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(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
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 At least two nuclear genes, RTG1 and RTG2, are required for
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 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 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 In this paper we show that
the full-length Gal4-Rtg1 wild-type fusion protein is able to mediate
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
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
Figure 2:
A
Pro
Figure 3:
Western blot analysis of Rtg1p levels.
Equal aliquots of total cell proteins prepared from 3 ml of OD
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
Whole cell
extracts were prepared from SFY526 and SFY526
Figure 4:
Gal4-Rtg1p binds to the 76-bp CIT2 UAS
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
Figure 5:
Transactivation by Gal4-Rtg1p. Wild-type RTG1 (A) and
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 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
Surprisingly, the proteins
encoded by the three mutant constructs carrying the Leu
Figure 6:
RTG2 is required for efficient
transactivation by Gal4-Rtg1p. SFY526
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
Volume 270,
Number 49,
Issue of December 8, 1995 pp. 29476-29482
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
°
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.
° 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) .
°/
-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.
) 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) . 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. 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.
°/
-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.
Yeast Strains and Growth Conditions
The S.
cerevisiae strains used in this study are listed in Table 1.
All strains were derivatives of either COP161 U7 (8) or SFY526
(a gift from Stan Fields and Paul Bartel, SUNY, Stony Brook, NY). All
nuclear genotypes exist both as respiratory competent strains
containing wild-type mtDNA and as respiratory
incompetent
° derivatives lacking mtDNA. The
°
derivatives were obtained by several passages through rich dextrose
medium containing 20 mg/ml ethidium bromide, then checked for the
presence of mtDNA by staining with 4`,6`-diamino-2-phenylindole. Cells
were grown at 30 °C on YP medium (1% yeast extract, 2%
Bacto-peptone) and either 2% glucose (YPD), 2% raffinose (YPR), or 3%
glycerol (YPG) as a carbon source. Plasmids were selected for by growth
on minimal YNB medium (0.67% yeast nitrogen base without amino acids)
and 2% dextrose (YNBD) or 2% raffinose (YNBR) supplemented with
casamino acids or individual amino acids as required.
Recombinant Plasmids and Site-directed
Mutagenesis
Standard molecular biology techniques were
used(22) . The Escherichia coli strain XL1-Blue
(Stratagene, La Jolla, CA) was used to produce recombinant plasmids.
The plasmids used in this study are listed in Table 2.
Production of Antisera against Rtg1p
PCR primers
were used to construct a pMAL-RTG1 plasmid encoding a full-length
Rtg1-maltose-binding protein fusion. The pMAL-RTG1 plasmid was
expressed in E. coli and protein purified following the
protocol provided by New England Biolabs for their pMAL-c2 vector
system. The purified fusion protein was used to immunize two rabbits
following standard procedures(23) . Crude serum from these
animals was subsequently purified in two steps. First, the serum was
preabsorbed against maltose-binding protein (MBP) coupled to
CNBr-Sepharose (Pharmacia Biotech Inc.) to remove MBP-specific
antibodies. The serum was then affinity-purified using pMAL-RTG1 fusion
protein coupled to CNBr-Sepharose. The Rtg1p-specific antibodies were
eluted with 0.1 M glycine HCl, pH 2.3.Western Blot Analysis
Trichloroacetic acid
precipitates of total yeast cell proteins were prepared by pelleting
cells from 3 ml of an OD = 1 culture grown in YPR
following the method of Riezman and Schatz(24) . Equal volumes
of extract were fractionated on a 12% SDS-PAGE gel, transferred to
nitrocellulose and probed with Rtg1p- and actin-specific antibodies
following standard procedures(22) . Monoclonal actin-specific
antibodies were obtained from Amersham Corp. Secondary antibodies were
visualized using the ECL Western blotting system (Amersham).
Yeast Transformation and Gene Disruptions
Yeast
strains were transformed using a lithium acetate
procedure(25) . The pUCrtg1::LEU2 plasmid contained a 2.2-kb LEU2 fragment inserted into the HindIII-SacI
site of pUCRTG1, thus deleting amino acids 1-163 of Rtg1p. SFY526 
rtg1 strain was made by transforming SFY526 with a 4.8-kb
linear BglII fragment from pUCrtg1::LEU2. The pUCrtg2::LEU2
disruption plasmid contained a 2.2-kb LEU2 fragment inserted
into the SalI-XbaI site of pUCRTG2, thus deleting
amino acids 23-573 of Rtg2p. The SFY526 rtg2 strain
was made by transforming SFY526 with a 4.5-kb linear PstI
fragment from pUCrtg2::LEU2. Plasmid pRTG2-416 was made by cloning a
2.4-kb PCR fragment containing RTG2 plus approximately 350 bp
upstream of the ATG and 300 bp downstream of the stop codon into
pRS416. To form the disruption plasmid prtg2::HIS3, a 1.15-kb HIS3 fragment was inserted into the internal EcoRI site of
pRTG2-416, truncating the reading frame at amino acid 273, thus
deleting over 50% of the protein. The SFY526 rtg1 rtg2 strain was made by transforming SFY526 rtg1::LEU2 with a 2.8-kb fragment from p416RTG2::HIS3. Each of the disruption
strains was checked by Southern blot analysis. To verify that the
integrated GAL1-LacZ reporter gene was still functional, each
of the SFY526 disruptions was crossed to a wild-type tester.Electrophoretic Mobility Shift Assays
Preparation
of whole-cell extracts and gel retardation assays were as described
previously (8) with the following modifications. A DNA fragment
containing the 76-bp UAS was prepared by PCR off the CIT2 promoter using the following 5` and 3` flanking primers:
5`-GCATTAGGATCCGCTACGGAAAAGGTCACA and
GCATTAGGATCCCCGGTGGTCATCGACTAG-3`. The final concentration of the
reaction buffer was 25 mM Tris-HCl (pH 7.5), 5 mM MgCl, 0.125 mM EDTA, 300 mM KCl, 10%
glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, 0.5 µg/ml aprotinin, and 0.5
µg/ml leupeptin. Two mg of single-stranded salmon sperm DNA were
used as blocker. Reactions were incubated at room temperature for 20
min and then applied to a 4% polyacrylamide gel (40:1 in 0.5 
Tris borate-EDTA) and run at 150 V for 3 h at 4 °C.
Cell
extracts and assays were carried out as described by Rose and
Botstein(26) . Each culture was inoculated with a pool of
15-20 independent transformants. A minimum of three independent
extracts were made for each plasmid-strain combination, and each
extract was assayed in triplicate.
-Galactosidase Extracts and Assays
The rtg1-1 Mutant Allele Contains Two Changes Relative
to the Wild-type Allele
We previously identified a mutant allele
of RTG1 (rtg1-1) that blocked CIT2 expression in both and
°
cells(8) . The rtg1-1 allele was cloned by PCR
amplification from genomic DNA of the mutant strain. DNA sequence
analysis revealed that the mutant allele contained two base pair
changes relative to the wild-type allele (Fig. 1A): one
was a C
T transition, changing a CCC Pro codon to a CTC Leu
codon at amino acid 39 at the end of the first conserved helical domain
of the HLH motif; the second was a G
A transition changing a GTG
valine codon to an ATG methionine codon at amino acid 112 in the
carboxyl-terminal domain of Rtg1p.
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.
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.
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.
Rtg1p Levels Are Comparable in
Since RTG1 is required for basal and retrograde
regulation of CIT2 expression, a simple explanation for
elevated CIT2 expression in °/
RTG1, rtg1-1, and
rtg2
Cells° cells would be a change
in the level of Rtg1p itself. To test this, the level of Rtg1p was
monitored in whole cell extracts from
and
° derivatives of the wild-type and mutant forms of COP161 U7
cells by Western blot analysis. The strains analyzed included the
wild-type, rtg1-1 mutant, and two strains described
previously(8) , harboring a deletion of either RTG1 (
rtg1) or RTG2 (rtg2).
Polyclonal Rtg1p-specific antiserum detected a specific band of about
20 kDa, the expected size for the Rtg1p, which was not detected in
extracts from the COP161 rtg1 cells (Fig. 3, lanes 1 and 2). Relative to actin, there was no
significant difference in the level of the 20-kDa species between
° and
wild-type cells (Fig. 3, lanes 3 and 4), suggesting that the large difference
in CIT2 expression between these cells types is not due to
changes in Rtg1p levels.
= 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.''
or
° cells.
Rtg1p Is Present in a DNA-Protein Complex with the CIT2
UAS
Since the CIT2 UAS does not
contain a consensus E box or an N box, it was therefore important to
determine whether Rtg1p was actually a part of the DNA-protein complex
seen in EMSA of the 76-bp UAS. To do this we took advantage
of the construct, p2H1-177, encoding the DNA binding domain of Gal4p
fused to full-length Rtg1p (Fig. 1B). This plasmid was
constructed for the transactivation studies described in the next
section, but proved useful for the analysis of Rtg1p binding to the
76-bp UAS in EMSA. The Gal4-Rtg1p encoded by p2H1-177 was
327 amino acids long compared to 177 amino acids for endogenous Rtg1p.
If the Gal4-Rtg1p was incorporated into the EMSA DNA-protein complex,
the complex would run more slowly due to the presence of the larger,
recombinant protein. The Gal4-Rtg1 fusion protein was used both to
substitute for and to compete with Rtg1p in EMSA.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.
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.''
Rtg1p Can Mediate
To
assay the ability of the Rtg1p to activate transcription, various
chimeric plasmids were constructed encoding fusion proteins containing
the DNA binding domain of Gal4p fused to the NH°/
-dependent Transactivation of a
Reporter Gene when Fused to the Gal4p DNA Binding Domain
terminus of
the full-length and truncated versions of Rtg1p (Fig. 1B). The base plasmid was pGBT9 (GenBank(TM)
accession number U07646), a multi-copy 2-µm plasmid carrying TRP1 as a selectable marker and sequences encoding amino acids
1-147 of Gal4p under a constitutive
promoter(27, 28) . These constructs were transformed
into and
° SFY526 cells. which carried
an integrated LacZ reporter under the control of a GAL4-responsive GAL1 UAS
promoter(29) . The Gal4p DNA binding domain would direct
binding of the fusion protein to the UAS, so that
individual domains of Rtg1p could be tested for their ability to
transactivate the LacZ reporter independent of their ability
to bind to the CIT2 UAS. In addition, any
accessory proteins required for gene activation would be available to
interact with or modify Gal4-Rtg1p.-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.
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.
°/
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. 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.
Gal4-Rtg1p Is a Potent Transactivator in a Strain Deleted
for Endogenous RTG1
To eliminate possible competition for
factors required for transcriptional activation that might interact
with the endogenous Rtg1p and thus skew the results with the GAL4-RTG1 fusion constructs, a null allele of RTG1 (rtg1) was constructed in strain SFY526. Activation
of the GAL1-LacZ reporter gene by the various GAL4-RTG1 constructs was then tested in and
° derivatives of the
rtg1 strain. As shown in Fig. 5B, -galactosidase activity in both
and
° cells transformed p2H1-177 and
p2H26-177 was much higher than in the wild-type SFY526 background.
(Note the change in scale of the
-galactosidase activity in Fig. 5, panel A versus B). The COOH-terminal domain of
construct p2H99-177, however, was still unable to activate expression
of the LacZ reporter gene in either or
°
tg1 cells. 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.The Gal4-Rtg1 Fusion Protein Is Not a Strong
Transactivator when Expressed in Cells Deleted for RTG2
RTG2 is required for UAS-dependent transcription of CIT2(8) . However, the pattern and intensity of the
EMSA using the 76-bp UAS and extracts from rtg2 cells is identical to wild-type(8) , suggesting either
that Rtg2p influences CIT2 transcription independent of Rtg1p,
or that Rtg2p affects a putative Rtg1p-dependent DNA-protein complex in
a manner that does not alter the binding of factors to the
UAS. We have examined this point using the Gal4-Rtg1p
transactivators in a rtg2 and a rtg1 rtg2 SFY526 background, whereby transactivation by the Gal4-Rtg1p does
not require binding to the UAS. Fig. 6compares the
transactivation activity of p2H1-177, p2H1-177m, and p2H26-177
constructs in wild-type, rtg1, rtg2, and rtg1 rtg2 strains. The data show that
-galactosidase activity in both the and
° strains is very low in the
rtg2 and rtg1 rtg2 backgrounds when compared with the
wild-type and rtg1 strains, respectively. These findings
suggest that Rtg2p regulates CIT2 transcription by affecting
Rtg1p, or associated proteins, rather than via an independent
interaction with the UAS. Both and
° activities in the
rtg2 strain were in the
range of the control Gal4 DNA binding domain plasmid, pGBT9, making it
difficult to assess a °/
response with
any certainty. However, the higher activity in the
rtg1
rtg2 strain clearly showed a
°/
response for all three constructs,
although the -fold induction of the
°/
response was not as great as in the single
rtg1 disruption strain.
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.
Rtg1p Is Not Limiting in the
The bHLH transcription factor encoded by the RTG1 gene is required for both basal expression of the CIT2 gene as well as for its elevated expression in cells with
dysfunctional mitochondria, such as in °/
-responsive Regulation of CIT2
Transcription
° petites(8) .
One possible explanation for the dramatic difference in CIT2 expression between
and
° cells is
a change in the level of Rtg1p itself. That mechanism of regulation
would be analogous to tissue-specific expression of a variety of
neurogenic and myogenic HLH proteins that correlates with
transcriptional activation or repression of specific target genes
during development(31, 32, 33) . However,
using Rtg1p-specific antisera to compare protein levels, we found that
the amount of Rtg1p did not vary significantly between
and
° cells, nor was its amount affected by a deletion
of RTG2, whose product is also required for CIT2 expression (Fig. 2). These data suggest that Rtg1p is
constitutively expressed along with other components that might be
required for its interaction with the CIT2 UAS
.The UAS
The full-length wild-type Gal4-Rtg1p construct proved
useful in demonstrating that the 76-bp UAS Is Bound by a Complex Containing
Rtg1p upstream of the CIT2 gene is a target for Rtg1p(8) . In EMSA, the
fusion protein expressed in yeast could compete with endogenous Rtg1p
in binding to the UAS, forming a slower migrating complex (Fig. 4). This demonstration was important, since the 76-bp
UAS does not contain an E box or N box, the target sites
for most bHLH and bHLH/Z proteins that have been studied to
date(17, 18) . For notable exceptions, see Refs. 34
and 35. Work is presently under way to define the exact binding site
requirements for Rtg1p. Taken together, our data suggest the
possibility that some post-translational modification of one or more
components in the Rtg1p-UAS complex is responsible for the
/
° transcriptional switch.
The HLH Domain of Rtg1p Is Important for
Transactivation
The bHLH and bHLH/Z transcription factors form
homo- or heterodimers through hydrophobic interactions of the H1 and H2
or H2-leucine zipper amphipathic helices(36) . This
dimerization is required for binding of the factors to a specific DNA
template and for subsequent transcriptional activation of a target
gene(37, 38, 39) . In the Gal4-Rtg1p
transactivation assays, the presence of the Gal4 DNA binding domain
fused to Rtg1p would obviate the requirement of Rtg1p dimerization per se for interaction of the fusion protein with the
UAS target site upstream of the LacZ reporter
gene. Nevertheless, deletion of the HLH domain of Rtg1p in construct
p2H99-177 resulted in a loss in the ability of the fusion protein
to transactivate (Fig. 5). These data are consistent with the
view that Rtg1p forms a homo- or heterodimer required for
transactivation. Several constructs were made encoding only the HLH or
bHLH domain of Rtg1p fused to the Gal4 DNA binding domain, to test
whether the HLH domain was sufficient for interaction with a
transactivating partner. Unfortunately, however, none of these produced
stable proteins.A Model for Target Site Interactions
The model
shown in Fig. 7accounts for most of the observations presented
in this paper. We propose the existence of an interacting factor X,
most likely another bHLH protein, that is required for transcriptional
regulation. Thus, by analogy with other bHLH and bHLH/Z proteins, dimer
formation is a requisite for site-specific DNA
interactions(36) . Formation of the Rtg1p-X complex is required
for efficient binding to the CIT2 UAS (reaction 1)
and for transcriptional activation of CIT2 (reaction 4). The
full-length Gal4-Rtg1 wild-type fusion construct binds independently of
X to the GAL1 UAS (reaction 3) and also to the CIT2 UAS in a reaction dependent on complexing with X
(reaction 2). When the fusion construct is bound to the
UAS, transcriptional activation of LacZ (reaction
5) occurs as a result of complex formation with X.
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.
RTG2 Potentiates Transactivation by Gal4-Rtg1p
The
levels of CIT2 mRNA in rtg1 and rtg2 strains are at the threshold of detection in both and
° cultures(8) . It was therefore impossible
to determine epistasis between RTG1 and RTG2 by
analysis of CIT2 expression. The dramatic decrease in
transactivation by the Gal4-Rtg1 fusion construct in the
rtg2 backgrounds (Fig. 6) clearly shows that the Rtg1p requires
the RTG2 product in either or
° cells for full activity. It also suggests that RTG2 influences CIT2 transcription through Rtg1p rather than
via an independent pathway. Extracts from cells deleted for RTG2 are still able to form the RTG1-dependent EMSA complex
with the 76-bp UAS
(8) . Therefore, RTG2 appears not to influence complex formation but may act to modify
one or more of the components associated with the CIT2 UAS transcriptional complex, perhaps in response to
signals from mitochondria.
)
We thank Stan Fields and Paul Bartel (SUNY, Stony
Brook, NY) for generous gift of plasmids.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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