From the Department of Biological Sciences, Columbia University, New York, New York 10027
Received for publication, January 19, 2003, and in revised form, February 14, 2003
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ABSTRACT |
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aE280/U1 is a pet mutant of
Saccharomyces cerevisiae partially deficient in cytochromes
a, a3, and cytochrome b. The ability of this
mutant to respire is restored by RIB3, a gene previously shown to code for 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBP
synthase), an enzyme of the riboflavin biosynthetic pathway. The
sequences of RIB3 from wild type and aE280/U1 indicated a single base change resulting in an A137T substitution. The alanine 137 is a conserved residue located in a cavity on the surface of the
protein distant from the active site and from the subunit interaction
domain involved in homodimer formation. The respiratory defect elicited
by this mutation cannot be explained by a flavin insufficiency based on
the following evidence: 1) growth of the aE280/U1 on respiratory
substrates is not rescued by exogenous riboflavin; 2) the levels of
flavin nucleotides are not significantly different in the mutant and
wild type. We proposed that in addition to its known function in
riboflavin synthesis, RIB3 also functions in expression of
mitochondrial respiration. Restoration by riboflavin of growth of a
rib3 deletion mutant on glucose but not glycerol/ethanol also supported this conclusion. An antibody against the N-terminal half
of DHBP synthase was used to study its subcellular distribution. Most
of the protein was localized in the cytosolic fraction, but a small
fraction was detected in the mitochondrial intermembrane space.
Saccharomyces cerevisiae pet mutants
have been used to study a broad range of problems related to
mitochondrial function and biogenesis (1). Such mutants have been
assigned to the following phenotypic classes based on their
compositions of respiratory chain components: 1) mutants defective in
individual carriers of the respiratory chain, 2) mutants having a
normal set of respiratory enzymes (e.g. cytochrome spectra
and ATPase), and 3) mutants with partial or severe pleiotropic
deficiencies in respiratory chain components. Functional analysis of
the latter is difficult because the phenotype fails to provide clues as
to the nature of the biochemical lesion.
In the present study we have characterized a pet mutant
(aE280/U1) with partial pleiotropic defects in respiratory chain
components. The mutant has been determined to have a point mutation in
RIB3, a conserved gene of which the product,
3,4-dihydroxy-2-butanone-4-phosphate synthase
(DHBP1 synthase), catalyzes
the first step in the riboflavin biosynthetic pathway (2).
Complementation of aE280/U1 by RIB3 was unexpected in view
of previous studies indicating that rib3 mutants require riboflavin for growth (3). The lack of correspondence in the phenotype
of the rib3 point mutant and a mutant with a complete deletion of RIB3 prompted us to examine the biochemical
properties of the mutant further. We present evidence that the
phenotype of aE280/U1 is not related to a flavin nucleotide deficiency
and propose that in addition to its involvement in riboflavin
synthesis, DHBP synthase has another function essential for expression
of respiration in yeast.
Strains and Media--
The strains of yeast used in this study
are listed in Table I. The
respiratory-deficient mutant E280 (MAT Disruption of RIB3--
The linear
SacI-PstI fragment containing RIB3
(Fig. 1) was transferred to pUC18 (5). Two divergent primers each
containing a BamHI site were used to amplify the entire
plasmid except for the RIB3 coding region. The amplified
plasmid was ligated to a 1-kb BamHI fragment containing the
yeast HIS3 gene. The null allele, recovered as a linear
SacI-PstI fragment, was introduced into the wild
type diploid strain W303 by the one-step gene substitution procedure
(6). Dissection of tetrads obtained from several histidine prototrophic
diploid transformants indicated that growth of one-half of the meiotic
progeny on glucose depended on the presence of riboflavin. The
riboflavin auxotrophy co-segregated with the histidines prototrophy.
Preparation of an Antibody against DHBP Synthase--
The region
between the BclI and StyI sites coding for the
amino-terminal half of DHBP synthase was ligated in frame to the E. coli expression vector pATH2 (7) digested with
BamHI. This ligation required the introduction of a
BamHI linker at the StyI site. E. coli
RR1 cells harboring the plasmid were used to produce the
trpE/RIB3 fusion product as described elsewhere
(6). Following cells lysis, the overexpressed protein was recovered in
the insoluble protein fraction and was dissolved in buffer containing
10 mM Tris-Cl, pH 7.5, 2% SDS, 10 mM
Cloning of the Wild Type and Mutant RIB3--
A recombinant
plasmid (pG164/T3) containing RIB3 was obtained by
transformation of aE280/U1 with a yeast genomic library by the method
of Schiestl and Gietz (8). This library, consisting of partial
Sau3A fragments of yeast nuclear DNA cloned in the URA3-bearing shuttle plasmid YEp24 (9), was generously
provided by Dr. Marion Carlson, Department of Human Genetics, Columbia University. The chemical method of Maxam and Gilbert (10) was used to
sequence the ends of the nuclear DNA insert in pG164/T3.
To clone the mutant rib3 gene, nuclear DNA prepared from
aE280/U1 was digested with a combination of PstI and
SacI. Fragments averaging 1.9 kb were cloned in YEp352 (11)
and used to transform E. coli. The resultant
library was screened by colony hybridization with a
32P-labeled 1.9-kb PstI-SacI fragment
containing RIB3 (12). The identical procedure was
used to clone RIB3 from the wild type strains W303-1A and
D273-10B/A1. The two wild type and the mutant RIB3 genes
were sequenced by the Sanger method (13) with a commercial sequencing
kit (United States Biochemical, Cleveland, OH).
Miscellaneous Procedures--
Standard methods were used for
plasmid manipulations and colony hybridization (14). Yeast mitochondria
were prepared by the method of Faye et al. (15) except that
Zymolyase 20T (ICN Laboratories, Aurora, OH) instead of glusulase was
used to convert cells to spheroplasts. Spectral analyses of
mitochondrial cytochromes were performed as described previously (16).
Flavin nucleotides were extracted and separated by HPLC on a C18
reverse phase column as described previously (17). Protein
concentrations were determined by the method of Lowry et al.
(18).
Phenotype of E280 and Identification of the Mutant Gene--
E280
is a respiratory-deficient strain of S. cerevisiae
previously assigned to complementation group G164 of a pet
mutants collection (1). Like other pet mutants, E280 grows
on glucose but not on nonfermentable substrates such as
glycerol/ethanol. Some growth of the mutant, however, is observed on
glycerol/ethanol after incubation of plates for prolonged periods. The
mutant is complemented by
To identify the gene responsible for the respiratory defect of E280,
the uracil-requiring derivative strain aE280/U1 was transformed with a
yeast genomic plasmid library. Two independent respiratory competent
clones were obtained from the transformation. The plasmids had
overlapping nuclear DNA inserts, one of which (pG164/T3) was used to
localize the gene (Fig. 2). The results
of subcloning indicated that the gene conferring respiration on
aE280/U1 is located between a SacI and a PstI
site (Fig. 2). This region of the insert contains RIB3, a
gene of chromosome IV, previously shown to code for DHBP synthase (3).
This enzyme catalyzes the conversion of ribose-5-phosphate to
3,4-dihydroxy-2-butanone-4-phosphate, an early precursors in riboflavin
synthesis (2). The failure of pG164/ST31 and pG164/ST32, each
containing different halves of RIB3, to restore respiration
confirmed the correct identification of the gene. Site directed
integration of a single copy of RIB3 in the chromosomal DNA
of aE280/U1 restored normal growth on nonfermentable carbon sources
(Fig. 3A), suggesting that
RIB3 acts by complementation rather than suppression the
mutant.
aE280/U1 Is a rib3 Mutant--
Mutations in
RIB3 have been shown to elicit a riboflavin auxotrophy on
glucose (3). This phenotype was confirmed in the present study by
deleting the gene in the diploid strain W303 (see "Materials and
Methods" for the construction of the
The discrepancy in the growth properties of the rib3 null
mutant and aE280/U1 on glucose raised the possibility that
RIB3 might be suppressing rather than complementing the
respiratory defect. To test linkage of the Sequence of the Mutant RIB3 Gene--
To sequence the mutation,
the rib3 gene was cloned from aE280/U1. Because this strain
was derived from a cross of two non-isogenic strains (E280 and
W303-1A), the gene was also isolated from D273-10B/A21 (parent of E280)
and W303-1A. The genes obtained from the three strains were sequenced
and compared with the previously reported sequence of
RIB3.2 The
sequences of the D273-10B/A21 and W303-1A genes were identical to the
sequence number reported in GenBankTM. The rib3 gene
isolated from the aE280/U1 mutant, however, had a single G to A
transition at nucleotide 409 of the gene, resulting in the substitution
of alanine 137 by threonine.
The mutant gene isolated from aE280/U1 was cloned into the
integrative plasmid YIp352. Uracil-independent clones
obtained by transformation of the rib3 null mutant
a/ Flavin Content of Subcellular Fractions from Wild Type and Mutant
Cells--
The flavin nucleotides and riboflavin compositions of
mitochondria and cytosolic fractions (post-mitochondrial supernatant) were compared in the wild type, the mutant aE280/U1, and the mutant transformed with RIB3 on a high copy plasmid. Flavin
nucleotides were extracted and separated by HPLC on a C18 column.
Although some differences were noted in the amount of riboflavin and
flavin nucleotides in the three strains, there was no indication of any significant deficit of FMN or FAD in either the mitochondrial or
post-mitochondrial fractions of the mutant (Fig.
4). Some reduction in riboflavin
concentration is seen in the post-mitochondrial supernatant fraction.
The increased ratio of FMN relative to FAD in mitochondria of the
mutant and transformant may be because of strain differences, as this
is also true of mitochondria in the D273-10B/A21 wild type strain (not
shown). These results argue strongly against any significant effect of
the A137T mutation on riboflavin synthesis.
Localization of DHBP Synthase--
The subcellular localization of
DHBP synthase was studied with an antibody raised against the protein
expressed from a trpE/RIB3 fusion gene. The
antibody detected a protein of ~28 kDa in the post-mitochondrial
supernatant fraction of wild type yeast (Fig. 5A). The identity of this
protein as DHBP synthase is confirmed by its absence in the
rib3 null mutant and its presence at much higher
concentrations in the mutant transformed with the wild type gene on a
multicopy plasmid. The mass of DHBP synthase derived from the sequence
of RIB3 is 22.5 kDa. The reason for the apparent larger mass
obtained by SDS-PAGE is not clear.
Although most of the DHBP synthase was detected in the
post-mitochondrial supernatant fraction corresponding to the soluble cytosolic proteins of yeast, a small fraction was also present in
mitochondria. This was true both for the transformant overexpressing the protein (Fig. 5A) and for wild type (see longer exposure
in Fig. 5B). Several lines of evidence indicated that this
was not an artifactual association. The mitochondrial fraction isolated from an over-expresser was further purified on a Nycodenz gradient (23). The gradient fractions were probed with antibody against DHBP
synthase and cytochrome b2, used here as a
mitochondrial marker. The antibody detected two peaks, one of which
co-sedimented with the mitochondrial cytochrome
b2 marker (Fig. 5C). The second peak
was well separated in the upper part of the gradient. No attempts were
made to identify the nature of the material in this fraction.
Mitochondria purified on a Nycodenz gradient were diluted under
isotonic and hypotonic conditions. Even though the hypotonic treatment
causes the outer membrane to burst, it remains attached to and
fractionates with the mitoplasts. Both mitochondria and mitoplasts were
treated with proteinase K (24). The results of this experiment indicate
that disruption of the outer membrane released most of the DHBP
synthase and of the intermembrane marker cytochrome
b2 (Fig. 5D). The small fraction of
the two proteins remaining in the mitoplasts was completely susceptible
to the protease. In contrast, DHBP synthase and cytochrome
b2 were largely resistant to the protease in
unlysed mitochondria (Fig. 5D). The loss of DHBP synthase
from the mitoplasts suggests that the protein is located in the
intermembrane space. The possibility that DHBP synthase may be
associated with the outer membrane and is dissociated from it under
hypotonic conditions, however, cannot be excluded.
The mutation in aE280/U1 does not affect the steady-state concentration
of DHBP synthase in the cytoplasm (data not shown). This result
excludes the possibility that the respiratory deficiency is due to a
lower cytosolic concentration of the enzyme. It is interesting to note
that the mitochondrial and cytoplasmic concentrations of DHBP synthase
are higher in transformants harboring an integrated copy of the gene
than in wild type (Fig. 6). This suggests
that the chromosomal context (URA3 site in the case of the
integrated genes) influences the expression of the gene. The estimated
4-5-fold increase of DHBP synthase in the strain with the integrated
mutant allele (aW303 In the present study, the RIB3 gene encoding DHBP
synthase, an enzyme of the riboflavin biosynthetic pathway, was cloned
based on its ability to restore respiration in the pet
mutant, aE280/U1. The rescue by RIB3 is the result of
genetic complementation. This is supported by tight linkage of the
mutation in aE280/U1 to a rib3 null allele and sequence
analysis of the rib3 gene in aE280/U1, which confirmed the
presence of a mutation in the gene. The single base mutation causes a
relatively innocuous substitution of a threonine for an alanine at
residue 137 of the protein.
The rib3 null mutant used in this study shows an auxotrophic
requirement of riboflavin for growth on glucose as reported previously (3). In contrast, the rib3 point mutant, like other
pet mutants, grows on glucose in the absence of added
riboflavin. Moreover, riboflavin does not restore the growth of the
mutant on nonfermentable substrates. This suggests that the phenotype
of the rib3 point mutant is unrelated to the activity of
DHBP synthase in riboflavin synthesis. Instead it must have another
function required for expression of mitochondrial respiration. This is
also supported by the following evidence. 1) Analysis of FAD and FMN
failed to show any substantive decrease in the mitochondrial and
cytosolic concentrations of these nucleotides in the mutant aE280/U1.
Some decrease in the riboflavin concentration was seen in the cytosolic fraction of the mutant. This, however, could be because of the different genetic background of the two strains used for the analysis. 2) The rates of oxidation of NADH or succinate in isolated mitochondria were not increased when the point mutant was grown in the presence of
riboflavin in the growth medium. 3) Riboflavin rescued growth of the
rib3 null mutant on glucose but not on nonfermentable substrates.
Transformation of the rib3 null mutant with the
rib3 allele of E280 either in single (integrated into
chromosomal DNA) or in multiple copies (episomal plasmid) conferred
growth on glucose. Surprisingly, the transformants also grew on the
nonfermentable substrates glycerol/ethanol. We attribute this to
the elevated level of the mutant protein in the integrant as a result
of increased expression of the gene in the new chromosomal context
(URA3 locus). This explanation is consistent with the
partial restoration of growth of aE280/U1 on glycerol/ethanol when
transformed with an extra copy of the mutant gene. These observations
together with the slow growth phenotype of aE280/U1 on glycerol/ethanol
suggest that the E280 allele reduces but does not abolish the activity of DHBP synthase needed for respiration. Overexpression of the mutant
protein, therefore, is able to compensate for the partial loss of function.
The role of DHBP synthase in expression of mitochondrial respiration is
not clear. The presence, even at somewhat elevated levels, of
riboflavin in the mitochondria of the point mutant excludes DHBP
synthase from involvement in riboflavin transport. The presence of
riboflavin in the mutant also excludes 3,4-dihydroxy-2-butanone 4-phosphate, the precursor of riboflavin formed by the synthase, from
being needed in some other pathway essential for respiration. The more
likely alternative is that mitochondrial respiration depends on a
function of DHBP unrelated to riboflavin synthesis. The small fraction
of DHBP synthase detected in the mitochondrial intermembrane space
suggests that this function is likely to occur in mitochondria.
On the basis of the reported structure of the Magnaporthe
grisea DHBP synthase (25), the A137T mutation in aE280/U1 occurs in a region distant from the binding sites for the substrate and metal
cofactor. The mutation is also removed from the interface of the
homodimer (25). The alanine is located in a depression on the surface
of the protein with the methyl group pointing outward. The threonine,
with an extra carbon and hydroxyl, can be accommodated without
interfering sterically with the neighboring residues in this surface
structure. Although the alanine is conserved in the M. grisea enzyme, in some organisms the corresponding position is
occupied by serine (26). Conceivably, this domain may be involved in
the binding of a molecule that is delivered to mitochondria by DHBP
synthase and is required for biogenesis/maintenance of the respiratory chain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
met6 rib3) was derived from S. cerevisiae D273-10B/A21 by mutagenesis with
nitrosoguanidine (1). The media used for the growth of yeast have been
described elsewhere (4). Escherichia coli RR1 was used for
amplification of plasmid DNA and for expression of fusion proteins.
Genotypes and sources of yeast strains
-mercaptoethanol, and 5 µg/ml phenylmethylsulfonyl
fluoride. The solubilized fusion protein was further purified by
sizing on a Bio-Gel A 0.5 (BioRad) with 10 mM
Tris-Cl, pH 7.5, 0.1 mM EDTA, and 5 mM
-mercaptoethanol as the eluting buffer. Fractions enriched for the
fusion protein were pooled, concentrated by precipitation with acetone,
and dissolved in 0.1% SDS, 1 mM
-mercaptoethanol for
immunization of rabbits.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
o testers indicating that the
growth defect on respiratory substrates is due to a recessive mutation
in a nuclear gene. E280 displays normal translation of the
mitochondrial gene products (Fig.
1A). Spectra of mitochondrial
extracts, however, indicate a partial deficit of cytochromes a,
a3, and b relative to cytochrome
c (Fig. 1B). A 2-fold reduction of cytochrome
oxidase in the mutant was measured enzymatically (Table
II). The NADH and succinate oxidase activities are reduced, respectively, to 25 and 16% of wild
type mitochondria. The different oxidase activities were not increased in mitochondria of the mutant grown in the presence of
riboflavin.
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Fig. 1.
Phenotype of E280. A,
in vivo labeling of mitochondrial translation products. The
wild type strain D273-10B/A1 and the rib3 mutant E280 were
labeled in the presence of cycloheximide (18). Mitochondria were
prepared and separated on a 7.5-15% polyacrylamide gel (20). The
ribosomal protein Var1, COX subunits 1 (Cox1), 2 (Cox2), and 3 (Cox3), cytochrome b
(Cyt. b), and ATPase subunits 6 (Atp6), and 9 (Atp9) are identified in the margin. B, spectra
of mitochondrial cytochromes in the wild type and mutant yeast.
Mitochondria were prepared from the respiratory competent parental
strain D273-10B/A1 and from the respiratory defective mutant E280. The
mitochondria were extracted at a protein concentration of 6 mg/ml in
the presence of 1% potassium deoxycholate and 1 M KCl
(19). The difference spectra of the extracts oxidized with potassium
ferricyanide versus those reduced with sodium dithionite
were recorded at room temperature.
Respiratory activities of mitochondria
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Fig. 2.
Restriction map of pG164/T3 and
subclones. The restriction sites for BglII
(G), PstI (P), SacI
(S), XbaI (X), and StyI
(St) are indicated on the nuclear DNA insert of
pG164/T3. The solid arrow shows the location and direction
of the RIB3 open reading frame. The different regions of the
insert subcloned in YEp352 are denoted by the bars in the
upper part of the figure. The plus and
minus signs in parentheses indicate complementation and lack
thereof, respectively, of aE280/U1.
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Fig. 3.
Growth of wild type and rib3
mutants on different carbon sources in the presence and absence
of riboflavin. The following strains were used to compare growth
on the different media: the respiratory competent strains W303-1A
(WT), the rib3 null mutants aW303 RIB3
(
rib3) and D273
RIB3 (
rib3*), the
rib3 point mutant aE280/U1 (rib3), the
rib3 point mutant aE280/U1/ST22U with RIB3
inserted at URA3 (rib3/i-RIB3), the
rib3 point mutant aE280/U1/E280U with the mutant
rib3 gene of E280 inserted at URA3
(rib3/i-rib3), and the null mutant aW303
RIB3
with the mutant rib3 gene of E280
(
rib3/i-rib3) or of the wild type gene
(
rib3/i-RIB3). The different strains were
spotted on rich glucose (YPD) or rich
glycerol/ethanol (YEPG) plates without and without 25 µg/ml riboflavin (+ribo). The plates were incubated at
30 °C for 36 h.
rib3:HIS3 null
allele). Growth of the haploid strain W303
RIB3 containing the
deletion allele on glucose is strictly dependent on the addition of
riboflavin. This phenotype is different from that of E280 or aE280/U1,
both of which grow on glucose in the absence of riboflavin. An
unexpected property of the null mutant is its failure to grow on a
nonfermentable carbon source supplemented with riboflavin (Fig
3A). Like the rib3 null mutant, growth of the
point mutants on glycerol/ethanol also fails to be rescued by
riboflavin (Fig. 3A)
rib3:HIS3 null
and E280 point alleles, the 1.98-kb SacI-PstI
fragment containing RIB3 was transferred to the integrative
vector YIp352 (11) yielding pG164/ST22. This plasmid was linearized at
the BstXI site internal to RIB3 and was used to
transform aE280/U1. A respiratory competent and Ura+
clone obtained from the transformation (aE280/U1/ST22) were crossed to
W303-1B. Integration of RIB3 at its homologous locus in
chromosomal DNA was confirmed by dissection of 12 complete tetrads
issued from the resultant diploid cells. In each case all four meiotic spore progeny were ascertained to be respiratory competent. For the
allelism test aE280/U1/ST22 was crossed to the rib3 null
mutant W303
RIB3. Ten complete tetrads were analyzed following
sporulation of the diploid cells. In every case the requirement of
riboflavin for growth on glucose segregated 2:2. The ability of a
single copy of RIB3 to restore the respiratory deficiency of
aE280/U1 and the tight genetic linkage between the mutant allele of
aE280/U1 and the rib3 null allele constitute strong evidence
that the respiratory deficient phenotype of E280/U1 stems from a point
mutation in RIB3.
W303
RIB3 with the E280 gene on the integrative plasmid
were sporulated and the growth phenotype of the meiotic products
analyzed. The presence of the mutant gene in a single copy
restored normal growth of the null strain on glucose and glycerol in
the absence of added riboflavin (Fig. 3B). The ability of
the mutant gene to rescue the growth of the rib3 null strain
is consistent with the growth properties of E280 and suggested that the
A137T mutation does not affect riboflavin synthesis. Surprisingly,
growth of the point mutant on glycerol was also partially restored when
a single copy of the mutant gene was integrated into chromosomal DNA of
the null mutant (Fig. 3C).
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Fig. 4.
Riboflavin and flavin nucleotide compositions
of subcellular fractions of wild type and mutant yeast.
Mitochondria and the post-mitochondrial supernatant (PMS)
fractions were prepared from the wild type strain W303-1A, the
rib3 mutant aE280/U1, and the rib3 mutant
transformed with RIB3 on a high-copy plasmid
(aE280/U1/T3). Approximately 350 µg
of mitochondrial or post-mitochondrial supernatant proteins were
extracted and analyzed on a C18 reverse phase column as described
previously (17). The elution of FAD, FMN, and riboflavin
(RIBO) from the C18 column is indicated in the upper
left panel.
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Fig. 5.
Subcellular localization of DHBP
synthase. A, immunodetection of DHBP synthase.
Mitochondria and the post-mitochondrial supernatant fraction were
prepared from W303-1A (WT), the rib3 null mutant
aW303 RIB3 (
RIB3), and aW303
RIB3/ST15
(ST15), the null mutant transformed with a multicopy plasmid
containing RIB3. The mitochondria (M) and the
post-mitochondrial supernatant (S) fractions representing 20 µg of protein each were separated on a 12% polyacrylamide gel (22),
transferred to nitrocellulose, and probed with an antiserum against the
trpE-DHBP synthase fusion protein. Antibody-antigen complexes were
detected by a secondary reaction with 125I-protein A. The
arrow on the right points to DHBP
synthase. The migration of molecular size markers is shown on
the left. B, wild type mitochondria and
post-mitochondrial supernatant proteins were separated and probed as
described in A, but the Western blot was exposed to x-ray
film for a longer time. C, purification of mitochondria on a
Nycodenz gradient. The gradient was prepared by layering 0.9 ml of 5, 10, 15, 20, and 25% Nycodenz containing 0.6 M sorbitol, 20 mM K-MES, pH 6, in a 5-ml Beckman Ultracentrifuge tube
4 h before usage. Mitochondria (5 mg protein in 0.5 ml) from the
transformant aW303
RIB3/E280, prepared by the method of Glick
and Pon (23), were layered on the gradient and centrifuged at
75,000 × g for 90 min in a Beckman SW 65 rotor. A
total of 15 fractions were collected, separated on a 12%
polyacrylamide gel, and assayed for DHBP synthase and the mitochondrial
intermembrane space marker, cytochrome b2. DHBP
synthase (DHBP Synth.) and cytochrome
b2 (b2) are identified by the
arrows on the right. D, localization
of DHBP synthase in the intermembrane space of mitochondria.
Mitochondria of aW303
RIB3/E280 were purified on a Nycodenz gradient
as described in B. The mitochondria (Mt) at a
protein concentration of 13.5 mg/ml were diluted to 1 mg/ml in either
0.6 M sorbitol or 20 mM K-Hepes pH 7.4 or under
hypotonic conditions in 20 mM K-Hepes pH 7.4 to prepare
mitoplasts (Mp). One-half of the mitochondria and mitoplasts
were exposed to proteinase K (0.1 mg/ml) for 45 min on ice. The
proteinase-treated and nontreated controls were centrifuged at
12,000 × g for 20 min, and the pelleted particles were
resuspended and analyzed for DHBP synthase and cytochrome
b2 as described in A. DHBP synthase
and cytochrome b2 are identified by the
arrows on the right.
RIB3/i-E280) may explain the ability of
this transformant to grow on nonfermentable substrates (Fig.
3B).
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Fig. 6.
Levels of DHBP synthase in cells transformed
with the mutant gene on integrative and episomal plasmids.
Mitochondria were prepared from W303-1A (WT) and from the
rib3 null mutant transformed with the mutant gene from
sE280/U1 either on integrative plasmid
( RIB3/i-E280) or episomal plasmid
(
RIB3/E280). The mitochondrial (M)
and post-mitochondrial supernatant (S) fractions (20 µg
protein) were separated on a 12% polyacrylamide gel (22), transferred
to nitrocellulose, and probed with the antibody against DHBP synthase
as described in the legend for Fig. 5.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This research was supported by National Institutes of Health Research Grant HL2274 and by Postdoctoral Fellowship MDACU01991001 from the Muscular Dystrophy Association (to A. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biological
Sciences, Columbia University, New York, NY 10027. Tel.: 212-854-2920; Fax: 212-865-8246; E-mail: spud@cubpet2.bio.columbia.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M300593200
2 J. J. Garcia-Ramirez, M. A. Santos, and J. L. Revuelta, GenBankTM accession number Z21619.
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ABBREVIATIONS |
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The abbreviations used are: DHBP, 3,4-dihydroxy-2-butanone-4-phosphate synthase; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid.
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