet123-I mutation reduces mitochotldrial translation in cellsgrownatnonpermissivetenlperature.
T o p panel:
Wild-type(DRY947) and prf123-I mut;tnt (PTH2O) cells were
grown and r;tdioactiveIy labeled (in the presence of cycloheximide)
at permissive (30") o r non-permissive (35") tempe~tturesas described i n MATERIAIS ANI) METHODS. Crude mitochondri;t were
prepared, and equal aliquots of protein from each sample ( I 6 pa)
were subjected to electrophoresis i n SDS-polvacrvl;l~nide( I 0- 15%
gradient) gels. T h e gels were dried and autor;ldiogr;lplied. Rottonl
panel: T h e pet123-I mutation has no effect on the level of cosIll
mRNA at non-permissivetemperature. Tot;~lcellular R N A was
isolated from wild-tvpe (DBY947) and prt123-I nlutant (PTHPO)
cells gro\vn in YPD nledium at permissive (30") or non-pernjissive
(37")temperatures.Followingelectrophoresis of 2 0 pg of each
s;tmple and blotting to nitrocellulose. the RNA was probed with
r;tdio;lctivelv labeledpZHS3 DNA, a probespecific for coxlll
(MULLERet al. 1984). and the filter w a s autoradiographed. A
parallel gel containing equal ;11110~1nts
of RNA w a s stxined with
ethidium bronlide and visualized under the U V light to confirm,
that equal anlounts of R N A were loaded. T h e I .6 kb coxlll m R N A
is indicated by the ;Irrowhe;ld. Lanes 1, Wild type (DBY947) grown
a t pertnissivetemperature: Lanes 2. pet123-Illlutant
(I"I'H20)
grown a t permissivetemperature:Lanes3,
Wild type grown a t
nonpermissivetemperature; I.anes 4. prtl23-I mutantgrown at
nonpermissive temperature.
comparable to wild type. However in cells grown at
the nonpermissive temperature, translation of all mitochondrial gene productsin the petl23-l mutant was
dramatically reduced relative to wild type although
some translation was still apparent. In contrast,
petl23-1 had no effect on the level ofthe 1.6-kb cox111
mRNA in cells grown at non-permissive temperature
(Figure 2; bottom panel), confirming that mitochon-
500
P. Haffter, T. W. McMullin and T. D. Fox
-114
URA3
.
CTCGAGGTATATAAffiAATGTAATTCAATG~AGACCCGATC~~CGT~TGC~C~TATCffiCffi
.
-514
.
AATTGGACCTTGTGTTGATCTTGTATCTGCTGCAATCACCACTCCGTTA~GAAT~AC~CTACAATG
-644
CGGTACCGGCACCAGCACACCATATTTTffi~~~CTATGCAATTTTGCGCTCACAATTCTTATCT~G~
Kpnl Kpnl
Ecll
Ssp1
Ghol
L
Dral
H~hcll
+/-
Xbal
EcoRl
-"
Cdl
Dral
BamHl
&oRI
"
"
+
Xmnl
"
"
"
orf of 318 aminoacids
PET123
FIGURE3,"Restriction map and sequence determination strategy of a 1 .l)-Lb D N A fragtnent carrying the PET123 gene. Arrows
indicate the extent and direction of nucleotide sequence detertninations. 'The position and direction of the PET123 coding sequence
is indicated by a thick arrow. Black bars indicate fragments tested
I i w their ability to complement achromosomal pet123-1 allele when
carried 011 the replicating vector YCp50: indicates completnentation, - indicates noncomplementation. The site of disruption of
the I'BT123 gene by insertion of (IRA3 is indicated by a triangle.
+
.
-504
GTGGTACCCGTGWV\GTTGCTCACCTTAGGTTGTGTGTGCGAGTGCGAGTTTTCC~T~G~TTATTTCTTTGATAAT
.
-434
TGTCGAACGATAAACCTGCCATGCTCACTCACTCAATTCTATTTTCTACAATCAGG~TATGT~~AGTAT
-364
.
TCCTGAARAAATTTAACGCTTTACTATACAAACTCTCTCCACCTCAACTGATCACCC~CT~TGAATTATT
-294
.
AGTTTATCCTTTTCATCTTTCTTTTTGGTATTTTGCCACTCGGCCGGGTTAT~TACGAAACATGGCC
-224
.
CTAATTTACCCGCTTTTACAATATAAAGGGTTTGTAATGAGffiT~TATTGGCCCTAATG~TCAC
-154
.
TAGCAACGGAAGTGCAAGTAGAACCCTGAAGTC~CGAGTAffi~TGATTCCCAAGAffiAGTAGAATA
.
-84
AAGAAGAGAATCTACATATGATTATTTAGGTAATAGTTTCATT~TGATTGTCAACAACAATAATAACA
-14
.
AGGATAATTGAAGTATCGGGAARffiTGCTCACGGCCAAGTATGGTTTTALAAGTG~TTTTCCCCACAACAAG
MetGlyLysGlyAlaAlaLysTyrGlyPheLy~SerGlyValPheProThrThrAr
57
GTCCATTCTGAAGAGCTCACCCCACTACAAAGCAGRCTGATATTATTAATAAffiT~TCACCCAAGCCCAAG
gSerIleLeuLysSerProThrThrLysGlnThrAspIleIleAsnLysValLysSerProLy~ProLys
121
.
GGTGTTCTTGGTATTGGGTATGCCAAAGGTGTAAAACATCC~ffiATCACATAGATTATCACCCAAffi
GlyValLeuGlyIleGlyTyrAlaLysGlyValLysHisProLysGlySerHi~ArgLeuSerProLysV
197
.
TAAACTTTATCGACGTGGATAATCTCATTGCGAAGACCGTTGCTCACTGAACCCCAGAGTATALAATCCAGT~
alAsnPheIleAspValA~snLeuIleAlaLysThrValAlaGluProGlnSerIleLysSerSerAs
267
.
CGGTTCAGCACAGAAAGTWATTACAAAAAGCTGAATTATTCGC
nGlySerAlaGlnLysValArgLeuGlnLysAlaGluLeeuArgArgLysPheLeuIleGluAlaPheArg
drialgene expression was blocked atthe level of
translation.
Interestingly,althoughmitochondrialtranslation
was defective in a pet123-1 mutant grown for several
generations at nonpermissive temperature,mutant
cells grown at permissive temperatureandthen
shifted to nonpermissive temperature shortly before
labeling hadnormalmitochondrial
translation (our
unpublished observations). This finding suggests that
the petl23-1 mutation may cause thermolabile assembly of a complex, rather than temperature sensitive
function, per se.
Primarystructure
of the PET123 gene: The
PET123 gene was cloned by selection of a plasmid
that complemented the Pet'" phenotype of pet123-1
from a bank ofyeast genomic DNA in thevector
YCp50 (ROSEet al. 1987). A1.9-kb XhoI-BamHI DNA
restrictionfragment was isolated from this plasmid
that complemented petl23-1 when carried on a replicating vector. Genetic data, described below, confirmed that this fragment carried the PET123 gene.
The 1.9-kb fragment was subcloned and sequenced
(Figure 3). The sequence contains one long open
readingframe, 954 nucleotides in length, which is
within a smaller SspI-BamHI fragment that also complements the pet123-1 mutation (Figure 3). This reading frame predicts that the PET123 protein is basic
(net charge, +26),with a molecular mass of 41,650 D
(Figure 4). However,thepredictedamino
acid sequencehadnostrong
homology to any sequences
found in the SWISS-PROT, PIR, MIPSX or EMBL
databases or in a collection of about 400 ribosomal
331
.
AAG~GAffiCTAGGCTATTACACAAACACGAGTATTTGCAGAAGAGGAC~GGAA~ffi~GC~
LysGluGluAlaArgLeuLHisLysHisGluTyr~uGlnLysAr~hrLysGlu~uGluLysAlaL
401
.
AAGAACTGGAGCTAGATMATAAGGAU.AATCTTCAGACTTGACTATTATGACTCTAGACALAAT
ysGluLeuGluLeuGluLysLeuAsnLysGluLysSerSerAJpLeuThrIleMetThrLeuAspLysMe
.
417
GATGTCTCAACCTCTTTTAAACAGATCACCAGAAGA~~TTGTT~GTTG~GAATTAC
tMetSerGlnProLeuLeuArgAsnArgSerPrffiluGluSerGluLeuLeuLysLeuLysArgAsnTyr
547
.
AATCGATCGTTGTTGAACTTTCAGGCGCACAA~GCT~CGAATTATT~CCTCTACCA~TCG
A s n A r g S e r L e u L e u A s n P h e G l n A l a H i s L y s L y s L y s I l A
611
.
CTAATGAATTCATCGTTACAGAATCCCCAACTACT~~TTGATAAGGT~TTAACGATG~CffiA
laAsnGluPheIleValThrGluSerGlnLeuteuLysLysIleAspLysValPheAsnAspGluThrG1
681
.
GGAATTCACTGATGCCTATGATGTAACTTCTAACTTCACGTTTCA
uGluPheThrAspAlaTyrAspValThrSerAsnPheThrGlnPhffilyAsnArgLysLeuLeuLeuSer
151
.
GGTAACACCACCCTACAAACCCAAATTAACAACGCTCACAATAAT~GCTC~TGTC~C~GTTTTTCG
GlyAsnThrThrteuGlnThrGlnIleAsnAsnALaIleMetGlySerLeuSerAsnGluLysPhePheA
a21
.
ATATTTCTTTAGTGGATTCATATCTGAACAA~ATTT~CATTTCCAAT~TTGATTCC~TT
spIleSerLeuValAspSerTyrLeuAsnLysAsp~uLysAsnIleSerAsnLysIleAspSerLYsLe
891
.
AARCCCCACCTCTAATGGAGCTGGAAATAAT~TAATAATAATAATACAACCAACTTGT~TAACTAAT
uAsnProThrSerAsnGlyAlaGlyAsnAsnGlyAsnAsnAsnAsnThrThrAsnLeu*'*
961
.
ATATATACTACTTCAACATGTCATGTATTCGAAAGTGCCTTAGACGCA~ACAACT~G~GAAG~
1037
.
AAAAAAATCAAATCGAGAATAAATATGCCCATGTATATACGTCACTT~GAATTGTCTATTTC~TCGTT
1101
.
CCCCTTTTTCTTTCAGTCCGCTGATGTTATCAAT~GATATTATCGCCTTTC~CA~ATC
FIGURE4.-Nucleotide sequence of PET123 and derived amino
acid sequence of its product.
protein sequences (I. WOOL, personal communication).
Disruption of PET123 yields viable cells that become rho- or rho': Mutations that completely block
mitochondrial translation have been shown to destabilize the wild-type mtDNA and lead to the produc-
Translation in Yeast Mitochondria
tion of cells that contain large or complete deletions
of mtDNA (rho- and rho', respectively) (MYERS,PAPE
and TZAGOLOFF
1985; MYERS,CRIVELLONE
and TZAGOLOFF 1987; FEARON andMASON 1988). The PethS
mutation pet123-I did not cause cells to become rhoduring growth at the nonpermissive temperature (MATERIALS AND METHODS). However, the pet123-1 mutation is slightly leaky, and residual protein synthesis
couldbe sufficient to maintain rho+ mtDNA. T o
determine whether a pet123 null mutation would prevent the maintenanceof rho+ mtDNA, theURA? gene
was inserted at theXbaI site in the open reading frame
of PET123 (Figure 2;MATERIALS AND METHODS). This
construct was then used to transform toUra+, a
Pet+,ura?/ura?, rhof diploid strain (PTH39), disrupting one chromosomal copy of PET12? (ROTHSTEIN
1983). Sporulation of a transformed diploid resulted
in 2:2 cosegregation of Pet- and Ura+ in all tetrads.
As expected, this pet12?::URA? allele failed to complement thePethSphenotype of pet123-I, and was
genetically linked to ade2 and his?.
None of the haploids carrying the pet12?::URA?
allele (including, for example, PTH78) were able to
complement the respiratory defect of Pet+, rho' tester
strains, demonstrating thatthey were unable to maintain wild-type mtDNA. However, like the translation
defective null mutants previously studied by others,
the petl2?::URA? strains couldmaintain rho- mtDNA:
some subclones from each of the pet12?::URA? haploids studied were able to marker-rescue mitochondrial mutations in the genes oxil, 0x22, oxi? and cob.
A strain (PTH211) carrying a chromosomal deletion of virtually the entirePET123 reading frame was
made as described in MATERIALS AND METHODS. Like
the pet12?::URA? disruptiondescribedabove, cells
carrying this deletion, termed pet12?-12, were viable
but could not maintain rho+ mtDNA.
Null mutations in the mitochondrialRNA polymerase gene, rpo41, reportedly cause the complete
lossof mtDNA, resulting in rho' cells (GREENLEAF,
KELLYand LEHMAN 1986).
However, in the course of
control experiments we found that a strain carrying
an rpo41-disruption (kindly provided by M. WOONTNER and J. JAEHNING) was also able to maintain rhomitochondrial genomes over several generations, as
judged by the ability to marker-rescue mitochondrial
mutations (our unpublished observation).
DISCUSSION
Translation of the mitochondrialmRNAcoding
cox111 is specifically activated by the productsof three
nuclear genes PET122, PET494 and PET54 (CABRAL
and SCHATZ1978; MULLER et al. 1984; COSTANZO
SEAVERand Fox 1986;
and FOX 1986; COSTANZO,
COSTANZO et al. 1988; KLOECKENER-GRUISSEM,
Mc-
50 1
EWENand POYTON1987, 1988)which must somehow
communicate with the mitochondrial translation system. This study provides strong genetic evidence for
adirectinteraction
between one of these specific
activators, the PET122 protein, and a component of
the generalmitochondrialtranslation
system coded
by the newly discovered gene P E T I 2 3 .
We sought to identify proteins that interact with
PETl 22 by looking for unlinked allele-specific suppressors of a pet122 mutation. Of particular interest
were suppressor mutationsthat, by themselves, caused
a temperature sensitive defect in mitochondrial gene
expression. Two such PethS suppressor mutations
were
isolated, which (along with eight other suppressors
having no intrinsic phenotype) mapped to the same
locus onchromosome XV, identifying a previously
unknown gene, termed P E T I 2 3 .
These suppressors (petl2? mutations) were selected
to act on a pet122 mutation that deleted 67C-terminal
amino acid residues of the PET 122 protein. Interestingly, they also suppressed a mutation that truncated
the PETl 22 protein by 24 C-terminal amino acids.
However, the suppressormutations did not simply
bypass the requirement for PET122 function, since
they were unable to suppress both a complete deletion
of PET122 and a missense mutation affecting the Nterminal regionof the PETl22 protein. Furthermore,
suppression did not appear to involve increased levels
of either the wild-type PETl 23 protein or the Cterminally truncated PET 122 protein: the presence
of genes coding eitherof these proteins on multicopy
plasmids did not suppress the pet122-6 mutation (our
unpublished results). Taken together, these data are
most easily explained by the hypothesis that the wildtype PET122 and PET123 proteins interact to promote the translationof coxIII. Accordingto this interpretation, the interaction would be disrupted by removal of the PET 122C-terminus, but would be partially restored by compensating mutations affecting
the structure of the PET123 protein. However we
cannot exclude an alternativepossibility, whereby the
suppressor mutations in PET123 would change the
structure of part of the mitochondrial translation system such as the ribosome. These structural changes
might then allow translation of the cox111 mRNA to
be promoted by a truncated version of PET 122 protein without involving adirectcontact
between
PET122 and PET123.
The finding that the Pethsallele, pet123-I, dramatically reduces the synthesis of all mitochondrially
coded proteins incells grown at the nonpermissive
temperature while the level of cox111 mRNA remains
normal, clearly suggests that the PET123 gene product is required for general mitochondrial translation.
Further support for this conclusion comes from the
finding thatpet123 null alleles cause instability of wild-
502
P. Haffter, T. W. McMullin and T. D. Fox
type (rho’) mtDNA and lead to the production of rhovide important clues to themechanism of this mRNAand rho’ cells. This phenotype has been previously
specific translational activation system.
found to be characteristicfor null alleles of genes
We thank S. PASSMORE,
C. CHANand P. LINDERfor performing
required for mitochondrial translation, such as mitocomputer searches, and I. WOOLfor searchinghis ribosomal protein
chondrial aminoacyl tRNA synthetases and mitochonsequence library. We also thank E. C . SEAVERfor cloning PET122,
drial ribosomal proteins (MYERS, PAPEand TZAGO- and M. CULBERTSONfor gifts of strains. P.H. was a recipient of an
LOFF 1985; MYERS, CRIVELLONE and TZACOLOFF EMBO long-term fellowship and T.W.M. was a Post-doctoral Associate of theCornell Biotechnology Program.This work was
1987; FEARON and
MASON 1988). Clearly, the viability
supported by a grant (GM29362) from theU.S. National Institutes
of strains carryingpet123 null mutations indicates that
of Health.
PET123function is notrequiredfor
vital cellular
Note added in proof: The PET123 sequencehas been
processes other than mitochondrial translation.
entered
in the EMBL Data Library, accession number
The phenotypes of the pet123 mutants described
X52362.
here are consistent with the possibilities that PET123
could be a mitochondrial ribosomal protein or a miLITERATURECITED
tochondrial translation initiation factor required for
ATTARDI,G., and G. SCHATZ,1988 Biogenesis of mitochondria.
translation of many (or all) mitochondrial mRNAs.
Annu. Rev. Cell Biol. 4: 289-333.
The open reading frame of PET123 encodes a basic
BOEKE,J. D., F. LACROUTEand G . R. FINK, 1984 A positive
protein (net charge, +26) with a predicted molecular
selection for mutants lacking orotidine-5’-phosphate decarboxmass of approximately 42 kD. The predicted aminoylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen.
Genet. 197: 345-346.
terminus could serve as a signal for import into mitoCABRAL,
R., and G. SCHATZ,1978 Identification of cytochrome c
chondria, since, like signals for othernuclear-encoded
oxidase subunits in nuclear yeast mutants lacking the functional
mitochondrial proteins (reviewed in HAY,BOHNIand
en7,yme.J. Biol. Chem. 253: 4396-4401.
GASSER1984; DOUGLAS,MCCAMMON
and VASSACOSTANZO,M . C., a n d T . D. FOX, 1986 Product of Saccharomyces
ROTTI 1986), it is relatively rich in basic amino acids
cereuisiae nuclear gene PET494 activates translation of a specific mitochondrial mRNA. Mol. Cell. Biol. 6: 3694-3703.
and in serine and threonine residues. The predicted
COSTANZO,M. C., and T. D. FOX, 1988a Specific translational
amino acid sequence of PET1 23 exhibited no strong
activation by nuclear gene products occurs in the 5’ untranshomology to any other proteinsequence we have
lated leader of a yeast mitochondrial mRNA. Proc. Natl. Acad.
searched. However in this connection, it is worth
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M. C., and T . D. FOX, 1988b Transformation of yeast
notingthat several S . cerevisiae mitochondrial riboby
agitation
with glass beads. Genetics 1 2 0 667-670.
somal proteins are not closely homologous to any
COSTANZO,
M . C., E. C. SEAVER
and T . D. FOX, 1986 At least two
other known polypeptides (MYERS, CRIVELLONE and
nuclear gene products are specifically required for translation
TZACOLOFF
1987;PARTALEDIS and MASON 1988;
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CHOLI
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COSTANZO,M. C . , E. C . SEAVERand T. D. Fox, 1989 The PET54
mRNA-specific translational control may be a comgene of Saccharomyces cereuisiae: characterization of a nuclear
mon feature in yeast mitochondria, since translation
gene encoding a mitochondrial translation activator and subof the mitochondrial mRNAs codingboth
cox11
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(POUTRE and Fox 1987;STRICKand Fox 1987) and
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1988 Multiple nuclear gene products are required toactivate
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and
1985; ROtranslation of a single yeast mitochondrial mRNA, pp. 3 7 3 DEL, KORTE and KAUDEWITZ1985; RODEL1986; RO382 in Genetics of Translation: New Approaches, Vol. 14, edited
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Communicating editor: M. CARLSON