(Received for publication, September 20, 1995; and in revised form, January 17, 1996)
From the
Mitochondrial NAD-dependent isocitrate dehydrogenase catalyzes a
rate-limiting step in the tricarboxylic acid cycle. Yeast isocitrate
dehydrogenase is an octomer composed of two subunits (IDH1 and IDH2)
encoded by different genes and possessing independent mitochondrial
targeting presequences. Oligonucleotide-directed mutagenesis was used
to remove the presequences from each gene and from both genes carried
on centromere-based expression plasmids. Effects on cellular
localization were examined in a yeast strain containing chromosomal
disruptions of IDH1 and IDH2 loci. Each subunit was
found to be dependent upon its presequence for mitochondrial
localization, and the subunits are independently imported into
mitochondria under most growth conditions. Furthermore, an active
holoenzyme can be assembled in the cytosol and this
``cytosolic'' form of isocitrate dehydrogenase can reverse
the acetate growth phenotype characteristic of the
IDH1/
IDH2 disruption strain, indicating functional
replacement of the mitochondrial enzyme. However, transformants
containing plasmids lacking either the IDH1 or IDH2 presequence coding regions were unexpectedly found to be capable
of growth on acetate medium. Further investigation demonstrated that
cellular localization of the IDH1 subunit can be biased by this
stringent growth pressure.
NAD-dependent isocitrate dehydrogenase in eucaryotic organisms
is a key enzyme in cellular energy metabolism. The oxidative
decarboxylation of isocitrate is considered an important control point
for flux through the tricarboxylic acid cycle since isocitrate
dehydrogenase is a complex oligomeric enzyme subject to extensive
allosteric regulation by energy charge(1) . The active form of
isocitrate dehydrogenase in Saccharomyces cerevisiae is an
octomer with subunits designated
IDH1 and IDH2(2, 3) . The deduced amino acid sequences
from the cloned genes and amino-terminal sequence analysis (4, 5) indicate that IDH1 and IDH2 are synthesized as
precursors of 360 and 369 amino acids, respectively, and are processed
upon mitochondrial import to yield mature polypeptides of 349 and 354
amino acids. IDH1 and IDH2 share 42% residue identity, and both
subunits share an approximate 32% residue identity with the
NADP-dependent isocitrate dehydrogenase of Escherichia
coli(6) . For the latter enzyme, crystallographic analyses
to 2.5 Å have been completed(7, 8) . Based on
similarity with the procaryotic enzyme, mutagenesis of the putative
isocitrate binding sites of the yeast subunits and kinetic analyses led
to a model for IDH1 function as a regulatory subunit and IDH2 function
in catalysis(9) .
The presequences of IDH1 and IDH2 have
features characteristic of many mitochondrial
presequences(10, 11) ; each contains several
positively charged and hydroxylated but no acidic amino acid residues.
The presequences of many mitochondrial proteins have been shown to be
essential and sufficient for import. Some exceptions include yeast
mitochondrial malate dehydrogenase, which has a dispensable presequence (12) , and the subunit of yeast F
-ATPase,
which contains redundant import signals, two residing within the
presequence and the third within the mature protein(13) . In
this report, we examine the requirement for presequences for
mitochondrial localization of IDH1 and IDH2 subunits and also examine
the independence of import of the two subunits of this multimeric
enzyme.
Compartmentalization of different metabolic pathways in eucaryotic cells frequently involves isozymes which catalyze similar reactions in different cellular compartments. The two mitochondrial and single cytosolic isozymes of isocitrate dehydrogenase in S. cerevisiae are good examples of this phenomenon. The mitochondrial tricarboxylic acid cycle enzyme is distinguished from the other two homodimeric NADP-specific isozymes (14, 15) by cofactor specificity, oligomeric composition, and allosteric sensitivity. Yeast strains containing disruptions in either or both IDH1 and IDH2 chromosomal loci share a dramatic growth phenotype, an inability to grow with acetate as a carbon source(4, 5) , with some other tricarboxylic acid cycle mutants including those lacking mitochondrial malate dehydrogenase (16) or citrate synthase(17) . This phenotype for strains lacking NAD-specific isocitrate dehydrogenase indicates that the residual mitochondrial NADP-specific isocitrate dehydrogenase cannot compensate for tricarboxylic acid cycle function, suggesting that NADH is the essential product of this reaction. In this report, we test whether a catalytically active NAD-specific enzyme can be assembled in the cytosol and whether reducing equivalents produced in that compartment can compensate for loss of the mitochondrial reaction.
Both IDH1 and IDH2 genes were previously subcloned into a multicopy 2-µm expression vector(9) . For the current study, to express the subunits at normal cellular levels, a 3.8-kilobase pair BamHI/HindIII fragment containing both genes was removed from the 2-µm plasmid and subcloned into pRS316, a centromere-based plasmid with a yeast URA3 gene for selection(21) . To insure a full-length promoter for the IDH1 gene, it was necessary to restore a BamHI restriction site 100 base pairs 5` of the coding region for insertion of an adjacent genomic DNA fragment. This involved use of a 24-mer oligonucleotide (5`-TCTAGAGGATCCATTTCAACAGTA) and resulted in a single nucleotide change (T to C at position -98).
Introduction of
the BamHI restriction site into IDH1 and mutagenesis
to remove the mitochondrial targeting presequences of IDH1 and IDH2 were conducted simultaneously. Two 30-mer
oligonucleotides complementary to regions flanking the presequence
coding regions (5`-ATTGTAAGAGAAAAATGGCCACTGCCGCTC for IDH1 and
5`-AATATTTTTTAATAATGGCTACTGTAAAGC for IDH2) were synthesized
for loopout mutagenesis and a 28-mer oligonucleotide
(5`-AGGGCGAATTGGCATGCCACCGCGGTGG) to change a SacI restriction
site in the multicloning region to an SphI site was
synthesized for use as a selective primer. All four mutagenic primers
were used in a single synthesis reaction with single stranded template
plasmid DNA prepared from the pRS316 plasmid carrying IDH1 and IDH2 genes. The ratio of loopout primers, BamHI
primer, and selective primer was 10:2:1. After strand synthesis and
transformation of mutS E. coli cells, the mixed plasmid
population was subjected to two rounds of amplification and digestion
with SacI to eliminate the template plasmid. The final plasmid
population was transformed into E. coli strain
DH5F`(22) . Colony hybridization was used to identify
three types of mutant plasmids, all bearing the BamHI
restriction site, plus (a) both wild type IDH1 and IDH2 genes, (b) the presequence deleted form of IDH1 with the wild type IDH2 gene, or (c)
the presequence deleted form of IDH2 with the wild type IDH1 gene. All mutations were confirmed by nucleotide sequence
analysis.
A 2.5-kilobase BglII restriction fragment
containing the IDH1 promoter region was subcloned from the
original genomic isolate (4) into the BamHI site
upstream of either the wild type or the presequence deleted form of IDH1, generating plasmids, respectively, designated
pIDH1/IDH2 and pIDH1/IDH2. The
correct orientation of the BglII promoter fragment was
confirmed by nucleotide sequence analysis. XbaI restriction
fragments from the two plasmids carrying the promoter and IDH1 coding regions were used to replace XbaI fragments in a
plasmid bearing the presequence deleted form of IDH2,
generating plasmids, respectively, designated
pIDH1/
IDH2 and
p
IDH1/
IDH2.
For oxygen consumption experiments,
mitochondrial pellets were resuspended to 10 mg/ml in a buffer
containing 0.65 M mannitol, 10 mM KPO (pH
6.5), 10 mM Tris maleate (pH 6.5), 10 mM KCl, and 0.1
mM EDTA. Rates of oxygen consumption were measured with a
Clarke-type polarographic oxygen electrode as described by Ohnishi et al.(25) . State III respiratory rates were measured
in the presence of 334 µM ADP with 13.3 mM succinate, citrate, or pyruvate (12.0 mM) plus malate
(1.3 mM). All respiratory substrates and ADP solutions were
adjusted to pH 6.5.
For
activity staining of native enzyme, whole cell protein extracts (100
µg) were electrophoresed on 10% polyacrylamide gels and the gels
stained for 20-30 min with an isocitrate dehydrogenase activity
stain containing 50 mM Tris-HCl (pH 7.6), 5 mM MgCl, 5 mM isocitrate, 0.5 mM NAD
, 0.4 mg/ml nitro blue tetrazolium, and 40
µg/ml phenazine methosulfate. Staining was terminated by immersing
the gel in 7% acetic acid.
For current studies of
localization and function, the IDH1 and IDH2 genes
were subcloned into the polylinker site of pRS316(21) , a
centromere-based plasmid containing a yeast URA3 gene for
selection. To determine requirements for mitochondrial localization of
the subunits, deletions of the presequence coding regions were
constructed by oligonucleotide-directed mutagenesis as described under
``Experimental Procedures.'' These deletions preserve the
initiator methionine codons and the coding regions for the mature
polypeptides. A complete set of four plasmids was constructed by
subcloning. These include a plasmid carrying both wild type genes
(pIDH1/IDH2), a plasmid carrying both presequence deletions
(pIDH1/
IDH2),
and plasmids lacking either the IDH1 or IDH2 presequence (p
IDH1/IDH2 and
pIDH1/
IDH2, respectively).
The
pRS plasmids were transformed into a haploid yeast strain (IDH1/
IDH2) containing a deletion/LEU2 insertion disruption of the IDH1 chromosomal locus and a
deletion/HIS3 insertion disruption of the IDH2 chromosomal locus(5) . Resulting Ura
transformants were isolated and cultivated in rich medium with
glycerol plus lactate or ethanol, carbon sources permissive for growth
of the
IDH1/
IDH2 disruption mutant. Immunoblot
analysis of whole cell protein extracts was conducted to examine
plasmid-borne expression of IDH1 and IDH2. As shown in Fig. 1,
IDH1 and IDH2 levels are comparable with each other under both growth
conditions in extracts from transformants expressing both wild type
genes (pIDH1/IDH2, lanes 1) or the IDH2 presequence deletion gene
(pIDH1/
IDH2, lanes 3). This
is also true for transformants expressing both presequence deleted
genes
(p
IDH1/
IDH2, lanes 4) but overall cellular levels of both subunits are
approximately 50% lower than wild type. In contrast, levels of IDH1 are
reduced relative to IDH2 in transformants expressing the IDH1 presequence deletion gene
(p
IDH1/IDH2, lanes 2); in
fact, long exposure times are necessary to visualize IDH1 in extracts
from the ethanol-grown transformant. No larger forms of IDH1 or IDH2
are immunochemically detectable in any of the samples, indicating no
accumulation of precursor forms of either subunit.
Figure 1:
Cellular expression of IDH1 and IDH2 in
yeast transformants. Whole cell protein extracts (25 µg) prepared
from a yeast IDH1/
IDH2 disruption strain transformed
with pIDH1/IDH2 (lanes 1),
p
IDH1/IDH2 (lanes 2),
pIDH1/
IDH2 (lanes 3), or
p
IDH1/
IDH2 (lanes 4) were used for immunoblot analysis of IDH as
described under ``Experimental Procedures.'' The
transformants were cultivated in YP medium with the indicated carbon
sources.
Enzyme assays of
the whole cell extracts (Table 1, A) show measurable IDH activity
in the pIDH1/IDH2 transformant (the levels are approximately
2-fold higher than those measured for the parental strain) and in the
pIDH1/
IDH2 transformant with both carbon sources. No activity was measurable
for the pIDH1/
IDH2 transformant
despite the presence of both subunits, suggesting that the subunits may
be localized in different cellular compartments preventing formation of
the holoenzyme in vivo. This result, along with mixing
experiments performed with extracts from
IDH1 and
IDH2 disruption mutants, also indicates that assembly of
holoenzyme does not occur in vitro under these conditions.
Traces of activity with glycerol plus lactate and with ethanol were
detected in the p
IDH1/IDH2 transformant, perhaps due to differential localization or simply
to low cellular levels of IDH1 (cf. Fig. 1, lanes
2).
In another sensitive test for formation of holoenzyme, the
whole cell extracts from glycerol/lactate-grown transformants were
electrophoresed on a nondenaturing polyacrylamide gel and the gel
treated with an IDH activity stain as described under
``Experimental Procedures'' (Fig. 2A). With
this method, a trace of IDH activity, relative to strong staining for
the extracts from pIDH1/IDH2 and
pIDH1/
IDH2 transformants (lanes 1 and 4, respectively), is
detected in the extract from the
p
IDH1/IDH2 transformant (lane
2). Again, no activity is observed for the
pIDH1/
IDH2 transformant (lane
3). Importantly, the activity stains indicate similar
electrophoretic mobilities suggesting formation of native, presumably
octomeric, holoenzyme in three types of transformants.
Figure 2:
Electrophoretic analysis of IDH in yeast
transformants. Whole cell protein extracts (100 µg) prepared from
the parental yeast strain (P) or from a yeast IDH1/
IDH2 disruption strain transformed with
pIDH1/IDH2 (lanes 1),
p
IDH1/IDH2 (lanes 2),
pIDH1/
IDH2 (lanes 3), or
p
IDH1/
IDH2 (lanes 4) were electrophoresed on non-denaturing
polyacrylamide gels and stained for enzyme activity as described under
``Experimental Procedures.'' Transformants were cultivated in
YP medium with the indicated carbon
sources.
Similar patterns of relative expression were obtained for transformants grown on glucose (data not shown), which is also a permissive carbon source; however, overall immunochemical levels and activities were 3-5-fold lower due to glucose repression of IDH subunit levels (27) .
Figure 3:
Localization of IDH1 and IDH2 in yeast
transformants cultivated with glycerol/lactate. Cytosolic (C,
100 µg) and mitochondrial fractions (M, 10 µg)
prepared from a yeast IDH1/
IDH2 disruption strain
transformed with pIDH1/IDH2 (lanes 1),
p
IDH1/IDH2 (lanes 2),
pIDH1/
IDH2 (lanes 3), or
p
IDH1/
IDH2 (lanes 4) were used for immunoblot analysis of IDH, of
mitochondrial malate dehydrogenase (MDH1), and of cytosolic malate
dehydrogenase (MDH2) as described under ``Experimental
Procedures.'' Reduced mitochondrial levels of IDH1 in lane 2,
M, relative to whole cell levels (Fig. 1, lanes 2)
are attributed to instability of the subunit during the period of
fractionation.
Cellular fractionation experiments indicate that no holoenzyme is
present in mitochondria from pIDH1/IDH2 or
p
IDH1/
IDH2 transformants. As an additional physiological test, relative
respiratory rates, measured as oxygen consumption with various
oxidative substrates, were determined with mitochondria prepared from
transformants as well as from parental and
IDH1/
IDH2 disruption strains. As shown in Table 3, and as previously
reported(27) , mitochondria from strains containing
IDH1 and/or
IDH2 gene disruptions are
characterized by an inability to use citrate as a respiratory
substrate, whereas pyruvate plus malate and succinate are adequate
substrates. Mitochondria from pIDH1/
IDH2 and
p
IDH1/
IDH2 transformants are similarly deficient, indicative of the absence
of IDH holoenzyme in these organelles. Mitochondria from the
p
IDH1/IDH2 transformant, however,
retain some capacity for oxygen consumption with citrate, again
providing evidence for residual activity in these mitochondria despite
the immunochemical absence of IDH1.
To better evaluate phenotypic
changes conferred by IDH expression, liquid culture growth rates were
measured. As shown in Table 4, all four transformant strains as
well as the disruption strain grew well with ethanol as a permissive
carbon source, with cell doubling times ranging from 5.3 to 6.4 h. With
acetate, which is non-permissive for the disruption strain, culture
doubling times for the transformants ranged from 6.0 to 12.0 h. Based
on these growth rate values, the cytosolic form of IDH does permit
growth of the
pIDH1/
IDH2 transformant on acetate but does not completely restore parental
rates of growth. Growth of the p
IDH1/IDH2 and pIDH1/
IDH2 transformants on
acetate suggested formation of active enzyme in these strains and this
was confirmed by enzyme assays (Table 1, B) and electrophoretic
staining (Fig. 2B) conducted with the whole cell
extracts.
To determine the basis for growth on acetate, cellular
fractionation was conducted to define the cellular localization of IDH
subunits and activity in acetate-grown transformants. Immunochemical
analysis (Fig. 4) demonstrates that for acetate-grown
transformants carrying pIDH1/IDH2 (lanes 1) or
pIDH1/
IDH2 (lanes 4), localization of both subunits is similar to
that previously observed with glycerol/lactate or ethanol, i.e. mitochondrial in the former and cytosolic in the latter. The
relative specific activities for cellular fractions from these
transformants (Table 2, B) demonstrate compartmental distribution
of activity compatible with co-localization of the subunits. In the
acetate-grown p
IDH1/IDH2 transformant, IDH2 is primarily mitochondrial (lanes 2)
as previously observed; however, significant levels of IDH1 are now
detectable in the mitochondrial fraction. A significant enrichment in
mitochondrial specific activity is also apparent (Table 2, B),
suggesting that formation of a mitochondrial holoenzyme accounts for
growth of this transformant on acetate. In the acetate-grown
pIDH1/
IDH2 transformant, the
presequence-deleted form of IDH2 is primarily cytosolic (lanes
3), as previously observed with glycerol/lactate; however, IDH1
levels in the mitochondrial and cytosolic fractions now appear to be
equivalent. Co-localization of IDH1 and IDH2 in the cytosol and the
enrichment in IDH specific activity in that compartment (Table 2)
suggest that it is formation of a holoenzyme in the cytosol which
supports growth of this transformant on acetate. Mitochondria isolated
from acetate-grown transformants containing
pIDH1/
IDH2 or
p
IDH1/
IDH2 were also found to be unable to utilize citrate as a respiratory
substrate (values in parentheses in Table 3), supportive of
primary cytosolic localization of the holoenzyme in these strains.
Figure 4:
Localization of IDH1 and IDH2 in yeast
transformants cultivated with acetate. Cytosolic (C, 50
µg) and mitochondrial fractions (M, 50 µg) prepared
from a yeast IDH1/
IDH2 disruption strain transformed
with pIDH1/IDH2 (lanes 1),
p
IDH1/IDH2 (lanes 2),
pIDH1/
IDH2 (lanes 3), or
p
IDH1/
IDH2 (lanes 4) were used for immunoblot analysis of IDH, MDH1,
and MDH2 as described under ``Experimental
Procedures.''
In this study we have examined the structural requirements
for localization and assembly of the subunits of a multimeric
mitochondrial enzyme. The factors which determine localization of the
IDH1 and IDH2 subunits of yeast mitochondrial NAD-dependent isocitrate
dehydrogenase appear to be quite different. For IDH2, irrespective of
growth conditions, the presence or absence of its mitochondrial
targeting presequence appears to be the single determinant for
respective mitochondrial or cytosolic localization. The subunit is
stably expressed to similar cellular levels in either compartment. For
IDH1, localization and stability are affected by the presence or
absence of its presequence; however, other important factors appear to
be growth conditions and the compartmental location of IDH2. In
acetate-grown cells, for example, IDH1 is mitochondrial even in the
absence of its presequence (the
pIDH1/IDH2 transformant) whereas
cytosolic levels of IDH1 are significant even in the presence of its
presequence when IDH2 is cytosolic (the
pIDH1/
IDH2 transformant). These
observations suggest that holoenzyme assembly and selective pressure
for NAD-isocitrate dehydrogenase activity may override IDH1
localization signals.
In terms of mechanisms for aberrant IDH1
localization in acetate-grown cells, it is important to note that in
the pIDH1/IDH2 transformant, the
sizes of cytosolic and mitochondrial forms of IDH1 are similar (cf. Fig. 4, lanes 3). Since the precursor and
mature forms of IDH1 are easily distinguishable electrophoretically, (
)it appears that the cytosolic form has been processed. One
possible explanation is suggested by a recent report describing
partitioning of yeast fumarase into two cellular
compartments(28) ; the cytosolic form results from partial
insertion of the precursor form into the mitochondrion, normal removal
of the amino-terminal presequence, and release from the membrane as a
folded cytosolic polypeptide. If this is also the case for IDH1 in the
pIDH1/
IDH2 transformant, cytosolic
retention may be facilitated by the presence of and interaction with
IDH2 in that compartment. Mitochondrial accumulation of IDH1 in the
p
IDH1/IDH2 transformant with acetate
growth would suggest that either the presequence of IDH1 is not
essential for localization or that some interaction with the precursor
form of IDH2 may promote import. That these aberrant patterns of
localization of IDH1 are observed only with growth on acetate may be
due to some shift in equilibrium to favor formation of the holoenzyme
which in turn seems to stabilize IDH1. In support of this notion, we
have found that the presence of cytosolic IDH2 is essential for
detection of cytosolic IDH1 (data not shown).
The reason for
differences in stability of cytosolic forms of IDH1 and IDH2 subunits
is not clear. The mature polypeptides are similar in sequence and have
identical amino-terminal residues in positions one and two, so the same
N-end rules for cytosolic turnover (29) should apply to both.
Our results suggest that IDH2 may be the nucleus for formation of
holoenzyme. If so, it might be postulated that this subunit can form a
homo-oligomeric species which might afford greater stability than a
monomeric form. However, a previous study indicated that IDH2 isolated
from a IDH1 disruption mutant behaves as a monomer in gel
filtration studies(5) . Also, with the yeast two-hybrid system,
we find no evidence for IDH2/IDH2 or for IDH1/IDH1 interactions under
conditions where IDH1/IDH2 interactions are very strong. (
)
Deletion of both IDH1 and IDH2 presequences results in
cytosolic localization of both subunits and formation of active
holoenzyme. Thus, the mitochondrial environment and/or specific
mitochondrial factors are not essential for correct polypeptide folding
and subunit assembly. While we cannot rule out some accumulation of
mitochondrial enzyme, immunoblot analysis and oxygen consumption assays
suggest such levels would be very low. Restoration of the ability of a IDH1/
IDH2 disruption mutant to grow on acetate
suggests that the ``cytosolic'' form of IDH can supplement
metabolic requirements for tricarboxylic acid cycle function. Similar
results were recently found for a genetically engineered cytosolic form
of MDH1 which also complements the acetate
phenotype
of a
MDH1 disruption strain. (
)An energetic
requirement in both cases is NADH. In yeast, reducing equivalents from
cytosolic NADH may be supplied to the respiratory chain via an
externally directed mitochondrial NADH dehydrogenase(25) ,
perhaps explaining compensatory function by the cytosolic enzymes. In
any event, stoichiometric amounts of mitochondrial IDH are clearly not
essential for tricarboxylic cycle function. Along these lines, a very
interesting recent report by Elzinga et al.(30) presents evidence that yeast NAD-IDH can specifically
bind to sites within the 5`-untranslated leaders of several
mitochondrial mRNAs. Among possible functions served by this binding
are regulation of mRNA stability and/or control of translation. Our
results suggest that either this binding requires very low levels of
IDH or that loss of the function served by mRNA binding does not
dramatically reduce cellular growth with acetate.