(Received for publication, May 10, 1995; and in revised form, July 5, 1995)
From the
The malate dehydrogenase isozyme MDH3 of Saccharomyces cerevisiae was found to be localized to peroxisomes by cellular fractionation and density gradient centrifugation. However, unlike other yeast peroxisomal enzymes that function in the glyoxylate pathway, MDH3 was found to be refractory to catabolite inactivation, i.e. to rapid inactivation and degradation following glucose addition. To examine the structural requirements for organellar localization, the Ser-Lys-Leu carboxyl-terminal tripeptide, a common motif for localization of peroxisomal proteins, was removed by mutagenesis of the MDH3 gene. This resulted in cytosolic localization of MDH3 in yeast transformants. To examine structural requirements for catabolite inactivation, a 12-residue amino-terminal extension from the yeast cytosolic MDH2 isozyme was added to the amino termini of the peroxisomal and mislocalized ``cytosolic'' forms of MDH3. This extension was previously shown to be essential for catabolite inactivation of MDH2 but failed to confer this property to MDH3. The mislocalized cytosolic forms of MDH3 were found to be catalytically active and competent for metabolic functions normally provided by MDH2.
Differentially compartmentalized isozymes of malate
dehydrogenase in eucaryotic cells catalyze the NAD(H)-dependent
interconversion of oxaloacetate and malate. In mammalian cells this
reaction, catalyzed by mitochondrial and cytosolic isozymes,
respectively, is a critical step in the tricarboxylic acid cycle and in
gluconeogenesis. The two isozymes also participate in the
malate/aspartate shuttle cycle, a mechanism for exchange of reducing
equivalents between cellular compartments. In yeast and plant cells, a
third isozyme localized in peroxisomes catalyzes a step in the
glyoxylate pathway. This pathway allows formation of C metabolites from C
precursors. The malate
dehydrogenase isozyme family is therefore an ideal focus for analysis
of structural features responsible for differential compartmentation
and metabolic function.
To initiate molecular genetic studies, the three isozymes of malate dehydrogenase have been purified from Saccharomyces cerevisiae, and the corresponding genes have been cloned, sequenced, and disrupted(1, 2, 3) . The isozymes are all homodimers, and they exhibit similar kinetic properties(3) . The aligned amino acid sequences have residue identities ranging from 43 to 50%. Among conserved residues are those with catalytic functions or those that participate in cofactor binding(4, 5) . Salient differences include regions with putative functions in organellar targeting. The yeast mitochondrial isozyme (MDH1, subunit molecular weight = 33,500), for example, has a 17-residue amino-terminal extension not present on the other isozymes; this extension is removed upon mitochondrial import (6) . The peroxisomal isozyme (MDH3, subunit molecular weight = 37,200) has a unique carboxyl-terminal tripeptide sequence, Ser-Lys-Leu. Similar SKL termini on other peroxisomal proteins have been found to be necessary and sufficient for organellar localization(7, 8) . The importance of this carboxyl terminus for localization of MDH3 is a focus of this work.
Cellular levels of all three malate dehydrogenase isozymes are low in yeast cells cultivated with glucose as a carbon source(2, 3, 9) . For MDH1, this appears to be the result of glucose repression of gene expression as is the case for many mitochondrial proteins. Levels of MDH2, the cytosolic isozyme (subunit molecular weight = 40,700), exhibit more dramatic changes. This enzyme, along with others with functions in gluconeogenesis or in the glyoxylate pathway in yeast (10, 11, 12) , is subject to rapid glucose-induced inactivation and degradation(13) , a phenomenon termed catabolite inactivation. MDH2 has a 12-residue amino-terminal extension not present on aligned sequences of mature MDH1 or of MDH3 polypeptides, and we have shown that removal of this amino-terminal extension makes MDH2 refractory to catabolite inactivation(13) . In this report, we investigate changes in MDH3 expression with glucose addition and test the sufficiency of the MDH2 amino-terminal extension for catabolite inactivation.
Disruption of the chromosomal MDH loci in yeast and
analysis of resulting growth phenotypes have elucidated metabolic
functions of each isozyme. Disruption of the MDH1 gene in a
haploid strain produces an inability to grow with acetate as a carbon
source (9) . A similar growth phenotype is obtained with
disruption of genes encoding several other tricarboxylic acid cycle
enzymes including citrate synthase (14) and NAD-dependent
isocitrate dehydrogenase (15) and is attributed to an energy
deficiency with this carbon source. Disruption of the MDH2 gene produces an inability to grow with acetate or ethanol on
minimal medium(2) . Since such a mutant grows with C carbon sources on rich medium, this phenotype indicates an
auxotrophic requirement, perhaps for C
metabolites. Singh et al.(16) have reported that disruption of MDH3 eliminates growth with oleate as a carbon source, perhaps
indicative of a deleterious effect on peroxisomal
-oxidation.
These different phenotypes imply distinct and critical metabolic
functions for each isozyme.
Because of the unfavorable equilibrium
for formation of malate from oxaloacetate (G°` ≅ +7
kcal/mol), it has been proposed that malate dehydrogenase may
physically associate with other specific enzymes within a metabolic
pathway to ensure direct transfer of oxaloacetate(17) . One of
our goals in manipulating the yeast MDH isozymes is to assess the
ability of isozymes, which catalyze the same reaction but which have
structural dissimilarities, to function in alternative metabolic
pathways and cellular compartments. In this report, we examine the
ability of MDH3, normally found in peroxisomes, to function in the
cytosol.
Yeast strains used in this study were the parental haploid strain S173-6B (MATa leu2-3, 112 his3-1 ura3-52 trp1-289; (18) ) and a collection of mutants derived from that strain containing disruption mutations in MDH loci. The collection was obtained by a genetic cross of a haploid strain containing deletion/HIS3 insertion mutations in MDH1 and MDH2 chromosomal genes (1, 2) with a haploid strain of the opposite mating type containing a deletion/LEU2 insertion mutation in the MDH3 gene(3) . Haploid strains obtained following tetrad dissection were analyzed using protein immunoblots and Southern blots as described previously (1, 2, 3) to define segregation patterns for the disrupted loci. A complete collection of MDH strains was obtained; this includes the wild type, strains containing each single gene disruption, strains containing the three possible combinations of double disruptions, and a strain containing the triple gene disruption. The method of Ito et al. (19) was used for yeast transformations.
The yeast MDH3 gene on a 3.5-kilobase pair HindIII restriction fragment
was previously subcloned into a Bluescribe plasmid (designated
pBSMDH3; (3) ). This plasmid was used to prepare
single-stranded DNA for oligonucleotide-directed mutagenesis. A 66-mer
oligonucleotide
(5`-GAATTGCGACTTTGACCGAATCTTGTTCTATGGATGGTGTAACTGAGTGAGGCATGTTTATGATTG-3`)
with ends complementary to the 5`-coding region of MDH3 was
used for ``loop-in'' mutagenesis to insert 12 codons from the
amino-terminal coding region of MDH2 between the AUG
translational initiation codon and the codon for the amino terminus of MDH3. The resulting plasmid was designated pBSMDH2/3.
The Bluescribe plasmids, pBSMDH3 and pBSMDH2/3, were
used for subsequent mutagenesis to delete three codons (Ser-Lys-Leu)
from the carboxyl-terminal end of the MDH3 coding region. This
was accomplished using a 20-mer oligonucleotide
(5`-AATTGAAGTAGCTCAAGAGTCTAGGATGAA-3`) for ``loop-out''
mutagenesis. The resulting plasmids were designated pBSMDH3SKL and pBSMDH2/3
SKL.
Oligonucleotide-directed
mutagenesis was conducted as described by Zoller and
Smith(21) . Bacterial transformants containing the altered
sequences were identified by differential colony hybridization using as
probes appropriate oligonucleotides, which were 5`-end-labeled with P using polynucleotide kinase. Single-stranded DNA was
prepared from bacterial colonies, and mutations were confirmed by
nucleotide sequence analysis. The dideoxy method (22) was used
for sequence analysis with oligonucleotide primers previously used to
sequence the MDH3 gene(3) . Following confirmation of
nucleotide sequences, the MDH3 gene constructs on 3.5-kilobase
pair HindIII fragments were cloned into pRS316, a
yeast/bacterial shuttle plasmid containing a centromere cassette for
single-copy expression in yeast and a URA3 gene for
selection(23) . The resulting plasmids were designated
pRSMDH3, pRSMDH3
SKL, pRSMDH2/3, and
pRSMDH2/3
SKL.
Spectrophotometic enzyme assays for malate dehydrogenase (9) and catalase (27) were conducted as described previously. Protein concentrations were determined by the Bradford method (28) using bovine serum albumin as the standard. Immunoblot analyses were performed using polyclonal antisera against MDH1(9) , MDH2(2) , MDH3(3) , and mitochondrial NADP-dependent isocitrate dehydrogenase (26) as described previously. The MDH3 antiserum was found to cross-react with MDH1 and was used in some immunoblot analyses to identify both isozymes in the same extract.
Figure 1: Peroxisomal localization of MDH3. Cellular fractionation was conducted as described under ``Experimental Procedures'' using the parental yeast strain S173-6B cultivated on rich YP medium with ethanol plus oleate as carbon sources. A, protein extracts from the whole cell homogenate (100 µg; lanesH) and, following centrifugation, from the organellar pellet (25 µg; lanesP) and soluble supernatant (100 µg; lanesS) were electrophoresed and used for immunoblot analysis with antisera specific for MDH1, MDH2, or MDH3. The organellar pellet was further subjected to Nycodenz gradient centrifugation, and resulting fractions were used for immunoblot analysis (100 µl/lane; panelB) with antisera specific for MDH3, MDH1, or mitochondrial NADP-specific isocitrate dehydrogenase (IDP1; (26) ), and for enzyme assays (C) for malate dehydrogenase and catalase. Activities are expressed relative to peaks detected within the gradient. The large increase in malate dehydrogenase activity in fractions at the top of the gradient is due to the abundance of mitochondrial MDH1.
Most yeast peroxisomal enzymes that function in the glyoxylate pathway, including malate synthase (11) and isocitrate lyase(12) , are subject to rapid glucose-induced inactivation and degradation. We therefore investigated this phenomenon for MDH3. The parental strain was cultivated with acetate as a carbon source and then shifted to glucose medium as described under ``Experimental Procedures.'' As shown in Fig. 2and as previously reported(13) , levels of MDH2 in whole cell extracts are rapidly depleted after glucose addition and are immunochemically undetectable after 60 min, whereas MDH1 levels are gradually diminished due to glucose repression of expression. Changes in levels of MDH3 in the same extracts are seen to more closely resemble those for MDH1 (also cf. Fig. 6A) and not for MDH2, suggesting that MDH3 is subject to glucose repression but is not subject to the phenomenon of rapid glucose-induced degradation. With steady state growth conditions, we have also found that immunochemical levels of MDH1 and MDH3 are detectable in glucose grown cells at levels 5-10-fold below acetate growth levels(3, 6) , whereas MDH2 is not immunochemically detectable in extracts from glucose-grown cells.
Figure 2: Glucose-induced changes in levels of the MDH isozymes. Immunoblot analysis was conducted using whole cell extracts (100 µg) obtained from the parental yeast strain cultivated on rich YP medium with 2% acetate (lanes0) and at the time indicated (in minutes) following a shift to YP medium with 5% glucose. The antisera were specific for MDH2 (toppanel) or for MDH3 and MDH1 (bottompanel) as described under ``Experimental Procedures.''
Figure 6:
Glucose-induced changes in levels of
mutant forms of MDH3. Immunoblot analysis using antiserum for MDH3 and
MDH1 was conducted using whole cell extracts (100 µg) obtained from
a yeast strain containing a chromosomal MDH3 disruption and
transformed with pRSMDH3 (A), pRSMDH3SKL (B), or pRSMDH2/3
SKL (D). Extracts
were obtained from cells cultivated on YP medium with 2% acetate (lanes0) and at times indicated (in minutes)
following a shift to YP medium with 5% glucose. Panel designations
correspond with sequence designations in Fig. 4.
Figure 4:
Expression of mutant forms of MDH3.
Immunoblot analysis using antiserum against MDH3 and MDH1 was conducted
with whole cell extracts (100 µg) obtained from a yeast strain
containing disruptions of MDH1, MDH2, and MDH3 chromosomal loci and transformed with centromeric plasmids
pRSMDH3 (A), pRSMDH3SKL (B),
pRSMDH2/3 (C), or pRSMDH2/3
SKL (D) and from a yeast strain containing chromosomal
disruptions of MDH2 and MDH3 and transformed with
pRSMDH3 (A`) or pRSMDH3
SKL (B`). Amino- and carboxyl-terminal sequence differences
among the forms of MDH3 are illustrated at the bottom of the
figure, with letters corresponding to lane designations.
Figure 3: Aligned terminal sequences from yeast MDH isozymes. Amino acid sequences from amino and carboxyl-terminal regions of MDH1(1) , MDH2(2) , and MDH3 (3) are aligned to show identical (lines) and similar (dots) residues. The mitochondrial targeting presequence of MDH1 is shown in brackets. Total residue numbers for the mature polypeptides are indicated in parentheses.
To assess the importance of these structural features for
localization and turnover, the cloned MDH3 gene was used for
oligonucleotide-directed mutagenesis as detailed under
``Experimental Procedures'' to ``loop in'' codons
for the MDH2 amino-terminal extension and to ``loop-out''
codons for the carboxyl-terminal SKL tripeptide. Four variants of the MDH3 gene were constructed and cloned into
pRS316(23) , a centromere-based shuttle plasmid carrying the
yeast URA3 gene for selection. As shown in Fig. 4,
these variants encoded authentic MDH3 (MDH3) (A),
MDH3 lacking the carboxyl-terminal SKL (MDH3SKL) (B), MDH3 with the MDH2 amino-terminal extension (MDH2/3) (C), and MDH3 with the MDH2 extension and
lacking the carboxyl-terminal SKL (MDH2/3
SKL) (D).
For different experiments described in following sections, the plasmids carrying wild type and mutant forms of the MDH3 gene were transformed into haploid yeast strains containing various disruptions in the three MDH genomic loci. Construction of a complete collection of yeast MDH mutants by a genetic cross is described under ``Experimental Procedures.''
For initial tests of expression, a strain
containing genomic disruptions in all three MDH loci was used
for transformation with the pRS plasmids. Ura transformants were isolated and grown on rich medium with ethanol
as the carbon source; this carbon source has previously been shown to
be permissive for growth of various yeast MDH
mutants(1, 2, 3) . MDH3 levels in whole cell
extracts from representative transformants were examined by
immunoblotting. As shown in Fig. 4, MDH3 polypeptides are all
well expressed from the four plasmid-borne genes (lanesA-D), although levels of the wild type protein are
approximately 2-fold higher than mutant enzyme levels. Total cellular
MDH activities are also similar, with values ranging from 0.5 to 1.0
units/mg cellular protein as determined in three independent
experiments. These levels are similar to those obtained from a yeast
strain with an intact MDH3 locus and containing disruptions in MDH1 and MDH2 genes(3) , suggesting that the
plasmid-encoded wild-type and mutant forms of MDH3 are stably expressed
and fully active. The size differences among the MDH3 polypeptides are
also apparent. As shown in Fig. 4, polypeptides containing the
12-residue MDH2 amino-terminal extension (lanesC and D) migrate more slowly during SDS-polyacrylamide gel
electrophoresis than those with authentic MDH3 amino termini (lanesA and B). The polypeptides containing
carboxyl-terminal SKL deletions (lanesB and D) also migrate slightly more rapidly than their counterparts
retaining the MDH3 carboxyl terminus (lanesA and C). The latter difference is more apparent in extracts from
yeast transformants containing an MDH3 gene disruption but
with an intact MDH1 locus (lanesA` and B`).
Figure 5:
Cellular localization of mutant forms of
MDH3. Immunoblot analysis was conducted using fractions (100 µl)
collected following Nycodenz gradient centrifugation of organellar
pellets obtained from a yeast strain containing chromosomal disruptions
of MDH1, MDH2, and MDH3 genes and
transformed with pRSMDH3 (A), pRSMDH3SKL (B), pRSMDH2/3 (C), or
pRSMDH2/3
SKL (D). Panel designations correspond
with lane and sequence designations in Fig. 4. Each panel shows independent immunoblot analyses using antisera specific for
MDH3 and for IDP1. The trace of MDH3 in fraction18 of panelB is attributed to variable cytosolic
contamination of the mitochondrial fraction due to omission of an
organellar washing step as described under ``Experimental
Procedures.''
Figure 7:
Growth phenotypes of MDH3 mutant strains.
Yeast strains containing a chromosomal disruption of the MDH2 gene and transformed with pRSMDH3 (A),
pRSMDH3SKL (B), pRSMDH2/3 (C),
or pRSMDH2/3
SKL (D) were streaked onto plates
containing rich YP medium or minimal YNB medium with 2% ethanol as
carbon source and grown, respectively, for 2 or 4 days at 30
°C.
Two types of peroxisomal targeting signals, termed PTS1 and PTS2, have been identified and are described in recent reviews of organellar import and biogenesis(7, 8) . The SKL carboxyl-terminal tripeptide of the MDH3 isozyme of malate dehydrogenase in S. cerevisiae is a canonical type 1 PTS. In this report, we demonstrate that MDH3 is localized to microbody organelles isolated by density gradient centrifugation and that removal of the SKL tripeptide results in mislocalization of the enzyme to the soluble cytosolic fraction of cellular lysates. Interestingly, the plant homologue of yeast MDH3 contains a type 2 PTS, in this case an amino-terminal extension of 37 residues, which is proteolytically removed upon organellar import(31) . The plant malate dehydrogenase extension has also been demonstrated to be essential for import(32) . Distinct import mechanisms for proteins containing the two types of PTS motifs have been implicated by analyses of mutants with defects in peroxisome assembly(7, 8) . Thus, although the yeast and plant enzymes are structurally similar (43% residue identity; (3) ) and presumably functionally equivalent, it is likely that relatively recent evolutionary events involved acquisition of different import motifs.
The metabolic function of
yeast and plant peroxisomal malate dehydrogenases is believed to be
catalysis of a reaction in the glyoxylate cycle. This cycle is
important during plant seedling germination for conversion of storage
fatty acids to gluconeogenic precursors and in yeast allows growth with
fatty acids or C carbon sources such as acetate and
ethanol. The report by Singh et al.(16) that
disruption of the S. cerevisiae MDH3 gene results in an
inability to grow with oleate as a carbon source supports a role in
glyoxylate metabolism. However, we have been unable to duplicate this
observation with our disruption mutants, suggesting there may be some
strain-specific differences in this pathway.
The cytosolic form of
MDH3 obtained by removal of the carboxyl-terminal SKL appears to retain
full catalytic activity. This is not surprising given sequence homology
with mitochondrial MDH1 and cytosolic MDH2, which lack this carboxyl
terminus. Also, recent reports(33, 34) suggest that
proteins targeted to the peroxisomal matrix may attain a folded state
and form oligomeric complexes prior to peroxisomal import. Thus,
cytosolic formation of an active MDH3 dimer may be one step in the
normal import pathway. Because the cytosolic form of MDH3 is
catalytically active and stably expressed, we have been able to test
the competence of this isozyme for replacement of MDH2 in yeast strains
containing an MDH2 gene disruption. Cytosolic MDH3 was found
to restore the ability of these strains to grow on C carbon
sources. Thus, despite some significant differences in physical
properties of the two isozymes, e.g. respective pI values of
8.6 and 4.4 for MDH3 and MDH2(3) , function in gluconeogenesis
is apparently not dependent on unique structural attributes of MDH2.
However, cellular growth rate studies are a relatively gross measure of
function, and conclusions about equivalence of metabolic contributions
will require finer methods for measuring function in vivo as
well as reciprocal compartmental exchanges of MDH isozymes. In related
previous experiments, we attempted to obtain cytosolic localization of
mitochondrial MDH1 by removal of the 17-residue amino-terminal
targeting presequence. However, we found that this did not prevent
mitochondrial import in vivo(6) . We are currently
altering possible cryptic internal targeting signals with the intent of
testing metabolic functions of cytosolic MDH1 for comparison with our
results for MDH3.
We have found that, unlike gluconeogenic enzymes including MDH2 (13) and other glyoxylate pathway enzymes including malate synthase (11) and isocitrate lyase(12) , MDH3 is not subject to rapid glucose-induced degradation. Cytosolic localization of MDH3 following removal of the SKL tripeptide does not increase susceptibility to the phenomenon. This result was not unexpected since we previously found that removal of the amino-terminal 12-residue extension of MDH2 produces an active enzyme refractory to glucose-induced inactivation and degradation. Thus, catalytic activity and cytosolic localization are insufficient attributes to target malate dehydrogenase for degradation. Also, addition of the 12-residue amino-terminal extension of MDH2 to peroxisomal or cytosolic forms of MDH3 has no effect on turnover, suggesting that, while this extension is essential for rapid turnover of MDH2(13) , there are other unique structural features of MDH2 that are recognized by components of the inactivation/turnover pathway. A final conclusion from current and previous results is that the amino-terminal extension of MDH2 is not critical for cellular location since its removal has no effect on cytosolic localization of MDH2 and its addition does not hinder peroxisomal localization of authentic MDH3. In future studies, we plan to extend these studies to correlate unique structural features of the malate dehydrogenase isozymes with differential expression and compartmental function.