• No results found

Expression of the Saccharomyces cerevisiae Gene YME1 in the Petite-Negative Yeast Schizosaccharomyces pombe Converts It to Petite-Positive

N/A
N/A
Protected

Academic year: 2020

Share "Expression of the Saccharomyces cerevisiae Gene YME1 in the Petite-Negative Yeast Schizosaccharomyces pombe Converts It to Petite-Positive"

Copied!
8
0
0

Loading.... (view fulltext now)

Full text

(1)

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 thea- andg-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 F1component of mitochondrial ATP synthase are also petite-negative. Thus, the F1complex has an essential activity in cells lacking mitochondrial DNA and Yme1p can mediate that activity, even in heterologous systems.

M

ITOCHONDRIAL biogenesis requires the coor- et al. 1996). While Yme1p is envisioned to have a role dinated expression of genes encoded by mito- as an assembly/editing factor similar to Yta10p/Yta12p chondrial and nuclear genomes, as well as the regulated and mitochondrial Lon, this has not yet been demon-assembly of a number of multicomponent protein com- strated experimentally. YME1 was originally identified plexes. Recent work in the yeast Saccharomyces cerevisiae in a screen for mutations that increase the rate at which has revealed the importance of a related set of mito- mitochondrial DNA (mtDNA) is transferred to the nu-chondrial proteases in the assembly of energy transduc- cleus (ThorsnessandFox1993). yme1 strains are also tion complexes. Located in the inner mitochondrial heat sensitive for growth on nonfermentable carbon membrane is a hetero-oligomer, composed of the ho- sources (pet-ts) and cold-sensitive for growth on rich-mologous proteins Yta10p and Yta12p, that is necessary glucose media and have altered mitochondrial mor-for the assembly of cytochrome oxidase and ATP syn- phology (Thorsnesset al. 1993;Campbellet al. 1994). thase (Paul and Tzagoloff 1995; Arlt et al. 1996; S. cerevisiae strains in which YME1 has been inactivated

Gue´linet al. 1996). The yeast homolog of the Escherichia are petite-negative; that is, they are incapable of growth coli lon protease has also been implicated in the assem- in the absence of mtDNA (Thorsnesset al. 1993). Wild-bly of inner membrane protein complexes (Repet al. type S. cerevisiae is a petite-positive strain, capable of 1996). These proteases have an ATP requirement, and growing on fermentable carbon sources in the absence mutational analysis has led to a proposal that these pro- of mtDNA. As a consequence of dysfunctional mito-teins function largely as assembly factors with editing chondrial structure and function, yme1 strains have an capability. Misfolded and damaged proteins or proteins increased rate of mitochondrial compartment turnover present in excess that cannot be assembled into a by the vacuole (

Campbell and Thorsness 1998).

higher-order complex are degraded by these proteases.

Clearly, Yme1p is involved in a number of important Yme1p forms another inner mitochondrial membrane

mitochondrial processes that must extend beyond the complex with protease activity (Leonhardet al. 1996).

turnover of unassembled Cox2p. Yme1p determines the stability of cytochrome oxidase

In an effort to understand the physiological role of subunit II that is present in excess or is not assembled

Yme1p in mitochondrial biogenesis and function, a into the multicomponent cytochrome oxidase complex

number of suppressors of yme1 phenotypes have been (Nakaiet al. 1995;PearceandSherman1995;Weber

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

Corresponding author: Peter Thorsness, Department of Molecular

of the 26S protease (Campbellet al. 1994). Inactivation

Biology, University of Wyoming, Laramie, WY 82071-3944.

E-mail: thorsnes@uwyo.edu of YNT20, which encodes a putative 39-59exonuclease

(2)

below). The S. pombe strain PNY10 was a gift of Dr. Paul Nurse. located in mitochondria, suppresses the high rate of

The Kluveromyces lactis strain Y11401 was a gift of Dr. Claudia mtDNA escape in yme1 strains and also suppresses the

Abeijon. The genotypes of both strains are listed in Table 1. synthetic nonrespiring phenotype of yme1 yme2 strains Media: E. coli strains containing plasmids were grown in (HanekampandThorsness1998). Specific dominant Luria-Bertani (LB) medium (10 g bactotryptone, 10 g NaCl, mutations in the gene encoding the g-subunit of the 5 g yeast extract per liter;Maniatiset al. 1982) supplemented with 125 mg/ml of ampicillin. Yeast strains were grown in mitochondrial ATP synthase, ATP3, suppress the

petite-complete glucose medium (YPD) containing 2% glucose, 2% negative phenotype of yme1 yeast (Weberet al. 1995).

Bacto-peptone, 1% yeast extract; complete ethanol-glycerol For none of these suppressors has it been possible to

medium (YPEG) containing 3% glycerol, 3% ethanol, 2% demonstrate a substrate/product relationship between Bacto-peptone, 1% yeast extract; or minimal glucose medium the identified gene products and the Yme1p protease. (SD) containing 2% glucose, 6.7 g/liter yeast nitrogen base In the work presented here, a number of additional without amino acids (Difco, Detroit) and supplemented with the appropriate nutrients. Nutrients were uracil at 40 mg/ mutations that result in the suppression of various yme1

liter, adenine at 40 mg/liter, tryptophan at 40 mg/liter, lysine phenotypes are described, and another suppressor of

at 60 mg/liter, and leucine at 100 mg/liter. For agar plates, the petite-negative phenotype of yme1 yeast is fully

de-Bacto-agar was added at 20 g/liter. Where indicated, ethidium scribed. Additionally, the heterologous expression of bromide was added at 25mg/ml (Weberet al. 1995). Yme1p in the petite-negative yeast Schizosaccharomyces yme1D1::URA3suppressor isolation and analysis:Bypass

sup-pressors of yme1-D1::URA3 were isolated by screening for spon-pombe was used to define the genetic basis of yeast strains

taneous revertants of the pet-ts phenotype of PTY52. Overnight that require mtDNA for viability.

cultures grown in YPD at 308were plated on YPEG plates at z2000 cells/plate and incubated for several days at 378. Iso-lated revertants able to respire at high temperatures were MATERIALS AND METHODS

colony purified, rescreened on YPEG at 378, analyzed for sup-pression of other yme1-D1::URA3 phenotypes, and screened Strains:The E. coli strains used for preparation and

manipu-for recessive collateral phenotypes by scoring manipu-for growth on lation of DNA were DH5a[F2end, hsdR17 (rk2mk1), supE44,

different carbon sources at different temperatures. Each sup-thi-1, l recA, gyrA96, relA1, D(argF-lacZYA) U169, φ80,

pressed strain was mated to every other suppressed strain. lacZDM15] and XL1 Blue [recA1, endA1, gyrA96, thi-1, rsdR17,

Diploids were analyzed to identify complementation groups. supE44, relA1, lac (F9proAB, lacIqZDM15, Tn10 (tetr))]. The E.

Tetrad analysis of matings between the suppressed strains and coli strains ES1301 [lacZ53, mutS201::Tn5, thyA36, rha-5, metB1,

a wild-type strain provided confirmation that any collateral deoC, IN(rrnD-rrnE)] and JM109 [endA1, recA1, gyrA96, thi,

phenotype segregated with the suppressing mutation. Domi-hsdR17(rk2mk1), relA1, supE44, l-,D(lac-proAB), (F9, traD36,

nant suppressors were isolated in the same manner as the proA1B1, lacIqZDM15)] were used for in vitro mutagenesis

recessive suppressors except that the screen was performed and were obtained from Promega (Madison, WI). The

geno-using the diploid strain PTY523PTY60. Isolation of suppres-types of S. cerevisiae strains used in this study are listed in Table

sors of the yme1 petite-negative phenotype was performed as 1. Standard genetic techniques were used to construct and

analyze the various yeast strains (Sherman et al. 1986; see described (Weberet al. 1995).

TABLE 1

Yeast strains

Strain Genotypea Source

PTY44 MATaura3-52 lys2 leu2-3,112 trp1-D1 [rho1, TRP1] ThorsnessandFox(1993)

PTY52 MATaura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 [rho1, TRP1] Thorsnesset al. (1993) PTY62 MATaura3-52 lys2 leu2-3,112 trp1-D1 yme1-1 [rho1, TRP1] ThorsnessandFox(1993) PTY78 MATaura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ATP1-75 [rho0] Weberet al. (1995) PTY93 MATa ura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ATP1-75 [rho1, TRP1] Weberet al. (1995) DKY1 MATaura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 YNT9-1 [rho1, TRP1] This study

MATa ura3-52 ade2 leu2-3,112 trp1-D1 yme1-D1::URA3

DKY21 MATaura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ynt4-1 [rho1, TRP1] This study DKY22 MATaura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ynt5-1 [rho1, TRP1] This study DKY25 MATaura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ynt6-1 [rho1, TRP1] This study DKY26 MATaura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ynt7-1 [rho1, TRP1] This study DKY27 MATaura3-52 lys2 leu2-3,112 trp1-D1 yme1-D1::URA3 ynt8-1 [rho1, TRP1] This study DKY30 MATaura3-52 lys2 leu2-3,112 trp1-D1 atp1-D1::LEU2 [rho1, TRP1] This study

NTY1 MATaura3-52 lys2 leu2-3,112 trp1-D1 ynt1-1 [rho1, TRP1] Campbellet al. (1994) W303DATP12 MATaade2-1 his3-11,15 leu2-3,112 ura3-1 trp1-1 atp12::LEU2 [rho1] Bowmanet al. (1991)

PNY10b h2leu1-32 Paul Nurse

Y11401c MATa ura3 Claudia Abeijon

aThe mitochondrial genotype is bracketed.

bS. pombe strain.

(3)

Nucleic acid techniques and DNA sequencing:All manipula- Ackerman. The gene disruption was made by removing an internal 200-bp BglII fragment of the ATP1 gene and replacing tions of DNA were performed using standard techniques

(Maniatiset al. 1982). Restriction enzymes and DNA modifi- it with a 3-kb BglII fragment containing the LEU2 gene. The

chromosomal disruption was made by digesting pG50/ST6 cation enzymes were purchased from New England Biolabs

(Beverly, MA). Double-stranded DNA templates were pre- partially with KpnI and then digesting completely with HindIII. The 4-kb KpnI-HindIII fragment containing the atp1::LEU2 pared for sequencing by boiling lysis (Maniatiset al. 1982)

and were sequenced by the nucleotide chain termination construct was gel purified and used to transform PTY44. The resulting strain, DKY30, was then tested for the ability to re-method (Sangeret al. 1977) using the Sequenase Version 2.0

DNA sequencing kit (United States Biochemical, Cleveland). spire and whether it was petite-positive or petite-negative. Cre-ation of the null mutant was verified by performing PCR with DNA-blot hybridization analysis was performed using standard

techniques (Maniatiset al. 1982). Blots were washed in 23 the oligonucleotides 59-TAAAGGTCCTATTGACGCTGC-39 and 59-TATTGTAGAGGAGCGGCTTCA-39.

SSC with 0.5% SDS for 2 hr at 508. Random primed probes

corresponding to the entire open reading frames (ORFs) of S. pombe expression clones:The S. pombe expression con-structs were made using the plasmid pART1, a gift from Dr. YME1 and ATP1 from S. cerevisiae were prepared using the

NEBlot kit from New England Biolabs. Paul Nurse. To isolate the YME1 coding sequence, the plasmid pYME1-NdeI was digested with NdeI and the ends were made Isolation ofATP1-75:Briefly, genomic DNA was prepared

as described (RoseandBroach1991) from the yeast strain blunt by filling in the site with Klenow. After digestion with SacI, the 2-kb YME1 fragment was isolated and ligated into PTY78 bearing the ATP1-75 mutation. Genomic DNA was

par-tially digested with Sau3A; 6–10-kb DNA fragments were iso- pART1 that had been digested with SacI and SmaI, generating pART1-YME1. The plasmid pyme1Dm was constructed by di-lated and cloned into the yeast CEN-vector pRS316 (Sikorski

andHieter1989). Library DNA was prepared by large-scale gesting pART1-YME1 with MluI. This site was then filled in

using Klenow and the plasmid was religated. Transformation alkaline lysis (Maniatiset al. 1982) and was used to transform

PTY62 using the alkali cation treatment method (Itoet al. of S. pombe was performed by treatment of cells with alkali cations (Itoet al. 1983).

1983). Approximately 23105transformants were inoculated

into 10 ml of selective SD and incubated with agitation at 308 Preparation of cellular extracts and detection of Yme1p and Atp1p:Protein extracts were prepared as described (

Pil-for 2 hr. Aliquots (1 ml) of the SD culture were inoculated

into each of 10 tubes containing 10 ml selective SD media lus and Solomon 1986). Mitochondrial fractions were iso-lated by differential centrifugation (Daumet al. 1982). Protein plus ethidium bromide (25mg/ml) and grown to saturation

fractions were separated by SDS-PAGE, transferred to a nitro-at 308(8 days). Ten microliters of each culture was inoculated

cellulose membrane, and probed with antisera directed into 10 ml of fresh SD plus ethidium bromide media and

against Yme1p or Atp1p as described previously (Hanekamp

grown to saturation at 308(12 days). Dilutions of each culture

andThorsness1996).

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 RESULTS

from the YPD plates and the phenotype was rechecked.

Plas-mid DNA was prepared from four isolates. Three clones con- Isolation and characterization of bypass suppressors

tained identical inserts ofz2.4 kb. One of these, pDK1, was of yme1: To identify gene products that interact with sequenced and found to contain ATP1. This clone was sent Yme1p, suppressors of a yme1-D1::URA3 null allele were to Macromolecular Resources (Fort Collins, CO) for

sequenc-isolated. The yme1 null allele was used for this analysis ing of the entire ATP1-75 allele.

because suppressors of yme1-1, a missense mutation, In vitromutagenesis of plasmid DNA: In vitro mutagenesis

of plasmid DNA was performed using the Altered Sites in vitro were all intragenic. Mutants were isolated either as dom-mutagenesis system from Promega. Oligonucleotides were ob- inant or recessive suppressors of the yme1 pet-ts pheno-tained from Integrated DNA Technology. The ATP1-75 allele type or as suppressors of the yme1 petite-negative pheno-was cloned into pALTER-Ex1 as follows. pDK1 pheno-was digested

type. These mutants were then screened for their ability with KpnI and the ends were made blunt with mung bean

to suppress other yme1 phenotypes, as well as for inher-nuclease. After digestion with EcoRI, the 2.2-kb ATP1-75

frag-ent collateral phenotypes. Nine strains were idfrag-entified ment was isolated and inserted into pALTER-Ex1 that had

been digested with EcoRI and StuI. The resulting construct, with mutations that suppress various yme1 phenotypes pDK3, was the starting substrate for site-specific mutagenesis. (Figure 1). Complementation analysis indicates that The mutant ATP1 allele was restored to wild type using the

these suppressors represent mutations at distinct ge-oligonucleotide 59-GGCTTTGAACTTGGAGCC-39,

generat-netic loci. These mutations can be placed in classes ing the plasmid pDK5. The sequencing oligonucleotide 59

-based on the yme1 phenotypes suppressed and collateral GCAGTCGGTGATGGTATTGC-39was used to verify that the

wild-type ATP1 sequence was restored in pDK5. This plasmid phenotypes linked to the suppressing mutation. Class was then used as a substrate to recreate the ATP1-75 allele 1 contains the recessive suppressor ynt1, which sup-using the oligonucleotide 59-GGCTTTGATCTTGGAGCC-39. presses all of the yme1 phenotypes and has a recessive The sequence of the resulting plasmid, pDK6, was again

veri-collateral phenotype of cold-sensitive growth on the fied using the sequencing oligonucleotide described above.

nonfermentable carbon sources ethanol and glycerol The 2.2-kb EcoRI-HpaI inserts in pDK5 and pDK6 were

trans-ferred to the yeast CEN vector pRS316, creating the plasmids (Campbell et al. 1994). Class 2 includes the recessive pDK9 and pDK10, respectively. These constructs, along with mutants ynt4 and ynt5. These mutations suppress the pDK1, were transformed into PTY62 to verify that the ATP1- yme1 pet-ts, the high rate of mtDNA escape, and cold-75 mutation was able to suppress the petite-negative yme1

sensitive growth on rich-glucose phenotypes. These sup-phenotype.

pressing mutations display no collateral phenotypes. Creation of anATP1null allele:The ATP1 null mutant was

(4)

is the mutation YNT9-1, which is presumably a dominant suppressor of the yme1 pet-ts phenotype since it was iso-lated 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 suppres-sors includes the dominant mutations ATP3-1 and ATP1-75, previously referred to as YNT3-1 (Weberet al. 1995). These mutants suppress only the petite-negative pheno-type of yme1. Cells grown in the presence of ethidium bromide rapidly lose all DNA from their mitochondria (Slonimskiet al. 1968;Foxet al. 1991) and are termed rho0. Unlike wild-type yeast, which readily grows in the presence of ethidium bromide, yme1 rho0yeast 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

yme1rho0slow-growth phenotype:Previous work in our laboratory demonstrated that mutations in the ATP-synthaseg-subunit suppress the petite-negative pheno-type 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 (Chenand Clark-Walker1995, 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 li-brary 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

Figure1.—Suppression of yme1 phenotypes. Top, the indi- isolated from this screen. Figure 2 shows the growth of

cated yeast strains were scored for mtDNA escape by patching a rho0yme1-1 mutant after introduction of one of these on a YPEG plate, incubating at 308for 2 days, and then replica

plasmids, pDK1. DNA sequence analysis demonstrated plating to synthetic glucose lacking tryptophan. The plate was

that the plasmid insert contained the complete open then scored after 4 days of incubation at 308. Strains that have

reading frame of ATP1, the structural gene for the an increased rate of mtDNA escape have an increase in the

relative number of Trp1 papillae (Thorsness et al. 1993). a-subunit of mitochondrial ATP synthase. Sequence Middle, the indicated strains were patched to YPD, incubated analysis of the entire ORF identified a single nucleotide for 1 day and then replica plated to YPD and YPEG and

change that resulted in the conversion of the strictly incubated at the temperatures shown for 1–7 days, depending

conserved asparagine residue at position 102 to isoleu-on temperature and media. Bottom, the indicated strains were

cine. To demonstrate that the amino acid change at streaked on a synthetic glucose plate containing 25 mg/ml

ethidium bromide and incubated for 5 days at 308. Strains: position 102 in ATP1-75 was responsible for suppression yme-1D (PTY52), yme1D ynt1 (NTY1), yme1D ynt4 (DKY21), of the petite-negative phenotype, site-directed mutagen-yme1D ynt5 (DKY22), yme1D ynt6 (DKY25), yme1D ynt7

esis was performed. First, the residue was changed to (DKY26), yme1Dynt8 (DKY27), yme1DATP1-75 (PTY93), yme1D

the wild-type asparagine, followed by reversion of the YNT9-1 (DKY1), WT (PTY44).

asparagine residue back to the mutant isoleucine. As shown in Figure 2, a plasmid bearing the reverted wild-type ATP1 allele is unable to rescue the petite-negative mutants ynt7 and ynt8, which suppress the pet-ts and

cold-sensitive growth on rich-glucose media phenotypes phenotype, while both the original ATP1-75 allele and the reverted ATP1-75 allele restore growth on ethidium of yme1 strains. Additionally, these mutations have a

recessive collateral phenotype of temperature-sensitive bromide.

A requirement of functional mitochondrial ATP-syn-growth on rich-glucose media. The fourth class of

reces-sive yme1-D1::URA3 suppressors includes the mutant thase F1component in rho0yeast:It has been reported that null mutations in the ATP-synthase g-subunit ynt6, which suppresses only the pet-ts phenotype. This

mutation has a recessive collateral phenotype of pet-ts (Weber et al. 1995) and the d-subunit (Giraud and

(5)

Figure3.—Growth of rho0yeast bearing yme1, atp1, or atp12 null alleles. Yeast cells were streaked on a synthetic glucose plate (A) and incubated for 3 days at 308and on a synthetic glucose plate containing 25mg/ml ethidium bromide (B) and

Figure 2.—Complementation of the yme1 petite-negative

incubated for 5 days at 308. The indicated yeast strains are phenotype by cloned ATP1-75. Wild-type yeast (PTY44) or a

wild type (PTY44), yme1D(PTY52), atp1D(DKY30), and atp12D strain bearing the yme1-1 mutation (PTY62) was transformed

(W303DATP12). 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 25mg/ml ethidium

bromide (B) and incubated for 5 days at 308. The indicated andK. lactis:The results presented above suggest that plasmids are the vector (pRS316), the cloned ATP1-75 allele the activity of Yme1p in S. cerevisiae is essential to main-(pATP1-75), the cloned ATP1 allele reverted to wild type via

tain the petite-positive phenotype of this yeast. Because site directed mutagenesis (pATP1r), and the ATP1 wild-type

of the striking similarities between a yme1 mutant and clone reverted back to the ATP1-75 allele (pATP1-75r).

the petite-negative yeasts S. pombe and K. lactis, we won-dered if this growth characteristic was due to the absence of a Yme1p-like activity. To address this issue DNA-blot cerevisiae. These observations have been taken as

evi-hybridization analysis was performed to determine dence for a requirement of assembled and functional F1

whether the genomes S. pombe and K. lactis encode a subunits in mitochondria that lack DNA. To determine

YME1 homolog. Total DNA prepared from the strains whether an ATP1 null mutation would confer a similar

PTY44, PTY52, PNY10, and Y11401 was digested with phenotype, an ATP1-D1::LEU2 strain, DKY30, was

con-EcoRI or SspI, blotted, and probed with a random-structed and streaked on media containing ethidium

primed probe prepared from a PCR product corre-bromide. Additionally, another strain that had an

sponding to the YME1 ORF under low stringency condi-ATP12 null mutation was also cultured on media

con-tions. Figure 4 shows that a strong signal was detected taining ethidium bromide. Atp12p is involved in the

for the S. cerevisiae DNA (Figure 4, lanes 5, 6, 9, and assembly of ATP-synthase F1 subunits (Bowman et al.

10), corresponding to YME1. An additional weak signal 1991). As shown in Figure 3, both null mutant strains

was detected in the SspI-digested S. cerevisiae DNA at exhibit a petite-negative phenotype, demonstrating the

z6.5 kb (Figure 4, lanes 9 and 10). These fragments requirement for an assembled and functional F1portion

correspond to the AFG3 gene (data not shown). Diges-of the ATP-synthase complex, even in the absence Diges-of a

tion of the AFG3 locus with EcoRI produces a fragment functional F0 component.

(6)

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. Designa-tions for the DNA sources are as follows: Sc (lanes 1, 5, 9, 13, 16), wild-type S. cerevisiae (PTY44); Sc9(lanes 2, 6, 10),

yme1-D1::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 se- Figure5.—Heterologous expression of YME1 in S. pombe. S. cerevisiae and S. pombe were transformed with the indicated quences were used to probe genomic DNA (Figure 4,

plasmids and streaked on a synthetic glucose plate (A) and lanes 13–18).

incubated for 5 days at 308or streaked on a synthetic glucose To identify a closely related YME1 gene product in plate containing 25mg/ml ethidium bromide (B) and incu-S. pombe, whole cell extracts were prepared from a incu-S. bated for 7 days at 308. The indicated strains are as follows: pombe culture, proteins were separated on polyacryl- 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 amide gels and blotted to a nitrocellulose filter, and

PNY10. The indicated plasmids are as follows: pART1, S. pombe the filter was probed with polyclonal antisera directed

expression vector; pYME1, the vector plasmid pART1 bearing against Yme1p. No cross-reaction with the Yme1p anti- wild-type YME1; pyme1Dm, the vector plasmid pART1 bearing sera was observed (Figure 6, lane 4). In contrast, antisera an inactivated yme1 allele.

directed against the conserved S. cerevisiae Atp1p gene product detected S. pombe Atp1p (Figure 6, lanes 4

allows growth in the absence of mtDNA, while the and 5).

pART1 vector and the inactivated yme1 allele do not. Heterologous expression of YME1 in S. pombe: To

Thus, Yme1p provides an activity that is essential for determine if Yme1p activity was able to convert a

petite-viability of two yeast when they lack mtDNA. Western negative yeast to petite-positive, YME1 was expressed in

blot analysis using whole cell extracts from these strains S. pombe. The YME1 gene was cloned into the S. pombe

detected Yme1p in mitochondria of S. pombe trans-expression vector pART1. As an additional control, the

formed with pART1-YME1 (Figure 6). pART1-YME1 construct was digested with MluI, filled

in, and religated. This plasmid, pyme1Dm, has a frame-shift in the YME1 ORF that creates a nonfunctional gene

DISCUSSION product. These two plasmids, along with the vector, were

transformed into a S. cerevisiae yme1 mutant and the S. To identify possible Yme1p substrates and to elucidate the role of Yme1p in yeast mitochondria, we undertook pombe strain PNY10. As shown in Figure 5, expression

(7)

we surmised that these yeasts may be petite-negative because they lack a Yme1p-like activity. DNA-blot analy-sis indicated that neither K. lactis nor S. pombe encode a close YME1 homolog (Figure 4), nor is there an anti-genically related Yme1p homolog in S. pombe (Figure 6). Additionally, heterologous expression of YME1 in S. pombe converted this negative yeast to petite-positive. 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

Figure6.—Detection of Yme1p in S. cerevisiae and S. pombe. (Abrahamset al. 1994) to identify the relative location

Mitochondrial fractions prepared from the indicated strains of mutations in thea- andg-subunits of mitochondrial bearing the indicated plasmids were subjected to SDS-PAGE,

ATP synthase that convert negative yeast to petite-transferred to nitrocellulose filters, and probed with antisera

positive. The amino acid changed in ATP1-75, Asn102, directed against Yme1p and Atp1p. Lane 1, PTY44/pART1;

is located near the interface of thea- andg-subunits in lane 2, PTY52/pART1; lane 3, PTY52/pYME1; lane 4, PNY10/

pART1; lane 5, PNY10/pYME1. Whole cell extracts and post- the region referred to as the “dimple” of the F1complex, mitochondrial supernatants prepared from PTY52/pART1, distal to the stalk connecting F1to F0(Abrahamset al. PNY10/pART1, or PNY10/pYME1 do not produce a

cross-1994). The two amino acid positions mutated in the K. reacting signal with Yme1p antisera.

lactis a-subunit, Ala333 and Phe443, are also located along this interface, in direct contact with theg-subunit (ChenandClark-Walker1996). Thus, it is likely that genes were identified that, when mutated, lead to

sup-pression of various yme1 phenotypes (Figure 1). Because the interaction of the a- and g-subunits is altered in these mutant F1complexes.

mutation of different genes suppress different

spec-trums of yme1 phenotypes, we anticipate multiple roles The information presented here suggests that Yme1p must be involved in the regulation of mitochondrial for Yme1p in mitochondrial biogenesis and function.

Previous work in our laboratory identified mutations ATP synthase, although the nature of this regulation is unknown. The consequences of the absence of Yme1p in the ATP3 gene that suppress the petite-negative

phe-notype of yme1 strains (Weber et al. 1995). Here we in yeast that contain a complete mitochondrial genome is not deleterious at 308, as these cells can use nonfer-identify a mutation in the ATP1 gene that suppresses

the same phenotype. The F1complex of the mitochon- mentable carbon sources. Upon loss of mtDNA, how-ever, the absence of Yme1p leads to the petite-negative drial ATP synthase is a multi-subunit structure with a

subunit stoichiometry of a3b3gdε. Deletion of ATP1 phenotype. It has previously been shown that Yme1p is a metallo-protease that is involved in the turnover of leads to the inability to use nonfermentable carbon

sources (Takedaet al. 1986) and causes a petite-negative cytochrome oxidase subunit II (Nakai et al. 1995;

Pearce and Sherman 1995; Weber et al. 1996). One

phenotype (Figure 3). Mutational inactivation of the

g- and d-F1 subunits (Weber et al. 1995; Giraud and possibility is that Yme1p may also have a direct role in the turnover of F1 subunits. In mitochondria lacking

Velours1997) and of Atp12p, a protein involved in F1

subunit assembly (Bowmanet al. 1991), also cause this mtDNA, which encodes the F0subunits Atp6p, Atp8p, and Atp9p, excess F1subunits might be degraded in a phenotype (Figure 3). These results indicate that

Yme1p, as well as the F1portion of the ATP synthase, are Yme1p-dependent manner. Without Yme1p, F1subunits may accumulate, leading to aberrant complex forma-important for the growth of S. cerevisiae in the absence of

mtDNA. One extensively characterized mutation that tion. This scenario seems unlikely since the accumula-tion of Atp3p has been shown to be independent of causes S. cerevisiae to become petite-negative is op1, a

mutation in the major ADP/ATP translocator encoded Yme1p (Weber et al. 1995). Alternatively, Yme1p may affect the F1 complex indirectly by interacting with F0 by AAC2 (Kovacovaet al. 1968;LawsonandDouglas

1988;Kolarovet al. 1990). Both op1 and a null mutation subunits, at least six of which are encoded in the nu-cleus. In the absence of the mitochondrially encoded of ACC2 render cells unable to grow on nonfermentable

carbon sources (Kovacova et al. 1968; Lawson et al. F0subunits, the nuclearly encoded subunits may be sub-ject to degradation by Yme1p, allowing the F1portion 1990). The petite-negative nature of op1 strains has been

taken as evidence for the absolute requirement for ATP of the ATP synthase to function properly. In the absence of Yme1p, these F0subunits may accumulate and inter-in the matrix of mitochondria.

The yeasts K. lactis and S. pombe are petite-negative. fere with an F1activity that is essential for cells lacking mtDNA. The mutations that have been identified in Work in K. lactis has shown that specific mutations in

the ATP-synthasea-,b-, andg-subunits convert this yeast ATP1 and ATP3 would thus allow the F1 complex to assume a conformation that is not subject to interfer-to a petite-positive organism (ChenandClark-Walker

1995, 1996). Based on the similarities between these ence by inhibitory factors that exist in the absence of Yme1p activity.

(8)

ing oxidative phosphorylation with cytoplasmic mutation to respi-We thank Dr. Sharon Ackerman, Dr. Paul Nurse, and Dr. Claudia ratory deficiency. Biochim. Biophys. Acta 162: 157–163. Abeijon for generously providing plasmids and yeast strains. We also Lawson, J. E.,andM. G. Douglas,1988 Separate genes encode thank the members of the Thorsness laboratory for critical review of functionally equivalent ADP/ATP carrier proteins in

Saccharo-myces cerevisiae. J. Biol. Chem. 263: 14812–14818. this manuscript. This work was supported by Public Health Service

Lawson, J. E., M. Gawaz, M. KlingenbergandM. G. Douglas,1990 grant GM-47390.

Structure-function studies of adenine nucleotide transport in mitochondria. I. Construction and genetic analysis of yeast mu-tants encoding the ADP/ATP carrier protein of mitochondria. J. Biol. Chem. 265: 14195–14201.

LITERATURE CITED Leonhard, K., J. M. Herrmann, R. A. Stuart, G. Mannhaupt, W.

Neupertet al., 1996 AAA proteases with catalytic sites on

oppo-Abrahams, J. P., A. G. W. Leslie, R. LutterandJ. Walker,1994

site membrane surfaces comprise a proteolytic system for the Structure at 2.8 A resolution of F1-AtPase from bovine heart mito- ATP-dependent degradation of inner membrane proteins in

mi-chondria. Nature 370: 621–628. tochondria. EMBO J. 15: 4218–4229.

Arlt, H., R. Tauer, H. Feldmann, W. NeupertandT. Langer,1996 Maniatis, T., E. F. FritschandJ. Sambrook,1982 Molecular Clon-The YTA10-12 complex, an AAA protease with chaperone-like ing: A Laboratory Manual. Cold Spring Harbor Laboratory Press, activity in the inner membrane of mitochondria. Cell 85: 875–885. Cold Spring Harbor, NY.

Boeke, J. D., F. LacrouteandG. R. Fink,1984 A positive selection Nakai, T., T. Yasuhara, T. FujikiandA. Ohashi,1995 Multiple for mutants lacking orotidine-59-phosphate decarboxylase activity genes, including a member of the AAA family, are essential for in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197: degradation of unassembled subunit 2 of cytochrome c oxidase 345–346. in yeast mitochondria. Mol. Cell. Biol. 15: 4441–4452.

Bowman, S., S. H. Ackerman, D. E. GriffithsandA. Tzagoloff, Paul, M. F.,andA. Tzagoloff,1995 Mutations in RCA1 and AFG3

1991 Characterization of ATP12, a yeast nuclear gene required inhibit F1-ATPase assembly in Saccharomyces cerevisiae. FEBS Lett. for the assembly of the mitochondrial F1-ATPase. J. Biol. Chem. 373:66–70.

266:7517–7523. Pearce, D. A.,andF. Sherman,1995 Degradation of cytochrome

Campbell, C. L.,andP. E. Thorsness,1998 Escape of mitochon- oxidase subunits in mutants of yeast lacking cytochrome c and drial DNA to the nucleus in yme1 yeast is mediated by vacuolar- suppression of the degradation by mutation of yme1. J. Biol. Chem. dependent turnover of abnormal mitochondrial compartments. 270:20879–20882.

J. Cell Sci. 111: 2455–2464. Pillus, L., andF. Solomon, 1986 Components of microtubular

Campbell, C. L., N. Tanaka, K. H. WhiteandP. E. Thorsness,1994 structures in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA Mitochondrial morphological and functional defects in yeast 83:2468–2472.

caused by yme1 are suppressed by mutation of a 26S protease Rep, M., J. M. van Dijl, K. Suda, G. Schatz, L. A. Grivellet al., subunit homologue. Mol. Biol. Cell 5: 899–905. 1996 Promotion of mitochondrial membrane complex

assem-Chen, X. J.,andG. D. Clark-Walker,1995 Specific mutations in bly by a proteolytically inactive yeast Lon. Science 274: 103–106.

Rose, M. D.,andJ. R. Broach,1991 Cloning genes by complementa-alpha- and gamma-subunits of F1-ATPase affect mitochondrial

tion in yeast. Methods Enzymol. 194: 195–230. genome integrity in the petite-negative yeast Kluyveromyces lactis.

Sanger, F., S. NicklenandA. R. Coulson,1977 DNA sequencing EMBO J. 14: 3277–3286.

with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:

Chen, X. J.,andG. D. Clark-Walker, 1996 The mitochondrial

5463–5467. genome integrity gene, MGI1, of Kluyveromyces lactis encodes

Sherman, F., G. R. Fink andJ. B. Hicks, 1986 Methods in Yeast the beta-subunit of F1-ATPase. Genetics 144: 1445–1454.

Genetics. Cold Spring Harbor Laboratory Press, Cold Spring

Har-Daum, G., P. C. Bo¨ hni andG. Schatz,1982 Import of proteins

bor, NY. into mitochondria. Energy-dependent uptake of precursors by

Sikorski, R. S.,andP. Hieter,1989 A system of shuttle vectors and isolated mitochondria. J. Biol. Chem. 257: 13028–13035.

yeast host strains designed for efficient manipulation of DNA in

Fox, T. D., L. S. Folley, J. J. Mulero, T. W. McMullin, P. E.

Thors-Saccharomyces cerevisiae. Genetics 122: 19–27.

nesset al., 1991 Analysis and manipulation of yeast

mitochon-Slonimski, P. P., G. PerrodinandJ. H. Croft, 1968 Ethidium drial genes. Methods Enzymol. 194: 149–165.

bromide induced mutation of yeast mitochondria: complete

Giraud, M. F.,andJ. Velours,1997 The absence of the

mitochon-transformation of cells into respiratory deficient non-chromo-drial ATP synthase delta subunit promotes a slow growth

pheno-somal “petites.” Biochem. Biophys. Res. Commun. 30: 232–239. type of rho2yeast cells by a lack of assembly of the catalytic

Takeda, M., W. J. Chen, J. SaltzgaberandM. G. Douglas,1986 sector F1. Eur. J. Biochem. 245: 813–818.

Nuclear genes encoding the yeast mitochondrial ATPase

com-Gue´lin, E., M. RepandL. A. Grivell,1996 Afg3p, a mitochondrial

plex. Analysis of ATP1 coding the F1-ATPase alpha-subunit and ATP-dependent metalloprotease, is involved in the degradation

its assembly. J. Biol. Chem. 261: 15126–15133. of mitochondrially-encoded Cox1, Cox3, Cob, Su6, Su8, and Su9

Thorsness, P. E.,andT. D. Fox,1993 Nuclear mutations in Saccharo-subunits of the inner membrane complexes III, IV and V. FEBS

myces cerevisiae that affect the escape of DNA from mitochondria Lett. 381: 42–46.

to the nucleus. Genetics 134: 21–28.

Hanekamp, T.,andP. E. Thorsness,1996 Inactivation of YME2/

Thorsness, P. E., K. H. WhiteandT. D. Fox,1993 Inactivation of RNA12, which encodes an integral inner mitochondrial mem- YME1, a gene coding a member of the SEC18, PAS1, CDC48 brane protein, causes increased escape of DNA from

mitochon-family of putative ATPases, causes increased escape of DNA from dria to the nucleus in Saccharomyces cerevisiae. Mol. Cell. Biol. 16: mitochondria in Saccharomyces cerevisiae. Mol. Cell. Biol. 13: 5418–

2764–2771. 5426.

Hanekamp, T.,andP. E. Thorsness,1998 YNT20, a bypass suppres- Weber, E. R., R. S. Rooks, K. S. Shafer, J. W. Chaseand P. E. sor of yme1 yme2, encodes a putative 39-5-exonuclease localized in Thorsness,1995 Mutations in the mitochondrial ATP synthase mitochondria of Saccharomyces cerevisiae. Curr. Genet. 34: 438–448. gamma subunit suppress a slow-growth phenotype of yme1 yeast

Ito, H., Y. Fukuda, K. MurataandA. Kimura,1983 Transformation lacking mitochondrial DNA. Genetics 140: 435–442.

of intact yeast cells treated with alkali cations. J. Bacteriol. 153: Weber, E. R., T. HanekampandP. E. Thorsness,1996 Biochemical 163–168. and functional analysis of the YME1 gene product, an ATP and

Kolarov, J., N. KolarovaandN. Nelson,1990 A third ADP/ATP zinc-dependent mitochondrial protease from S. cerevisiae. Mol. translocator gene in yeast. J. Biol. Chem. 265: 12711–12716. Biol. Cell 7: 307–317.

Kovacova, V., J. IrmlerovaandL. Kovac,1968 Oxidative

Figure

TABLE 1

References

Related documents

The town of Teluk Intan, Perak was selected as the study site based on the street network structures, patterns, designs, and characters of a Malaysian small town.. In

Since the emphasis of this work is on the bias circuit, this section will focus mainly on the aspects that affect its implementation, namely the number of bits necessary for

Supplementary Materials: The following are available online at www.mdpi.com/2072-6643/9/4/359/s1 , Figure S1: Supernatant iron of digested NP-FePO 4 after in vitro simulated

Such a collegiate cul- ture, like honors cultures everywhere, is best achieved by open and trusting relationships of the students with each other and the instructor, discussions

The decision to launch the Ekofisk platform made way for the development, not only of offshore structures but also for a development of the concrete material,

Elective neck dissection versus observation in squamous cell carcinoma of oral cavity with clinically N0 neck: A systematic review and meta-analysis of prospective studies

Despite the unsatisfactory empirical results on the potential infl uence of human capital on internal migration, two variables that could appropriately represent the supply of