(Received for publication, August 2, 1995; and in revised form, September 26, 1995)
From the
Rtg1p is a basic helix-loop-helix transcription factor in the
yeast Saccharomyces cerevisiae that is required for basal and
regulated expression of CIT2, the gene encoding a peroxisomal
isoform of citrate synthase. In respiratory incompetent °
petite cells, CIT2 transcription is elevated as much as
30-fold compared with respiratory competent
cells. Here we provide evidence that Rtg1p interacts directly
with a CIT2 upstream activation site (UAS
) and
that the
°/
regulation is not due to a
change in the levels of Rtg1p. A fusion protein consisting of the DNA
binding domain of Gal4p fused to the NH
terminus of the
full-length wild-type Rtg1p was able to transactivate an integrated LacZ reporter under control of the Gal4p-responsive GAL1 UAS
in a
°/
-dependent
manner. Other Gal4p fusions to deletions or mutations of Rtg1p indicate
that the helix-loop-helix domain is essential for transactivation.
Regulated expression of CIT2 also requires the RTG2 gene product. The Gal4-Rtg1p fusion was unable to transactivate
the LacZ reporter gene in a strain deleted for RTG2,
suggesting that the RTG2 product does not act independently of
Rtg1p in the
°/
transcriptional
response.
Recent evidence indicates that the functional state of
mitochondria and chloroplasts can influence the expression of nuclear
genes(1, 2, 3, 4) . In Saccharomyces cerevisiae, one such response involves the
elevated expression of the nuclear gene CIT2 (encoding a
peroxisomal isoform of citrate synthase (5, 6) in
cells with dysfunctional mitochondria(7, 8) . For
example, in respiratory incompetent ° petites (cells that lack
mtDNA), (
)CIT2 mRNA abundance is as much as 30-fold
greater than in isochromosomal respiratory competent
cells. This regulation of CIT2 expression appears to be
a mechanism for adjusting metabolic interactions between the
peroxisomal glyoxylate cycle and the mitochondrial tricarboxylic acid
cycle(7, 9) .
At least two nuclear genes, RTG1 and RTG2, are required for
°/
-responsive transcription of CIT2(8) . Strains with null alleles of either of these
genes are unable to use acetate as a sole carbon source, and show
growth requirements for glutamate or aspartate, which are phenotypes
typical of cells deficient in both the tricarboxylic acid and
glyoxylate cycles(10) . We have recently found that RTG1 and RTG2 have functions in addition to regulation of CIT2 expression(11) ; they are together also required
for oleic acid-induced expression of genes encoding peroxisomal
proteins, as well as for general peroxisome proliferation, which is
known to be induced in yeast by oleic
acid(12, 13, 14) . Thus, these genes appear
to play a central role in a novel three-way organelle communication
between mitochondria, the nucleus and peroxisomes.
RTG1 encodes a 177-amino acid protein (Rtg1p) that is a member of the
basic helix-loop-helix (bHLH) family of transcription
factors(15, 16) . We have found that sequences
contained within a 76-bp MspI-AluI fragment
(UAS) in the 5` flanking region of CIT2 are both
necessary and sufficient to convey a
°/
response to a reporter gene(8) . Electrophoretic mobility
shift assays (EMSA) using the 76-bp UAS
as a probe and
extracts from a wild-type strain and a strain deleted for RTG1 reveal an Rtg1p-dependent DNA-protein complex, suggesting that
Rtg1p binds to the UAS
. However, the UAS
does
not contain either an E box (CANNTG) or an N box (CACNAG), which are
canonical DNA binding sites recognized by most bHLH
proteins(17, 18) .
Although RTG2 is
required for CIT2 expression, its precise function is unclear. RTG2 encodes a protein containing an HSP70 type of ATP binding
domain with similarity to bacterial phosphatases that hydrolyze ppGpp
and pppGpp(19) . In addition to the absence of any obvious DNA
binding motifs in Rtg2p, the EMSA pattern using the CIT2 UAS as a probe and extracts from cells with an rtg2 null allele is indistinguishable from wild-type extracts,
suggesting that the RTG2 product may act indirectly in the
regulation of CIT2 expression.
To investigate the role of
Rtg1p in transcriptional activation, we have tested the ability of
various domains and alleles of RTG1 to activate transcription
in and
° cells, which have in
combination either wild-type or null alleles of the chromosomal RTG1 and RTG2 genes. To assay for transcriptional
activation, we have constructed various chimeric protein fusions
between the DNA binding domain of the yeast Gal4p transcriptional
activator (20, 21) and Rtg1p. This allows the
determination of potential transactivation by Rtg1p independent of its
intrinsic DNA binding characteristics.
In this paper we show that
the full-length Gal4-Rtg1 wild-type fusion protein is able to mediate
°/
-responsive transactivation of the
reporter gene under UAS
control, and that transactivation
is dependent on the presence of a wild-type allele of RTG2. We
have also used this construct to show that Rtg1p binds directly to the CIT2 UAS
. Finally, results are presented
indicating that Rtg1p interacts, probably via its HLH domain, with
another factor, or factors, that are required for the
°/
transcriptional response.
The plasmid pRTG1-416 contained a 1.5-kb SphI-PstI genomic fragment containing the entire RTG1 reading frame plus 740 bp of upstream and 300 bp of downstream sequences cloned into pRS416 (CEN, URA3). Site-directed mutagenesis was carried out using the Bio-Rad Muta-Gene in vitro mutagenesis kit.
The pGBT9 plasmid was a gift from Stan Fields and Paul Bartel (Dept. of Microbiology, SUNY, Stony Brook, NY). PCR primers were used to amplify specific fragments of the RTG1 gene for the construction of the 2H series of recombinant pGBT9 plasmids encoding Gal4p DNA binding domain-Rtg1p fusion proteins (Gal4-Rtg1p). All newly constructed plasmids were verified by sequencing using the Sequenase kit (United States Biochemical Corp.).
Figure 1:
Panel A, structural
domains of Rtg1p including the location of the two point mutations at
amino acids 39 (Pro Leu) and 112 (Val
Met) in the rtg1-1 allele. The basic (b), HLH (H1, L, and H2), and COOH-terminal (C) domains
are indicated. The putative protein dimerization HLH motif extends from
amino acid 26 to 98. Panel B, coding domains of the GAL4-RTG1 fusion plasmids used in the transactivation assays.
PCR was used to insert portions of RTG1 into the EcoRI and BamHI sites of the pGBT9 polylinker fusing
Rtg1p to amino acids 1-147 of Gal4p DNA binding domain. The
fusions contain an additional 3 amino acids encoded by polylinker
sequences.
To determine whether one or both
of these mutations were responsible for the phenotype of the rtg1-1 allele, a complementation test was done in which RTG1, rtg1-1, and the Leu and Met
mutant
alleles were each expressed from a low copy centromeric plasmid. To
assay for function of the mutant rtg1 alleles, we used a
° strain described previously (8) in which a LacZ reporter gene under the control of the CIT2 promoter was
integrated into the ura3 gene of the rtg1-1 strain.
That strain is defined here as CIT2-LacZ-I (Table 1). As shown in Fig. 2, CIT2-LacZ-I cells had a high level of
-galactosidase activity when transformed with a plasmid containing
the wild-type RTG1 gene (pRTG1-416). The single mutation
pL112-M construct also restored activity, while the double mutant
(prtg1-1), single mutant (pP39-L), and control plasmid (pRS416) all had
low activity. These data demonstrate that the mutant phenotype of the
original rtg1-1 allele is due largely to the Pro
Leu
change at amino acid 39 at the end of helix 1 of Rtg1p.
Figure 2:
A
Pro
Leu mutation blocks Rtg1p function. CIT2-LacZ-I
° cells carrying an integrated LacZ reporter gene
under the control of the CIT2 promoter were transformed with a
control plasmid (pRS416), or a plasmid carrying the wild-type RTG1 allele (pRTG1-416), or plasmids with the mutant alleles rtg1-1 (prtg1-1), Pro
Leu (pP39-L) or
Val
Met (pV112-M). Cultures were inoculated with a
pool of 15-20 transformants and grown to mid-log phase in YNBR
supplemented with casamino acids. Extracts were made and specific
activity assayed as described under ``Materials and
Methods.'' Extracts were assayed in triplicate for
-galactosidase activity and expressed in the figure as nmol/min/mg
of protein.
Figure 3:
Western blot analysis of Rtg1p levels.
Equal aliquots of total cell proteins prepared from 3 ml of OD = 1 cultures of
and
°
rtg1 (lanes 1 and 2), RTG1 (lanes 3 and 4),
rtg2 (lanes 5 and 6), and rtg1-1 (lanes 7 and 8) strains were fractionated on a 12% SDS-PAGE gel,
transferred to nitrocellulose, and probed sequentially with Rtg1p- and
actin-specific antisera as described under ``Materials and
Methods.''
We also tested whether Rtg2p could affect CIT2 transcription by modulating the level of Rtg1p. Fig. 3(lanes 5 and 6) showed that Rtg1p levels
were unaffected by the absence of Rtg2p. Finally, the data of Fig. 3(lanes 7 and 8) showed that in the
orginal rtg1-1 mutant, the nearly complete loss of the
specific DNA-protein complex detected in EMSA assays(8) , was
not the result of any significant instability of the mutant Rtg1p in
either or
° cells.
Whole cell
extracts were prepared from SFY526 and SFY526 rtg1 strains with and without the p2H1-177 plasmid encoding the
full-length wild-type Gal4-Rtg1p fusion. An Rtg1p-dependent band was
identified by comparing the 76-bp EMSA from wild-type verses the
rtg1 strain (Fig. 4, lanes 1 and 3). When extracts from cells transformed with p2H1-177
encoding the full-length fusion protein were used, a larger, slower
migrating complex was formed indicating the binding of the larger
Gal4-Rtg1p. In the
rtg1 background only the slower
migrating complex containing the fusion protein was present (Fig. 4, lane 4). In the wild-type background, however,
both the endogenous Rtg1p and larger Gal4-Rtg1p-dependent complexes
were present (Fig. 4, lane 2) indicating that the
Gal4-Rtg1p was able to compete with the endogenous Rtg1p for the DNA
template.
Figure 4:
Gal4-Rtg1p binds to the 76-bp CIT2 UAS and competes with Rtg1p. Wild-type (WT)
and
rtg1 SFY526
° cells (
rtg1)
were transformed with a control plasmid (pGBT9) (-), or a plasmid
encoding Gal4-Rtg1p (p2H1-177) (+). Cultures were inoculated with
a pool of 15-20 transformants and grown to late log phase in YNBR
supplemented with casamino acids. Whole cell extracts were made and
tested in EMSA with the 76-bp UAS
as described under
``Materials and Methods.''
To ensure that the various chimeric plasmids produced stable fusion proteins, whole cell extracts from transformed SFY526 cultures carrying each of the plasmids were fractionated on SDS-PAGE, transferred to nitrocellulose, and probed with polyclonal antisera raised against Rtg1p. All of the Gal4-Rtg1p fusions described here accumulated to comparable levels regardless of nuclear or mitochondrial background, and were present at higher levels relative to endogenous Rtg1p (not shown).
SFY526 cultures were grown
in selective raffinose medium to maintain the GAL4-RTG1 plasmids.
Extracts were prepared from log phase cultures and assayed for
-galactosidase activity. Fig. 5A presents a
summary of these data. The full-length fusion construct (p2H1-177) not
only activated expression of the LacZ reporter but did so in a
°/
-dependent fashion; i.e.
-galactosidase activity was about 2-fold higher in
°
than in
cells. This difference, however, was
much less than the
°/
transcriptional
response of the endogenous CIT2 gene(7, 8) .
The lower magnitude of the retrograde response using the Gal4-Rtg1p
transactivators may be inherent in the heterologous system or, as
suggested by the data below, may reflect competition for endogenous
components important in the retrograde signaling pathway.
Figure 5:
Transactivation by Gal4-Rtg1p. Wild-type RTG1 (A) and rtg1 (B) SFY526
and
° cells were transformed with a
control pGBT9 plasmid or a Gal4-Rtg1p fusion plasmid as indicated.
Cultures were inoculated with a pool of 15-20 transformants and
grown to mid-log phase in YNBR supplemented with casamino acids. Each
of the pooled transformants was assayed in triplicate, and a minimum of
three independent experiments were preformed. Specific
-galactosidase activity is expressed as nmol/min/mg of protein.
Note the 10-fold difference in the scale of the
-galactosidase
activity in panels A and B.
The
p2H26-177 construct deleted for the putative basic DNA binding domain
of Rtg1p (amino acids 1-25) was considerably more effective in
transactivation of the LacZ reporter gene and showed a
somewhat greater °/
response (3-fold)
than the full-length, wild-type construct. A likely explanation for the
higher overall activity of p2H26-177 is that in the absence of the
putative Rtg1p DNA binding domain, there was less competition with the
UAS
for the Gal4-Rtg1 fusion protein by endogenous Rtg1p
DNA targets.
Cells transformed with the p2H99-177 plasmid encoding just the carboxyl-terminal domain of Rtg1p fused to the Gal4p DNA binding domain had no activity above background (Fig. 5A). Western blot analysis indicated that the fusion protein encoded by this construct was, nevertheless, abundant (data not shown). Thus, the COOH-terminal portion of Rtg1p is not likely to be a transcriptional activator region of Rtg1p that acts independently of the HLH domain.
The full-length mutant form of
Rtg1p fused to Gal4p (p2H1-177m) did not transactivate the LacZ reporter in a wild-type SFY526 background (Fig. 5A). The construct carrying the Leu mutation (p2H3P39-L) was also unable to activate transcription of
the LacZ reporter. However, the fusion construct carrying the
Met
mutation (p2HV112-M) transactivated in a
°/
-dependent fashion at a level similar
to the full-length wild-type construct. These results support the in vivo complementation experiments shown in Fig. 2that identified the Leu
mutation as the one
accounting largely for the mutant phenotype of the original rtg1-1 allele.
Surprisingly, the proteins
encoded by the three mutant constructs carrying the Leu mutation (p2H1-177m, p2HP39-L, and p2H26-177m), which were
inactive in wild-type SFY526 cells, were potent transactivators in the
SFY526
rtg1 strain and showed a
°/
retrograde response even greater than
the wild-type construct, p2H1-177. Among bHLH proteins, the Pro
at the end of helix 1 is a highly conserved residue(8) .
A change to Leu could affect the conformation of the HLH motif and
alter subsequent protein-protein interactions. However, in some cases
leucine is permitted at this location, as in myogenin(30) .
This may explain why the mutant GAL4-RTG1 fusions (p2H1-177m,
p2H26-177m, and p2HP39-L) expressed in the
rtg1 background were effective transactivators. The mutant proteins may
be able to interact with other factors required for transactivation but
could not effectively compete with wild-type Rtg1p for them or for the
endogenous UAS
sites.
Figure 6:
RTG2 is required for efficient
transactivation by Gal4-Rtg1p. SFY526 and
° wild-type (A),
rtg1 (B),
rtg2 (C), and
rtg1
rtg2 (D) cells were transformed with the Gal4-Rtg1p fusion
plasmid indicated. Extracts were prepared and assayed as in Fig. 5. Specific
-galactosidase activity is expressed as
nmol/min/mg of protein. Note the differences in the scale of
-galactosidase activity in panels
A-D.
Figure 7: A model for the interactions of Gal4-Rtg1p and Rtg1p with DNA target sites.
The analysis of
point and deletion mutants of Rtg1p is also consistent with the model
of Fig. 7. Removal of the basic domain of Rtg1p in the fusion
construct, p2H26-177, results in a higher level of activity of
the LacZ reporter gene compared with the full-length protein,
since the competition of reaction 2 is essentially eliminated. In the
absence of the endogenous Rtg1p, competition for X in reaction 1 is
reduced or eliminated accounting for the marked increase in LacZ expression (reaction 5). Removal of the HLH domain in
p2H98-177 prevents any interaction with X (reaction 2 or 5). The
Leu mutant fusion construct would be unable to compete
with endogenous Rtg1p for X (reactions 2 and 5 versus reaction
1). However, in the absence of endogenous Rtg1p, some complex formation
would occur to allow LacZ expression. Recently, we have
identified a third gene, RTG3, that is required for
°/
CIT2 regulation, and
experiments are in progress to determine whether it encodes
``X'' or some other factor.