(Received for publication, March 6, 1997, and in revised form, May 27, 1997)
From the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
The Leu3 protein of Saccharomyces
cerevisiae regulates the expression of genes involved in branched
chain amino acid biosynthesis and in ammonia assimilation. It is
modulated by -isopropylmalate, an intermediate in leucine
biosynthesis. In the presence of
-isopropylmalate, Leu3p is a
transcriptional activator. In the absence of the signal molecule, the
activation domain is masked, and Leu3p acts as a repressor. The recent
discovery that Leu3p retains its regulatory properties when expressed
in mammalian cells (Guo, H., and Kohlhaw, G. B. (1996) FEBS
Lett. 390, 191-195) suggests that masking and unmasking of the
activation domain occur without the participation of auxiliary
proteins. Here we present experimental support for this notion and
address the mechanism of masking. We show that modulation of Leu3p is
exceedingly sensitive to mutations in the activation domain. An
activation domain double mutant (D872N/D874N; designated Leu3-dd) was
constructed that has the characteristics of a permanently masked
activator. Using separately expressed segments containing either the
DNA binding domain-middle region or the activation domain of wild type
Leu3p (or Leu3-dd) in a modified yeast two-hybrid system, we provide
direct evidence for
-isopropylmalate-dependent
interaction between these segments. Finally, we use the phenotype of
Leu3-dd-containing cells (slow growth in the absence of added leucine)
to select for suppressor mutations that map to the middle region of
Leu3-dd. The properties of nine such suppressors further support the
idea that masking is an intramolecular process and suggest a means for
mapping the surface involved in masking.
The Leu3 protein of yeast belongs to a class of transcriptional
regulators whose members are characterized by a
Zn(II)2-Cys6 binuclear cluster in their DNA
binding domains (1, 2). Many of them also have the ability to be
modulated, i.e. to respond to metabolic signals. Well
studied examples of modulation include Gal4p, which is activated
("unmasked") by galactose in a process that involves changes in the
interaction between Gal4p and an auxiliary protein known as Gal80p (3,
4), and Hap1p, the activation of which by heme is thought to be brought
about by dissociation of cellular factors that allow the DNA binding
domain to become functional and its activation domain
(AD)1 to become exposed (5,
6). Leu3p is modulated by -IPM,1 an intermediate in
leucine biosynthesis (7). Leu3p binds to UASL elements in
the promoters of genes involved in the biosynthesis of branched chain
amino acids (8, 9) and, surprisingly, in ammonia assimilation in yeast
(10). The finding that the GDH1 gene is regulated by Leu3p
and
-IPM (10) led to the hypothesis that
-IPM serves as a signal
molecule that communicates the status of amino acid biosynthetic
activity (as represented by the leucine pathway) to more central points
of nitrogen metabolism. It is of interest in this context that the
LEU3 gene itself is controlled by Gcn4p (11).
Leu3p binds to target DNA irrespective of the presence or absence of
-IPM (9). In the absence of
-IPM, Leu3p represses gene expression
below the basal level seen in leu3 null cells (9, 12). In
the presence of
-IPM, it causes strong activation. The zinc cluster
part of Leu3p's DNA binding domain is located between amino acids 37 and 67 of the 886-residue protein (11, 13, 14), and a putative
dimerization domain is found between amino acids 85 and 99 (14). A
region responsible for transcriptional activation has been identified
within the C-terminal 30 amino acids of Leu3p (14, 15). Truncated Leu3p
molecules lacking the AD still bind to DNA. They are totally devoid of
activation potential and act as repressors (9, 15, 16). Deleting as many as 600 amino acids from the middle of Leu3p leaves DNA binding and
transcriptional activation functions intact but eliminates modulation
(16, 17). Such molecules have a constitutively high activation
potential that usually exceeds that of modulated forms of Leu3p. The
implication of these results is that Leu3p's middle region contains
information that is essential for modulation. In dealing with the
question of modulation, it may be helpful to break the modulation
process down into discrete steps, e.g. the binding of the
modulator
-IPM, conformational changes caused by the binding of
-IPM, and the eventual exposure of the AD that allows it to interact
with components of the transcription apparatus. Our current view is
that the unmasking of the AD as well as its masking in the absence of
-IPM occur without the participation of auxiliary proteins. The
strongest support for this notion comes from recent observations made
when full-length yeast Leu3p was expressed in mammalian cells (18). It
was found that the behavior of Leu3p in mouse cells was almost
indistinguishable from that seen in its native environment; Leu3p was
stable but inactive when
-IPM was absent and caused an apparent
severalfold repression of reporter gene expression. When
-IPM was
present in the cell culture medium, Leu3p was converted into a strong
activator. Since mammalian cells do not synthesize branched chain amino
acids, it appeared highly unlikely that they would elaborate
Leu3p-specific factors for masking, unmasking, or
-IPM
interaction.
In this study, we have used biochemical and molecular genetic
approaches to advance our understanding of the modulation process. We
show that modulation is exquisitely sensitive to mutational changes in
the AD of Leu3p. Using wild type Leu3p and a mutant form with
drastically intensified masking, we show that the AD and the remainder
of Leu3p interact and that this interaction depends on -IPM.
Isolation of intragenic suppressors of the slow growth phenotype of the
masking mutant shows the usefulness of this approach for identifying
regions or individual residues of Leu3p that are involved in
masking.
The Saccharomyces
cerevisiae strains used were DBY746 (MAT leu2-3
leu2-112 trp1-289 ura3-52
his3-
1; YGSC), DK1 (MAT
leu3-
::LEU2 leu2-3 leu2-112
trp1-289 ura3-52 his3
1; see below),
and XK157-3C (MAT
leu3-
2::HIS3
trp1-289 ura3-52 his3-
1; (16)). The
latter two contain total leu3 deletions. DK1 was constructed
as follows. Yeast shuttle vector pRS305 (19) was digested with
ScaI and BsaI. A fragment containing the
LEU2 gene was recovered and inserted into plasmid pGB12 (9)
that had been cut with BamHI and rendered blunt-ended with
T4 DNA polymerase. The resulting plasmid, pDW1, contained one copy of
the LEU2 gene flanked by 5
- and 3
-noncoding sequences of
LEU3. The orientation of LEU2 was the same as the original orientation of LEU3 at this locus. Plasmid pDW1 was
digested to completion with SphI and SacI. Among
the fragments generated was a 4.3-kbp fragment containing the
LEU2 gene and LEU3-flanking sequences. The
digestion mixture was used for integrative transformation of strain
DBY746, selecting for Leu±. (Since the desired
transformants carried an intact LEU2 gene, but no
LEU3 gene, the phenotype was expected to change from
Leu
(DBY746, no growth in the absence of added leucine)
to Leu± (slow growth, about 40% that of wild type cells,
Ref. 9).) Transformants that also had the His
,
Ura
, and Trp
phenotype were collected and
designated DK1. Their genotype was further confirmed by PCR using
purified yeast chromosomal DNA and primers complementary to the
LEU2 coding region and regions of the LEU3 locus
flanking the LEU2 gene. Unless stated otherwise, yeast cells
were grown on SD medium (20) supplemented with the required nutrients.
A supplement of 2 mM leucine plus 1 mM each of
valine and isoleucine was routinely used to generate low intracellular
-IPM levels. (The presence of valine was found not to be obligatory, and valine was therefore omitted in some experiments.) High
-IPM levels were generated by supplementing the medium with 0.2 mM leucine. Cells were grown at 30 °C and harvested at
an A600 of about 1.
Escherichia coli strains used for DNA manipulations were TG1
(K12 [lac-pro] supE hsd
S/F
tra
36
proA+B+
lacIq lacZM15), DH5
(Life Technologies,
Inc.), and XL-2blue (Stratagene). Strain CJ236 (dut1 ung1
thi-1 relA1/pCJ105[CMr]) was used for
isolation of uracil-containing single-stranded DNA. E. coli
cells were grown at 37 °C in L broth or 2 × YT media with the
addition of 100 µg/ml penicillin where needed.
The majority of the mutants
was generated by degenerate oligonucleotide incorporation. This
approach required the construction of an expression plasmid containing
a LEU3 gene with engineered restriction sites that would
allow precise cassette exchange of the AD. To achieve this, new
NgoMI and PmeI/SmaI sites were created by site-directed mutagenesis. E. coli CJ236 cells were
transformed with plasmid pRS316-LEU3 which was constructed by cloning a
LEU3-containing fragment into pRS316 (19). (The fragment
extended from 561 to about +4700 relative to the A at the beginning
of the open reading frame of LEU3 (11).) Transformed cells
were grown to mid-log phase and superinfected with helper phage M13K07
in the presence of uracil. Phage particles were collected and
single-stranded DNA isolated according to a protocol from Bio-Rad.
Equimolar amounts of two phosphorylated primers (5
CATCATGGCCGGCTGGGATAAC-3
for the 5
side of the AD and
5
-CCCAAGGTTTAAACCCGGGTTCTTTTTTTGCG-3
for the 3
side of
the AD (NgoI and PmeI/SmaI sites
underlined)) were then used to mutagenize pRS316-LEU3. Note that
changes made to generate the new restriction sites did not alter the
amino acid sequence of Leu3p. Potential mutants were screened for the presence of an additional SmaI site, and positives were
sequenced using a double-stranded DNA cycle sequencing kit from Life
Technologies, Inc. A 1-kbp fragment of DNA containing new
NgoMI and PmeI sites was amplified by PCR using
appropriate primers. A BlnI-SmaI piece was
excised from the fragment and cloned into
BlnI/SmaI-digested pPC62H/86T-LEU3 from which two
existing NgoM1 sites had been removed, resulting in plasmid
pYHA. It contains unique NgoMI and PmeI sites that define the AD of the Leu-3 protein. Its LEU3 gene is
flanked by ADC1 promoter and terminator sequences,
respectively. Plasmid pPC62H/86T-LEU3 had been constructed from
centromere-containing plasmid pPC62H/86T (a gift from E. Taparowsky,
Purdue University) and pT7-LEU3 (a gift from J.-Y. Sze of this
laboratory). pT7-LEU3 was digested with PstI and
SmaI, and the 3.1-kbp, LEU3-containing fragment
was inserted into pPC62H/86T that had also been cut with PstI and SmaI.
To mutagenize the AD of Leu3p, a 93-base pair oligonucleotide,
5-ATGGCCGGCtgggataactgggaatctgatatggtttggagggatgttgatattttaatgaatgaatttgcgttcaatcccaaGGTTTAAACC-3
(NgoMI and PmeI sites underlined) was
synthesized (Integrated DNA Technologies, Coralville, IA) such that the
misincorporation rate at the positions shown in lowercase letters was
4.5% (i.e. the proportion of each of the three non-native
nucleotides was 1.5%) or, in a separate experiment, 2.7% (the
proportion of each of the three non-native nucleotides was 0.9%). The
lowercase letters correspond to amino acid positions 861-885 of Leu3p
(11). The oligonucleotide mixture was incubated, heated to 85 °C,
then cooled to 4 °C over a 1-h period. Nucleotides and Klenow enzyme
were added to the annealed mixture which was then incubated on ice for
5 min, at 23 °C for 5 min, and at 37 °C for 20-30 min. The extended and now double-stranded DNA was digested with NgoMI
and PmeI. The final product was a mixture of monomeric DNA
fragments. These were ligated into plasmid pYHA that had been digested
with NgoMI and PmeI. The molar ratio of fragment
to plasmid was approximately 3. Aliquots of 10 ng of ligation mixture
DNA were used to transform E. coli DH5
cells. DNA was
purified from 4 ml of overnight cultures using a QIAprep spin column.
Sequencing was done by the double-stranded DNA cycle sequencing
procedure (Life Technologies, Inc.) using a
[33P]ATP-end-labeled primer (5
-CCCGTTACAACTACAATC-3
).
pYHA plasmids carrying mutations in the
NgoMI-PmeI region of LEU3 were used to
transform yeast strain XK157-3C/pYB1 (leu3 null; pYB1
contains a LEU2
-lacZ reporter gene (9)). Yeast cells were
transformed either with the help of a transformation kit (Zymo
Research, Orange, CA) or by the lithium acetate procedure (21).
Transformants were plated on SD medium (20). Single colonies were
suspended in 10 ml of SD medium supplemented with 2 mM
leucine plus 1 mM isoleucine and grown at 30 °C for
24-30 h. Aliquots of the subcultures were then inoculated into 10 ml
of SD medium supplemented with either 0.2 mM leucine (for
high intracellular concentrations of
-IPM) or 2 mM
leucine plus 1 mM isoleucine (for low
-IPM
concentrations). To determine reporter gene activity, harvested cells
were resuspended in 0.1 M sodium phosphate buffer, pH 7.0, containing 10 mM KCl, 1 mM MgSO4,
and 50 mM
-mercaptoethanol. Aliquots of the suspension (made up to a total volume of 1 ml) were mixed with 20 µl of a 0.1%
solution of sodium dodecyl sulfate and 50 µl of chloroform and
vortexed vigorously for 15 s. The
-galactosidase activity was
then measured following the procedure of Miller (22).
Several mutations in the AD of Leu3p were generated by site-directed mutagenesis, as described (15, 23). The two sets of data, i.e. those obtained by the degenerate oligonucleotide method and those obtained by site-directed mutagenesis, were normalized with respect to the wild type controls.
Construction of the Leu3-dd (D872N/D874N) MutantFollowing
the procedure of Kunkel et al. (24), uracil-containing
single-stranded DNA from pRS316-LEU3 was mutagenized using an
oligonucleotide (5-GTTTGGAGGAACGTTAATATTTTAATG-3
) that contained AAC
and AAT triplets in place of the native GATs. Plasmid DNA isolated from
several E. coli colonies was then used to transform a
leu3 null strain (XK157-3C). Colonies growing slowly on
leucine
plates were identified, and the original plasmid
preparations were sequenced. There was excellent correlation between
slow growth and the D872N/D874N double mutation. Plasmids of this type
were designated pRS316-LEU3dd.To transfer the LEU3dd DNA to a plasmid with a different marker and to place the gene behind the
ADC1 promoter, a cassette exchange was performed between
pRS316-LEU3dd and pPC62H/86T-LEU3, as follows: pRS316-LEU3dd was used
as template for a PCR reaction to synthesize a DNA fragment extending
from the SalI site to the end of the LEU3 gene.
The PCR primers were 5
-CCAACAGAAGACATACGGA-3
(for the 5
end of the
fragment) and 5
-GTAGCACCGCGGTCATTACATA AC-3
(for the 3
end of the fragment; KspI restriction site underlined). The
PCR product was digested with SalI and KspI and
then inserted into pPC62H/86T-LEU3 cut with the same enzymes. The
resulting plasmid was designated pPC62H/86T-LEU3dd. A derivative
encoding a Leu3-dd protein from which residues 174-773 were deleted
was created by digesting pPC62H/86T-LEU3dd with SalI and
AvrII, followed by Klenow enzyme treatment and re-ligation. It was designated pPC62H/86T-LEU3dd
12.
The DNA binding part of the two-hybrid system was designed to contain the extended DNA binding region of Leu3p (DB, residues 1-173) and the adjacent "middle region" (MR, residues 174-773). It was expressed behind the ADC1 promoter. An appropriate centromere-containing plasmid was constructed by digesting pPC62H/86T-LEU3 (see above) with AvrII, filling in the overhangs with T4 DNA polymerase, and re-closing the plasmid with T4 DNA ligase. This created an in-frame stop codon at amino acid position 775 of Leu3p and replaced the arginine at position 774 with a serine. The resulting plasmid was designated pPC62H/86T-DB-MR.
The activation domain constructs for use in the two-hybrid system were
also expressed behind the ADC1 promoter. Plasmid pNLVP16 (a
gift from E. Taparowsky, Purdue University) served as starting material
for pVP-LEU3-WT-AD. A pair of primers was used to clone, by PCR, a
fragment encoding the AD of VP16. The primers
5-CTGAGCTATTCCTGCAGTAGTGAAGAG-3
(5
end primer) and
5
TCGACGGATC GACCTAGGACCCGGGGAA-3
(3
end primer) were
designed to contain a PstI site and an AvrII
site, respectively (underlined). The PCR product was digested with
PstI and AvrII and then ligated into plasmid
pPC62H/86T-LEU3 that had also been cut with PstI and
AvrII, thus fusing the VP16 AD sequence to the N terminus of
and in-frame with the extended Leu3p AD sequence (residues 774-886).
This yielded pPC62H/86T-VP-LEU3-WT-AD. To transfer the VP-LEU3-WT-AD
sequence to plasmid pRS423 (multicopy, different marker; ref. 25),
pPC62H/86T-VP-LEU3-WT-AD was digested with ApaI and
PvuII. A 3-kbp ApaI-PvuII fragment
containing the VP-LEU3-WT-AD sequence was ligated into pRS423 that had
been cut with ApaI and SmaI. The resulting
plasmid was designated pVP-LEU3-WT-AD. Plasmid pVP-LEU3-dd-AD was
constructed in the same way except that pPC62H/86T-LEU3-dd (see above)
was used instead of pPC62H/86T-LEU3. To construct pVP, a plasmid coding
for the VP16 AD only, the above PCR product was first digested with
AvrII, filled in with T4 DNA polymerase, then digested with
PstI. The digested PCR product was inserted into plasmid
pPC62H/86T-LEU3 that had been cut with SpeI, filled in, and
then cut with PstI. The resulting plasmid, pPC62H/86T-VP,
contained an in-frame stop codon behind the VP16 AD sequence. Next, the
VP sequence was transferred to pRS423 in the way described above,
yielding pVP. The DNA sequence of all junction regions and of the
entire PCR-synthesized region of the VP16 AD was confirmed using the
SequenaseTM version 2.0 sequencing kit from Amersham Corp.
Transformation of yeast cells was performed by the lithium acetate
method (21). The recipient strain DK1 was transformed first with the
reporter plasmid pYB1 (9) and then with pPC62H/86T-DB-MR. The resulting
doubly-transformed strain was then further transformed with either
pRS423 (control), or pVP, or pVP-LEU3-WT-AD, or pVP-LEU3-dd-AD. The
transformants were purified and single colonies from different isolates
were used to inoculate 2 ml of SD medium supplemented with 1 mM each of leucine, valine, and isoleucine. After the
pre-cultures had grown to saturation, cells that originated from the
same colony were used to inoculate 10 ml of SD medium supplemented with
either 0.2 mM leucine (if high intracellular concentrations
of -IPM were desired) or 4 mM leucine and 2 mM each of valine and isoleucine (if low
-IPM
concentrations were desired). Preparation of cell-free extracts and
determination of
-galactosidase activity were done as described
above (see "Mutagenesis of the AD of Leu3p").
To
facilitate the identification of mutants (suppressors of the Leu3-dd
phenotype), the MR (encompassing residues 172-772) was divided into
three subregions defined by naturally existing restriction sites. The
first subregion (SubRI) extended from the SalI to the
SpeI site (corresponding to residues 172-469); the second
subregion (SubRII) from the SpeI to the NdeI site
(residues 470-607); the third subregion (SubRIII) from the
NdeI to the AvrII site (residues 608-772). The
subregions were subjected to mutagenic PCR (26) separately. The pairs
of primers used for SubRI, SubRII, and SubRIII, respectively, were
5-CCAACAGAAGACATACGGA-3
plus 5
-TTTCCAGCACTTTGGGAGG-3
,
5
-AAGTCAATTGGAGATTAGTC-3
plus 5
-TACCTCCACCTTCCTTTTG-3
, and
5
-GACGTTTAATGCCTCAGTT-3
plus 5
-GTGTCCTTGATGTCTGTAG-3
. The PCR
products (pools of mutated DNA fragments) were digested with the
appropriate restriction enzymes and inserted into pPC62H/86T-LEU3dd that had been cut with either SalI and SpeI
(SubRI), or SpeI and NdeI (SubRII), or
NdeI and AvrII (SubRIII). Thus, any one Leu3p molecule contained only one mutated subregion at most. XK157-3C/pYB1 cells (leu3 null with the LEU2
-
lacZ
reporter) were transformed with ligation solutions containing either
SubRI, SubRII, or SubRIII mutants. The transformed cells were plated on
SD medium (20) and selected for significantly increased growth rates.
Cell-free extracts were prepared and
-galactosidase activities were
measured as described above ("Mutagenesis of the AD of Leu3p").
Mutants with elevated
-galactosidase activities were isolated, and
the appropriate subregion was subjected to DNA sequence analysis.
To determine the phenotype of the MR mutants in the context of a Leu3p molecule with a wild type AD, wild type subregions were replaced with the corresponding mutated subregions by cassette exchange. For example, mutated SubRII's were isolated by cutting mutated pPC62H/86T-LEU3dd molecules with SpeI and NdeI and inserting the SpeI-NdeI fragments into pPC62H/86T-LEU3 that had been cut with the same enzymes. The resulting plasmids contained a mutated SubRII in an otherwise wild type LEU3 gene.
Electrophoretic Mobility Shift Assays and Western BlotsWhole-cell extracts used in electrophoretic mobility shift
assays and Western blots were prepared using the glass bead method (27). Specifically, cells from 10 ml cultures were harvested at an
A600 of about 1, washed once, resuspended in 300 µl of lysis buffer (0.2 M Tris-HCl, pH 8.0, containing
0.4 M [NH4]2SO4, 5 mM MgCl2, 50 µM
ZnSO4, 1 mM EDTA, 20% (v/v) glycerol, 0.1%
(w/v) Nonidet P-40, 3 mM dithiothreitol, 2 mM
benzamidine, 2 mM phenylmethanesulfonyl fluoride, and 2 µM pepstatin), and transferred to a 1.5-ml Eppendorf tube. About 150 µl of treated glass beads were added to the
suspension, and the tube was kept on ice. It was vortexed for 30 s
and then left on ice for at least 1 min. The procedure was repeated six times. The mixture was then centrifuged for 20 min at 4 °C
(Eppendorf table top centrifuge, 14,000 rpm). The supernatant solution
was stored at 80 °C. Protein concentration was determined with the Coomassie Plus Protein Assay Reagent (Pierce).
For electrophoretic mobility shift assays, whole-cell extract was
incubated for 15 min at 30 °C with 25 mM HEPES-NaOH
buffer, pH 7.9, containing 80 mM KCl, 5 mM
MgCl2, 1 mM EDTA, 4 mM
dithiothreitol, 5% (v/v) glycerol, 1 µg of
poly(dI-dC)·poly(dI-dC), 40 µg of bovine serum albumin, 1.4 ng of
32P-5 end-labeled UASLEU-30-mer DNA (9), and
280 ng of non-labeled, non-binding UASLEU-24-mer DNA (9) in
a total volume of 40 µl. The solution was then applied to a
pre-electrophoresed 4% non-denaturing polyacrylamide gel.
Electrophoresis was performed for 2.5 h at 30 mA in buffer
consisting of 90 mM Tris base, 90 mM
H3BO3, and 2 mM EDTA. Gels were
dried and autoradiographed. Western blotting was performed on 15%
polyacrylamide gels containing 0.1% sodium dodecyl sulfate. For
immunoblotting, the enhanced chemiluminescence kit from Amersham was
used, following the supplier's protocol. VP16 AD, VP16-Leu3-WT AD, and
VP16-Leu3dd AD were detected using LA2-3 antibody (anti-Gal4-VP16
rabbit serum, gift from S. Triezenberg, Michigan State University).
Earlier experiments had defined the AD of
Leu3p as being contained within the C-terminal 30 residues (14, 15).
This region is sufficient to cause transcriptional activation not only
in yeast but also in mammalian cells. Leu3p molecules lacking the AD
are inactive. Full-length Leu3p is subject to metabolic modulation, requiring the presence of -IPM to be transcriptionally active (9,
12). To gain a better understanding of the contribution of individual
amino acids of the AD to activation and modulation, we extensively
mutagenized the AD, both by random and site-directed mutagenesis
methods. The results are shown in Table
I. In this table, the permutated sequence
of the AD is followed by two columns that show the activity of a
LEU2-lacZ reporter gene at high and low intracellular
-IPM concentrations; the third column shows the ratio of these
numbers (modulation ratio). Since yeast does not take up
-IPM from
the medium, "low" and "high" levels of
-IPM were established
by supplementing the growth medium either with an excess of branched
chain amino acids (conditions that severely diminish the production of
-IPM (9)) or with a limiting amount of leucine. Under both
conditions, the specific activity of
-galactosidase was close to 10 when Leu3p was absent. This value therefore represents a basal level of
expression of the reporter gene. The long upper part of Table I shows
the effect of single amino acid mutations on the activation and
modulation functions of Leu3p. Twenty of the 26 C-terminal residues
were found to have been mutated at least once. Remarkably, mutations at
16 of the 20 positions had a significant effect on modulation. Mutations causing lower modulation ratios were in the majority and
mapped to both the N-terminal side (positions 861-864, 866, 867, 869)
and the C-terminal side (positions 875, 879, 880, 882-884) of the AD.
In all of these cases, the main reason for lower modulation ratios was
a sharp increase in reporter gene activation at low
-IPM levels.
Given that this effect was produced by mutations in so many different
places, it is very unlikely that it was brought about by improved,
i.e. tighter, binding of
-IPM to Leu3p; more likely, the
effect was due to impaired masking. Most of these mutants also showed a
substantial rise in activation potential at high
-IPM levels (with a
disproportionate increase at low
-IPM levels). The strongest
increase in activation potential was seen with mutant proteins that
also had the lowest modulation ratios (W864A, S866P, S866Y, V869F,
V869A, F882Y, P884
, P884R, and P884A), suggesting that loss of
modulation allowed these molecules to approach their maximal inherent
activation capacity.
|
A significant increase in the modulation ratio was caused by
mutations at positions 872, 874, 883, and 885. Of these, the D872X and D874X mutations (including a seemingly
conservative D872E change) caused a particularly sharp, 5-11-fold
decline of the activation potential at low -IPM levels. The
activation potential at high
-IPM levels also declined but to a
lesser extent (3-5-fold). This decline was not due to instability of
the mutant proteins, as judged by the results from electrophoretic
mobility shift assays (Fig.
1A; see Ref. 23). These
results are consistent with the idea that the mutations at position 872 and 874 prevent Leu3p from assuming an active configuration, by
strengthening the masking interactions. (Another possibility, namely
that the mutations at positions 872 and 874 reduce the affinity for
-IPM is considered unlikely (see "Discussion").) We wondered
whether mutating Asp-872 and Asp-874 simultaneously might amplify the
effect caused by the individual mutations and therefore constructed a
D872N/D874N double mutant by site-directed mutagenesis. Properties of a
strain expressing the D872N/D874N mutant protein (designated Leu3-dd) are shown in Table II. The Leu3-dd
protein had lost essentially all of its activation potential. Again,
the loss of the activation potential was not caused by protein
instability (Fig. 1B). To learn more about the intrinsic
activation potential of the AD of Leu3-dd, we modified Leu3-dd by
deleting the 600 amino acid residues between positions 173 and 774. The
same deletion, when performed on wild type Leu3p (designated
Leu3-
12), had resulted in a highly active, non-modulatable Leu-3
molecule (16). Table II shows that the Leu3-dd-
12 protein also
regained substantial activation potential which in this case amounted
to about one-third of that of wild type Leu3p. This value must be
considered minimal since Leu3-dd-
12 appeared to be somewhat
unstable. Like Leu3-
12, Leu3-dd-
12 had lost essentially all
modulatability. These results strongly suggest that the inability of
full-length Leu3-dd to activate was not caused by an elimination of the
activation function; they are consistent with the idea that the
D872N/D874N mutation caused a severe defect in modulation that keeps
the Leu3-dd AD in a masked configuration (e.g. through
stronger interactions with a complementary surface) and thus prevents
it from interacting with the transcription machinery.
|
Our random mutagenesis of the AD of Leu3p also yielded a number of
nonsense mutations, creating stop codons at various points along the
AD. As shown in the lower part of Table I, deleting the eight
C-terminal residues of Leu3p (mutant 879-886) totally eliminated
the modulation response while the activation potential was fully
retained. This result is consistent with the effect of point mutations
in this region, exemplified by F882Y, P884D, P884R, and P884A.
Shortening the AD further by deleting two or three additional residues
actually stimulated the activation potential. A decrease of the
activation potential to about 25% wild type was noted when the 17 C-terminal residues were deleted (Leu3p-
870-886). Deleting 23 C-terminal residues (
864-886) resulted in an apparently complete
loss of the activation potential. It is likely, however, that
Leu3p-
864-886 retained just enough potential to overcome the
repression of reporter gene expression that is typically seen with
Leu3p molecules that are totally devoid of activation potential. Finally, deleting 26 residues from the C terminus (Leu3p-
861-886) created a protein that did cause repression of reporter gene
expression, indicating that the activation function had been lost.
Again, electrophoretic mobility shift assays showed that low
activation potentials were not due to loss of mutant protein (Fig.
1C). For example, the level of Leu3-
864-886 protein (not
an activator) was at least as high as that of Leu3p-
876-886,
877-886, or
879-886 (all good activators).
The above data show that the region of Leu3p identified as
AD is also intimately involved in modulation. Earlier experiments had
revealed that deletion of part or all of the middle region of Leu3p,
defined as extending from residue 174 to residue 773, also led to a
loss of modulation; by contrast, deletion of a region adjacent to the
AD (residues 774-854) had practically no effect on modulation (15).
Since other experiments, e.g. the recent observation that
masking of the AD is not affected when LEU3 is expressed in
mammalian cells (18), had led to the conclusion that masking probably
does not require extraneous factors, we wondered whether there was
direct interaction between the Leu3p AD and the remainder of the
molecule. To answer this question, we turned to the yeast two-hybrid
system in which the interaction of two proteins, separately fused to a
DNA binding domain and a transcriptional activation domain, results in
the formation of a functional transactivator and subsequent activation
of a reporter gene (28). Since Leu3p was a DNA binding protein itself, we chose the following experimental setup. The two segments of Leu3p to
be tested for interaction, expressed from separate plasmids, consisted
of residues 1-773 and 775-886, respectively. The former contained the
DNA binding domain (DB) plus the middle region (MR) of Leu3p and was
used directly as the DNA binding part of the two-hybrid system; the
latter contained the AD of Leu3p and an apparently functionless
connecting peptide (15). Interaction between the two segments, expected
to occur at low -IPM levels, would by definition create a
silent activator since the AD would be masked. To be able to
recognize interaction, we therefore fused the AD of the herpes simplex
virus protein VP16 (designated VP) to the N terminus of the 775-886
segment. We expected that interaction between the Leu3p AD and the
remainder of Leu3p at low
-IPM levels would recruit VP to the
UASL-containing promoter and activate the reporter gene. At
high
-IPM levels, the two segments would not interact, VP would not
be recruited, and the reporter gene would not be activated. An
important additional consideration was that interaction between the two
separated segments of Leu3p would likely be considerably weaker than
interaction between the same regions in the intact molecule since the
contact between the regions would be diffusion controlled and no longer
directed by the shape of the intact protein. For this reason, we also
included the 775-886 segment of the Leu3-dd mutant protein (containing the Leu3-dd AD) in the analysis. If the interactions leading to AD
masking were indeed stronger for Leu3-dd than for wild type Leu3, we
would expect interaction of the severed Leu3-dd AD with the remainder
of Leu3p also to be stronger. The experimental design is illustrated in
Fig. 2.
All of the above expectations were fulfilled by experimental results.
Fig. 3 shows that a low basal level of
reporter gene expression occurred when the DB-MR segment of Leu3p was
expressed by itself; changing the intracellular -IPM concentration
had no effect (column pair 1). The same result was obtained when the DB-MR segment of Leu3p was co-expressed with the VP16 AD (column pair
2). When the DB-MR segment was co-expressed with the VP-Leu3-WT AD
segment, a weak but statistically significant (2-fold) activation of
the reporter gene was observed at low but not at high
-IPM concentrations (column pair 3). Finally, when the DB-MR segment was
co-expressed with the VP-Leu3-dd AD segment, a strong, approximately 11-fold activation occurred at low (but not at high)
-IPM levels (column pair 4). The effects were specific for the Leu3 ADs since no
activation was seen with VP alone. Specificity was also implied by the
fact that reporter gene activation depended on the
-IPM concentration. That is, the DB-MR segment showed no sign of interacting with the Leu3 ADs when the
-IPM concentration was high; the very same DB-MR segment did interact with the Leu-3 ADs when the
-IPM concentration was low. The
-IPM dependence was as predicted, i.e. activation was seen when the
-IPM level was low, a
condition which should promote AD-MR interaction. The results are
consistent with the idea that the Leu-3 AD interacts with the remainder
of the Leu3p molecule and that this interaction is intensified when aspartate residues 872 and 874 are mutated to asparagines (see also
Fig. 7). Importantly, the results also demonstrate that Leu3-dd is
capable of interacting with
-IPM.
Proof that the constructs used in this experiment were stably expressed
in the cells was obtained from electrophoretic mobility shift assays
and Western blots (Fig. 4,
A-C).
Intragenic Suppression of the Leu3-dd Mutation as a Means to Identify Residues in the Middle Region (MR) of Leu3p That Are Potentially Involved in AD Masking
Cells expressing the Leu3-dd
protein grew very slowly on plates lacking leucine (Fig.
5), probably because of a repressive effect by Leu3-dd on the expression of LEU2 and possibly
other LEU genes. Similar growth behavior and concurrent
in vivo repression of LEU2 expression had earlier
been observed with mutants of Leu3p that carried a stop codon at
position 772 or 812 (9, 29). The slow growth of Leu3-dd-containing
cells provided an opportunity to select for suppressors. We argued that
if the phenotype exhibited by the Leu3-dd cells was indeed caused by
unusually strong interactions between the AD and the MR of Leu3-dd,
second-site mutations should be found that lessen these interactions,
returning the Leu-3 molecule to a wild type-like or constitutively
active mode and thereby leading to increased growth rates. To test this
idea, we performed mutagenic (error-prone) PCR on three MR fragments of
Leu3p that consisted of residues 172-469, 470-607, and 608-772,
respectively. The mutated fragments were inserted into a cloned
LEU3-dd gene by cassette exchange, and plasmids carrying a
mutated LEU3-dd gene were then used to transform
leu3 yeast cells that contained a
LEU2-lacZ reporter gene (9). Fast-growing
transformants were isolated and analyzed for
-galactosidase activity. Mutants of interest, i.e. those that caused
reporter gene activation (at high
-IPM levels) corresponding to at
least 25% that caused by wild type Leu3p, were collected and subjected to DNA sequence analysis. A batch of nine such mutants is shown in
Table III (mutants 1A-9A). Suppression
of the phenotype of the Leu3-dd mutation was evident from the
activation potential of the mutants, which ranged from 29 to 118% wild
type value (Leu3-dd's activation potential is <2% wild type). The
modulation ratios of the suppressors also covered a wide spectrum,
ranging from relatively large values (mutants 1A-4A and 6A-8A) to a
value that was closer to normal (mutant 5A). The behavior of mutants
1A-8A is readily explained by assuming that, to a varying degree, their Leu3 molecules regained the ability to expose their AD in response to
-IPM. The modulation ratio of one mutant (9A) was close to 1. The
phenotype of this mutant (strong activation potential, essentially no
response to
-IPM) was identical to that of cells containing
constitutive Leu3p molecules with a permanently active (unmasked)
conformation. The behavior of all nine mutant proteins is therefore
consistent with a relaxation of tight AD masking interactions present
in the Leu3-dd parent molecule. If the masking interactions in Leu3-dd
proceeded through the same (or very similar) contact regions as in wild
type Leu3p, one would expect wild type protein containing the above
mutations also to show diminished AD-masking capabilities. This was
indeed found to be the case. When the MR mutations present in mutants
1A-9A were introduced into wild type Leu3 (by the same cassette
exchange that was used to introduce them into Leu3-dd), it was found
that all nine mutant proteins (1B-9B, right half of Table III) had
become essentially constitutive (modulation ratios <2). At the same
time, their activation potential was either significantly above or
close to that seen with wild type Leu3p. These results strongly suggest
that the underlying mutations occurred in residue(s) that are important for the masking of the AD. For most of the MR mutants listed in Table
III, identification of individual critical residues is not possible at
this point since the mutants are the result of multiple (double,
triple, or quadruple) residue changes (see legend of Table III). There
is, however, a single mutation (K664E, mutant 1) that points to lysine
in position 664 as a residue potentially involved in the masking
process. The region of Leu3p to which all of the sequenced mutations
map is shown in Fig. 6.
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In this paper, we have taken a first step toward understanding the
mechanism by which Leu3p responds to -IPM. The functional change
brought about by
-IPM is quite dramatic. At low concentrations of
the metabolite or in its absence, Leu3p acts as a repressor, causing a
4- to 5-fold drop in reporter gene expression below the level observed
in cells lacking Leu3p (12, 14). When the intracellular
-IPM
concentration rises above a threshold value, which is probably in the
10th-millimolar range (12, 18), Leu3p becomes a strong activator of
gene expression. This transition is thought to be accompanied by a
conformational change that somehow allows the sole AD of Leu3p, located
near the C terminus, to interact with elements of the transcription
machinery. The form of Leu3p that represses gene expression (by an as
yet unknown mechanism, see Ref. 30) is also the form that assumes a
masked configuration. However, masking and repression are clearly
different processes since repression is seen with relatively small
segments of Leu3p (e.g. the Leu3p-(17-147) peptide, Ref.
14), whereas masking requires much larger and different segments of
Leu3p. How is masking accomplished? In the case of Gal4p, the
best-studied member of this class of proteins, masking is achieved by
specific interaction between Gal4p and the negative regulator Gal80p.
In the presence of galactose, the Gal4p-Gal80p interaction is altered,
and Gal4p's AD becomes available for transcriptional activation (3).
The masking of the Leu3p AD likely proceeds in a different manner. The
main argument against the participation of a Gal80p-like protein in the
masking of Leu3p's AD has come from the observation that tight masking
of Leu3p takes place when the LEU3 gene is expressed in
cultured mammalian cells (18). Masking is reversed and an active form
of Leu3p is generated upon addition of
-IPM to the cell culture
medium. In those experiments, expression of the LEU3 gene
was directed by the human cytomegalovirus major intermediate early
promoter. No other yeast-specific genes were present. Since the leucine
biosynthetic pathway is absent from mammalian cells and such cells do
not normally contain a Leu3p-type regulator, they would not be expected
to contain a specific Leu3p-masking factor either. It is important to
note in this context that expression of LAC9 of Kluyveromyces
lactis (a Gal4p homolog) in mammalian cells produced a protein
that was not masked unless GAL80 was co-expressed (31). The argument
that Leu3p does not require a separate masking factor is further
supported by the observation that, in an in vitro
transcription system using yeast whole-cell extract from
Leu3p-deficient cells, purified Leu3p was unable to out-titrate a
presumptive masking factor and stayed in a masked mode even at
relatively high concentrations, as long as
-IPM was absent (12).
Finally, the demonstration in this paper that the Leu3 AD can directly
interact with the remainder of the protein provides a very strong
argument for the self-contained nature of Leu3p with respect to its
modulation.
If masking of the AD of Leu3p is achieved intramolecularly, the next
question is which parts of the Leu3p molecule are involved in this
process. Recent domain-swap experiments with the
serine/threonine-responsive activator Cha4p of yeast have shown that
the extended DNA binding domain of Leu3p (encompassing residues 1-173)
is not required for modulation by
-IPM.2 Also, deleting a
region adjacent to the AD of Leu3p (residues 774-854) had no effect on
modulation (15). In the present work, we therefore focused on the AD
and the MR of Leu3p as potentially holding the key to the modulation
process.
Our attempt to understand modulation was aided significantly by the
construction of the Leu3-dd mutant. This mutant not only proved useful
in the two-hybrid experiment but also led to the isolation of
modulation-related mutants that map to the MR of Leu3p. It is therefore
important to inquire about the mechanism by which the D872N/D874N
mutation keeps the regulator in a virtually inactive state. A
priori, at least three possibilities to explain the behavior of
Leu3-dd might be considered: (i) the D872N/D874N mutation drastically
reduces the binding of -IPM to Leu3p; (ii) the double mutation does
not affect
-IPM binding but essentially quells a conformational
change that, in wild type Leu3p, is a consequence of
-IPM binding
and eventually leads to the exposure of the AD; or (iii) the double
mutation allows the AD to interact more strongly with the remainder of
the molecule. There are several observations that argue against the
first possibility. First and foremost, Leu3-dd still responds to
changes in the
-IPM concentration; a strong dependence on
-IPM
was clearly evident in the two-hybrid experiment with cleaved Leu3-dd.
Second, if the D872N/D874N mutation had caused severe impairment of
-IPM binding, it would be quite improbable that a relatively large
number of second-site suppressor mutations would be found that would
repair a damaged
-IPM binding pocket. Also, if the mutations leading
to suppression of the Leu3-dd phenotype did so by repairing the
-IPM
binding pocket of Leu3-dd, those same mutations would not be expected
to create generally strong and nearly
-IPM-independent activators
when introduced into wild type Leu3p. Yet that is what is observed
(Table III). Turning to the second possibility, it is also unlikely
that the D872N/D874N mutation interferes with a conformational change
caused by
-IPM. If this were the case, one would expect the
intramolecular masking interactions (which by definition occur
before any
-IPM-induced conformational change could take
place) to be the same with wild type Leu3p and Leu3-dd. Yet these
interactions, as observed in the two-hybrid experiment, are much
stronger with Leu3-dd. We therefore conclude, in accordance with the
third possibility and based on the evidence from the two-hybrid
experiment, that the behavior of Leu3-dd is a consequence of stronger
interactions between the AD and the remainder (very likely the MR) of
Leu3p. In Leu3-dd, these interactions are so strong that the normal
exposure of the AD following
-IPM binding cannot occur. A different
behavior results when the AD of Leu3-dd is severed from the rest of the molecule. The now diffusion-controlled interactions are weaker than
those in the intact Leu3-dd molecule, yet are still strong enough to
resemble those of wild type Leu3p. A schematic representation of
interactions proposed to take place is shown in Fig.
7. The observation that the Leu3-dd
suppressor mutations studied so far not only reverse the behavior of
Leu3-dd but also lead to essentially permanent unmasking of the AD of
wild type Leu3p suggests that residues identified in this way are also
involved in the normal masking process. The question of whether a given
residue participates directly in masking (e.g. by making
contact with the AD) or has an indirect effect (e.g. by
stabilizing a configuration favorable for masking) will be difficult to
answer in the absence of structural information. However, we think that
Lys-664 is a good candidate for direct participation because the K664E
mutation (Table III) causes a change in side chain size and a drastic
change in side chain chemistry, yet the mutated protein is very active,
making it seem unlikely that the mutation caused a gross conformational change.
We now turn to the portion of Leu3p that is presumed to interact with
the MR, i.e. the AD. This region is remarkably sensitive to
mutation with respect to the modulation function, suggesting that
important secondary or tertiary structural configurations are present.
CD spectroscopy has indicated a propensity for -helical structure in
the 859-886-residue region,3
and it is possible that the intactness of helical structure is important for efficient masking. However, drastic effects on modulation are seen both when the effect of a mutation on
-helical stability is
expected to be strong (e.g. F882Y) and when it is expected to be minor (e.g. S866Y). Strong effects on modulation are
also seen when Pro-884 (which is very likely not part of an
-helix) is mutated. This indicates that a need to conserve
-helical
structure per se is not sufficient to explain the
sensitivity of the modulation function to mutation. Another interesting
feature of the AD is the presence of two types of amino acid residues:
those that loosen and those that tighten the masking interactions.
Mutations that appear to loosen the interactions the most are found at
positions Trp-864, Ser-866, Val-869, Phe-882, and Pro-884. It is
reasonable to assume that those same residues facilitate masking in
wild type Leu3p. Residues Asp-872 and Asp-874, which are located in the
center of the AD and which, when mutated, increase masking efficiency,
would not be expected to contribute much to masking in wild type
Leu3p and might even antagonize masking. This pattern of residues that
either favor or oppose (or are neutral toward) masking might be
important for achieving a physiologically desirable balance between
open and closed forms of Leu3p.
In striking contrast to their effect on the modulation function of Leu3p, mutations in the AD have a much smaller effect on the activation potential of the protein. This is most evident from the deletion analysis (Table I). A Leu3p molecule lacking 17 of the 26 residues of the AD still has about 25% the activation potential of full-length Leu3p. It should be noted that the remaining short region still contains three of the original six acidic residues, as well as four hydrophobic residues. In view of the well-known fact that even random (acidic) sequences can bestow transcriptional activation potential upon DNA binding regions to which they are fused (32), the apparent promiscuity of the Leu3p AD does not come as a surprise.
Although this work has provided important evidence supporting intramolecular interactions as a mechanism for masking the AD of Leu3p, we are not yet able to determine whether these interactions occur in cis or in trans. Since Leu3p very likely acts as a dimer (33, 34), masking could in theory be achieved either by intra-monomer or by intra-dimer interactions. Experiments addressing this question are underway.