(Received for publication, December 8, 1995; and in revised form, February 21, 1996)
From the
Peroxisomes have a central function in lipid metabolism, and it
is well established that these organelles are inducible by many
compounds including fatty acids. Peroxisomes are the sole site for the
-oxidation of fatty acids in yeast. The first and rate-limiting
enzyme of this cycle is fatty acyl-CoA oxidase. The gene encoding this
enzyme in Saccharomyces cerevisiae (POX1) undergoes a
complex regulation that is dependent on the growth environment. When
this yeast is grown in medium containing oleic acid as the main carbon
source, peroxisomes are induced and POX1 expression is
activated. When cells are grown in the presence of glucose, the
expression of POX1 mRNA is repressed, whereas growth on a
carbon source such as glycerol or raffinose causes derepression. This
rigorous regulation is brought about by the complex interactions
between trans-acting factors and cis-elements in the POX1 promoter. Previously, we characterized regulatory
elements in the promoter region of POX1 that are involved in
the repression and activation of this gene (Wang, T., Luo, Y., and
Small, G. M.(1994) J. Biol. Chem. 269, 24480-24485). In
this study we have purified and identified an oleate-activated
transcription factor (Oaf1p) that binds to the activating sequence
(UAS1) in the POX1 gene. The protein has a predicted molecular
mass of approximately 118 kDa.
Peroxisomal -oxidation is an important pathway in mammalian
metabolism for catabolizing long and very long chain fatty acids. In
many organisms, including yeasts, peroxisomal
-oxidation is the
sole mechanism for the breakdown of fatty acids(1) . The
enzymes involved in this pathway are regulated according to the growth
environment. Expression of genes encoding peroxisomal proteins in the
yeast Saccharomyces cerevisiae is repressed when the yeast
cells are grown in the presence of glucose, derepressed during growth
on a nonfermentable carbon source, and activated when a fatty acid such
as oleate is supplied for growth(2) . This control is achieved
through stringent transcriptional regulation of the genes encoding
these
proteins(3, 4, 5, 6, 7, 8) .
Over the past several years we have focused our attentions toward
understanding the mechanisms that regulate genes encoding peroxisomal
-oxidation enzymes in S. cerevisiae. In order to address
this question, we have concentrated on the regulation of POX1,
the gene encoding acyl-CoA oxidase, the rate-limiting enzyme of this
cycle. Previously we characterized two upstream repression sequences
(URS1 and URS2) (
)and one upstream activating sequence
(UAS1) in the promoter region of POX1(7, 9) .
We demonstrated that a protein or protein complex binds to UAS1 in an
oleate-dependent fashion, and this brings about the activation of POX1. A similar UAS sequence (termed oleate response element)
was identified in the upstream regions of genes encoding some of the
other peroxisomal proteins(3, 4, 10) .
Several factors have been shown to be involved in the glucose
repression of thiolase, the last enzyme in the peroxisomal
-oxidation cycle, which in S. cerevisiae is encoded by
the FOX3 gene. This gene also undergoes regulation when the
yeast is grown in glucose or oleate medium. The gene products of ADR1, SNF1, and SNF4 are all known
regulators of glucose-repressible genes and have been shown to be
positive regulators of FOX3 expression(5) . However,
mutations in the ADR1 or SNF1 genes appear to have
little or no effect on the expression of POX1(11) ,
suggesting that different and/or additional factors are involved in the
regulation of this gene.
Having identified some of the DNA elements that serve as binding sites for specific transcription factors involved in POX1 regulation, we have now turned our attentions to characterizing the biochemical properties of these trans-acting proteins. In order to achieve this goal it is necessary to purify and characterize these proteins. Here we describe the purification and molecular identification of a UAS1-binding oleate-activated transcription factor (Oaf1p). We demonstrate that the amount of Oaf1p that binds to UAS1 progressively increases during growth on oleate. Thus, oleic acid is required, either for the induction of Oaf1p itself or for its activation. We have utilized photo-affinity cross-linking to confirm the molecular weight of this protein. Furthermore, we have identified the gene (OAF1) encoding this transcription factor and have demonstrated that POX1 is not induced by oleate in a yeast strain that carries an OAF1 disruption.
The cell extract was
then passed through a cation exchange SP-Sepharose column (Pharmacia
Biotech Inc.). Proteins were eluted using a linear KCl gradient
(0.2-0.7 M prepared in buffer A). Fractions containing
the highest amounts of Oaf1p (as judged by specific binding activity in
a DNA band shift assay) were pooled and concentrated using a
centriprep-10 centricon (Amicon). The concentrate was adjusted to 0.1 M KCl and then loaded onto a calf thymus double-stranded DNA
cellulose column. Elution of proteins bound to the column was achieved
using a KCl gradient, as above, and fractions with highest levels of
Oaf1p activity were again pooled and concentrated. The concentrated
fractions were loaded onto a 2-ml UAS1-oligonucleotide affinity column
(100 µg DNA/ml Sepharose) pre-equilibrated with buffer B (25 mM Hepes-NaOH, pH 7.9, 0.1 mM EDTA, 12.5 mM MgCl, 10% glycerol, 1 mM dithiothreitol, 0.1%
Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride) containing
0.25 M KCl. The column was then washed with 30 ml of
equilibrating buffer. Bound proteins were eluted with a linear KCl
gradient (0.25-1.0 M in buffer A). Eluted fractions were
analyzed in a DNA band shift assay with a labeled DNA fragment
containing UAS1. Aliquots of the fractions were also run on an
SDS-polyacrylamide gel and silver-stained by the method
described(16) .
Figure 1:
DNA band shift assays
carried out with extracts from cells grown in oleate medium for
0-18 h. a, the band shift assay was carried out with 30
µg of cell extract and a 184-nucleotide P-labeled DNA
fragment that contains URS2 and UAS1. b, the assays were
carried out as for a except that the DNA used was an 80-mer
containing UAS1. C1 refers to the complex formed by protein
binding to URS2, and C2 refers to the complex formed when
Oaf1p binds to UAS1. The intensity of the shifted bands was quantified
by PhosphorImager analysis.
Figure 2:
Temporal expression of POX1 in
cells switched from glucose to oleate medium. a, Northern blot
analysis of POX1 and PGK1 mRNA isolated from cells
grown for 8 h in YPD and then transferred to YPGO and grown for the
number of hours indicated. b, the mRNA levels shown in a were quantified by PhosphorImager analysis, and the relative POX1 mRNA abundance was normalized to the level of PGK1 mRNA. POX1 promoter-dependent -galactosidase
activities were also measured in extracts prepared from the same cells
used in a.
Figure 3: Elution profile of Oaf1p obtained by thymus DNA cellulose chromatography. a, protein and DNA binding activity profile. The protein concentration of alternate fractions eluted from a thymus-DNA column were determined by measuring the absorbance at 280 nm. Oaf1p activity was measured by performing a DNA band shift assay with 1 µl of alternate fractions and quantitating the radioactivity in the shifted band using a PhosphorImager. b, elution profile of the double-stranded thymus DNA cellulose column as detected by a DNA band shift assay carried out with 1 µl out of a total of 40 ml of column loading sample (lane 1), 1 µl of flow-through (lane 2), and 1 µl of alternate fractions (1.3 ml each) eluted with a linear KCl gradient (0.2-0.7 M) and 10 fmol of labeled annealed UAS oligonucleotides (lanes 3-14).
Fractions 6-10 from the DNA cellulose column were concentrated and loaded onto a UAS1 affinity column. The bulk of the nonspecific proteins did not bind to this column, and Oaf1p DNA binding activity was eluted at a KCl concentration between 0.4-0.6 M in fractions 4-10 (Fig. 4a, lanes 6-9). SDS-polyacrylamide gel electrophoresis of the fractions eluted from the affinity column revealed by silver staining protein bands that appear to consist of a doublet, with an apparent molecular mass of approximately 110-120 kDa (Fig. 4b, lanes 7-9). We were unable to detect Oaf1p in fraction 4 by silver staining, even though we found DNA binding activity in this fraction (compare Fig. 4, a and b). The reason for this is not clear; however, much less protein sample was used in the DNA band shift assay than was loaded onto the SDS gel (1 µl compared with 20 µl). Thus, it is possible that the discrepancy is due to experimental error if a volume slightly greater than 1 µl was inadvertently added to the band shift assay. Nevertheless, the strongest silver-stained band was recovered in fraction 8, which was also the fraction with the greatest DNA binding activity. The protein concentration of these fractions was too low to measure by standard techniques, thus we are unable to give accurate and specific recoveries for this step of the purification. However, by comparing the intensity of the bands on the silver-stained gel with that of a known concentration of protein from the molecular weight standards and from the band shift assays, we estimate that approximately 7% of the Oaf1p from the original lysate was recovered following affinity chromatography. This gave approximately an 18,000-fold purification (Table 2).
Figure 4: Elution profile of Oaf1p from a UAS affinity column. a, DNA band shift assays were carried out with 10 fmol of labeled annealed UAS-oligonucleotides and 1 µl of each of the following: starting sample (concentrated samples eluted from the thymus DNA column) (lane 1), flow-through (lane 2), 0.25 M KCl wash (lanes 3 and 4),and alternate fractions eluted from the UAS affinity column by a linear KCl gradient (0.25-1.0 M KCl) (lanes 5-12). b, SDS-polyacrylamide gel electrophoresis of the protein components in fractions eluted from the affinity column. 20 µl of the starting sample (lane 1), flow-through (lane 2), 0.25 M KCl wash (lanes 3 and 4), and alternate eluted fractions were loaded on the gel, electrophoresed, and visualized by silver staining. Sizes were approximated by using ``perfect protein markers'' (Novagen).
Figure 5:
Verification of the specificity of Oaf1p
binding. a, DNA gel shift assays carried out with fractions
eluted from an SP column in the presence of P-labeled
BrdUrd-UAS1. The assay was carried out in the absence (lane 1)
or the presence (lanes 2-4) of competitor DNA. The
following DNA was used as a competitor (100-fold excess of unlabeled
DNA) for the binding of Oaf1p to UAS1: an 80-mer that contains the
whole UAS1 (lane 2), annealed oligonucleotides containing the
upstream palindrome of UAS1 (lane 3), and annealed
oligonucleotides containing a mutated version of the upstream
palindrome of UAS1 (lane 4). b, the gel shown in a and a second gel in which a DNA band shift assay was carried
out using purified Oaf1p from a UAS1 affinity column were exposed to
film, and the protein-DNA complexes were excised and electroeluted into
an 8% polyacrylamide gel. Autoradiographs of the resulting
SDS-polyacrylamide gels containing the cross-linked Oaf1p-UAS1
complexes are shown. Lanes 1-4 show the result of UV
cross-linking the corresponding material from the gel described in a. Lanes 5-7 show the result of UV
cross-linking with UAS1 affinity purified Oaf1p in the absence of
competitor DNA (lane 5) or in the presence of 100-fold excess
UAS1 (lane 6) or mutated UAS1 (lane 7). The band
shift reaction was also carried out and the products run directly on
the gel without prior cross-linking (lane 8, no
-l). The binding assay samples from the SP column were also
exposed to UV radiation in solution, and the products run directly on
the polyacrylamide gel (lanes 9-11). Competitor UAS1
oligonucleotides (lane 10) and mutated UAS1 (lane 11)
were used to determine the specificity. The specifically labeled
polypeptide is indicated by an arrow.
In order to verify the results obtained above, we carried out a similar cross-linking experiment in which the mixture of labeled DNA and a partially purified fraction containing Oaf1p activity was exposed to UV radiation in solution. This reaction was carried out in the presence or the absence of competitor DNA, and the mixture was then treated with DNase I to degrade any DNA that was not protected by Oaf1p. Following separation of the products by SDS-polyacrylamide gel electrophoresis, several cross-linked bands appeared (Fig. 5b, lanes 9-11). However, only the protein-DNA complex at approximately 136 kDa was specifically competed out with oligonucleotides containing UAS1 (lane 10) but not with mutated UAS1 (lane 11). Thus, the DNase I treatment did not affect the mobility of this protein-DNA complex, suggesting that most of this DNA sequence is protected by bound Oaf1p.
Taken together, these results strongly indicate that the 120-kDa protein purified by our affinity chromatography strategy is Oaf1p. We cannot exclude the possibility that Oaf1p consists of two different proteins or protein subunits that have similar molecular masses rather than of a single protein, which gives rise to a dimerized form of approximately 200 kDa.
The sequence of Oaf1p from amino acids 66 to 97 shows a high level of structural homology to the DNA-binding ``fingers'' found in many regulatory proteins. Fig. 6shows a comparison of this region of Oaf1p with two other yeast regulatory proteins, the products of CYP1(23) and GAL4(24) .
Figure 6: Comparison of the homologous amino acid sequences of the DNA-binding finger-like domains of Oaf1p, Cyp1p, and Gal4p. The numbers on the left refer to the positions of the first Cys residue shown in the respective proteins. Identical residues are boxed, and a dash indicates positions where a gap has been introduced for the alignment.
A 1.3-kb SphI fragment of the open reading frame of OAF1 was replaced with the yeast HIS3 gene as described under ``Materials and Methods.'' The successful disruption of OAF1 was confirmed by using genomic DNA from the parental and disrupted strain in a PCR assay with oligonucleotides YL3 and YL4, previously used to clone the gene. The DNA product from the transformants was approximately 0.5 kb larger than that obtained from the parent strain, confirming that the OAF1::HIS3 fragment was integrated into the genome.
The effect of disrupting OAF1 was tested in four ways. Firstly we tested whether the disrupted
strain could grow on plates containing oleic acid but no glycerol (YNO
medium). Yeast strains that lack functional -oxidation enzymes are
not able to grow on YNO plates(12, 15) . Our parental
strain grew, although slowly, on this medium, whereas the disrupted
strain was unable to grow (Fig. 7a), suggesting that
the
-oxidation pathway was not induced in the presence of oleate
in this strain. We then prepared extracts from both parental and
disrupted strains grown in YPGO medium and used the extracts in a DNA
band shift assay with labeled UAS1. No band shift was seen with
extracts from the disrupted strain grown in the presence of oleate (Fig. 7b, lane 4), whereas the expected shift
was present with extracts from the parental strain (Fig. 7b, lane 2). The second, lower band seen
in this gel is nonspecific because it is not competed out by excess
probe (data not shown, but see (9) ).
Figure 7: Disruption of the OAF1 gene prevents oleate induction of the POX1 promoter. a, YNO plate showing the growth of the parental (1 and 2) and OAF1-disrupted strains (3 and 4). b, DNA band shift assay with control (lanes 1 and 2) and OAF1::HIS3 (lanes 3 and 4) strains grown in YPG (G) or YPGO (O) medium.
As further confirmation that we had disrupted the gene encoding the transcription factor responsible for the oleate-induction of POX1, we transformed the disrupted strain with a centromeric plasmid containing the OAF1 gene. We then performed a Northern blot on the parental, disrupted, and rescued strains grown in glycerol or oleate medium and measured the level of POX1 mRNA (Fig. 8a). The levels of PGK1 mRNA were also measured as a control for RNA loading (Fig. 8b). The amount of RNA loaded in each lane is somewhat uneven; however it is clear that POX1 expression is not induced in the OAF1 disrupted strain (Fig. 8a, lanes 3 and 4). According to the level of PGK1 expression (measured by PhosphorImager analysis), the amount of RNA loaded in lane 6 was 1.5-fold greater than that loaded in lane 5. However, POX1 expression was 7.5-fold greater in the oleate-induced cells (lane 6) compared with cells grown in glycerol (lane 5). Thus, allowing for unequal loading, POX1 is induced approximately 5-fold in the strain rescued with the OAF1 gene.
Figure 8:
POX1 expression in parental (P), OAF1-disrupted (K-O), and OAF1-rescued (R) strains grown in YPG (G) or YPGO (O)
medium. a and b, Northern blot analysis of POX1 and PGK mRNA expression, respectively. c,
-galactosidase activities measured in extracts prepared from the
same cells used for RNA extraction in a and b.
Finally, the parental and disrupted
strains also carried an integrated copy of the lacZ gene under
control of the POX1 promoter. Therefore, in addition to the
Northern analysis, we also measured the -galactosidase activity in
each strain (Fig. 8c). The results confirmed that the POX1 promoter was not activated by oleate in the OAF1-disrupted strain but was activated by oleate in both the
parental and rescued strains. Together these results confirm that we
have cloned the gene encoding the UAS1-binding protein and that
disruption of this gene prevents the encoded protein from binding to
UAS1 and thus activating POX1.
In an earlier report we demonstrated that a protein or proteins bind to a UAS1 element in the POX1 promoter in an oleate-activated fashion and that this causes transcriptional activation of the POX1 gene(9) . Here we describe the use of a standard chromatographic strategy for the purification of the binding protein using as a final step a DNA affinity column containing the UAS1 binding site(18) . The oleate-specific binding protein(s) (Oaf1p) appears as a doublet or triplet on a silver-stained gel and has an apparent molecular mass of approximately 120 kDa. Photoaffinity cross-linking data confirmed the size and specificity of this DNA-binding protein.
We find that the amount of Oaf1p binding to UAS1 increases following a shift of cells from glucose to oleate medium, suggesting that this transcription factor is activated and/or induced continuously in the presence of oleate. The protein that binds to URS2 appears to be present in cell extracts regardless of the growth conditions tested. However, in this in vitro assay, it is likely that the two proteins are bound to different molecules of DNA because there is no evidence of a ``super-shift'' in the DNA band shift assay, and this would be expected if both proteins were bound to the same DNA molecule.
Oaf1p was recently identified as YAL051W, an open reading frame on the left arm of chromosome I(25) . The gene product was predicted to be a zinc finger protein and a HAP1 product homolog. We have compared the amino acid sequence of Oaf1p with the protein products of HAP1 (CYC1) and GAL4 and found that the highest homology is present in the DNA-binding finger-like domain (Fig. 6).
Many of the described characteristics of Oaf1p resemble those of the Gal4 protein, a ``universal activator'' that activates transcription of an array of genes required for galactose and melibiose metabolism (26, 27) . The Gal4 protein contains four functional regions; the DNA-binding motif, a dimerization region, a Gal80p recognition region and three acidic activation regions(28) . Purified Gal4 protein runs as a triplet on SDS gels, the bands having apparent molecular masses of 100, 105, and 108 kDa(29) . Western blot analysis and dephosphorylation of Gal4p in galactose-induced yeast cells show that one form of this protein arises by phosphorylation. The high molecular weight form of Gal4p is converted to a more rapidly migrating form in the presence of phosphatase(29) . Thus, phosphorylation of Gal4p is associated with its activation. Cyp1p (the product of the CYP1 gene) mediates both positive and negative effects on the expression of several genes whose transcription is heme-dependent in yeast. Whether the effect is positive or negative depends on the target gene and on the redox state of the cell. Thus, it appears that Oaf1p belongs to a family of fungal transcription factors that have complex mechanisms for regulating several genes.
We intend to determine the
mode of regulation controlling transcription of genes encoding
peroxisomal -oxidation enzymes. In glucose-grown cells an
as-yet-unknown protein is bound to the URS elements of the POX1 promoter, and this prevents transcription of the gene. When the
cells are shifted to an oleate medium, Oaf1p may become modified
(perhaps phosphorylated) and bind to the UAS1. This would over-ride the
repressing activity of the URS protein(s) and cause activation of the
gene.
The model outlined above is a much simplified version of the
mechanisms that we believe operate to control repression, derepression,
and activation of POX1. It is likely that expression of genes
encoding peroxisomal proteins is coordinately mediated by a specific
subset of proteins. It was recently shown that two genes, RTG1 and RTG2, are required for efficient growth of S.
cerevisiae on oleate medium(30) . These genes are involved
in the basal and induced expression of the CIT2 gene(31) . CIT2 encodes peroxisomal citrate
synthase, a glyoxylate cycle enzyme(32, 33) . The RTG1 product is a transcription factor that binds to UAS in the CIT2 promoter(31) . This protein does not
bind to the oleate response element of the FOX3 gene, and it
was suggested that the RTG genes may act at an early step in
the signal transduction pathway leading to peroxisomal
induction(30) .
We are conducting experiments in our laboratory to isolate and characterize the factors involved in the pathway that regulates POX1. Experiments are also underway to further characterize Oaf1p. We will use the strain in which Oaf1p is disrupted in order to gain important information regarding the role of Oaf1p in the regulation of other genes encoding peroxisomal enzymes.