(Received for publication, September 12, 1995; and in revised form, December 15, 1995)
From the
Putative transcriptional adaptor proteins are found in eukaryotes from yeast to humans and are required for full function of many eukaryotic acidic activators. To study their functional interactions, deletion mutations in the yeast adaptors ADA2, GCN5, and ADA3 were created. We defined a region within the middle of GCN5 required for interaction with ADA2 in vitro. We identified regions of ADA2 required for function in vivo and determined whether these same regions are involved in physical interaction of ADA2 with GCN5 or ADA3 in vitro. Two regions were crucial for ADA2 function in vivo, the amino terminus and a middle region. Immunoprecipitation analysis showed that the amino terminus of ADA2 was required for interaction with GCN5, while a region in the middle of ADA2 was necessary for interaction with ADA3. Deletions of the region that was required for interaction with ADA3 abolished dependence of lexA-ADA2 transcriptional activity on ADA3. Moreover, using coimmunoprecipitation analysis, physical interaction between ADA2, ADA3, and GCN5 was demonstrated in yeast extracts. Taken together, the physical interaction in vivo, along with the correlation observed between regions of ADA2 required for in vitro interaction with GCN5 and ADA3, and regions required for function in vivo, argue for the existence of a physiologically relevant adaptor complex.
Transcriptional activation of RNA polymerase II (RNAPII) ()genes requires the coordinated function of several classes
of proteins (Tjian and Maniatis, 1994). Transcriptional activators, or
activator complexes, bind to specific DNA sequences generally upstream
of the core promoter. A second class of more general factors together
constitute the basal transcription apparatus, which includes RNAPII and
other factors required for initiation and elongation (TFIIA, TFIIB,
TFIID, TFIIE, TFIIF, and TFIIH; for review, see Buratowski(1994) and
Zawel and Reinberg (1993)). Activators induce high levels of
transcription by stimulating the activity of the basal complex, either
increasing the rate of initiation or elongation by RNAPII.
Functional and physical interactions between activators and basal factors require a third class of cofactors, which are not well characterized, but may physically bridge or functionally modify interactions between activators and basal factors (Gill and Tjian, 1992; Lewin, 1990; Ptashne and Gann, 1990; Roeder, 1991). In general these cofactors are required for optimal responses to activators, but their particular mechanisms may be quite varied. These cofactors include coactivators (Gill and Tjian, 1992), adaptors (Berger et al., 1990; Berger et al., 1992), mediators/SRBs (suppressors of RNA polymerase B) (Koleske and Young, 1995), and SWI/SNFs switch/sucrose non-fermenting) (Carlson and Laurent, 1994; Winston and Carlson, 1992). Interestingly, most of these proteins are highly conserved in eukaryotes, since putative homologues have been found from yeast to humans (for example, see Candau et al. (1996), Kwon et al.(1994), and Reese et al. (1994)), suggesting that their roles are fundamentally important in transcriptional activation.
One commonality among these factors is that they may all constitute large multicomponent complexes, perhaps to provide a high degree of flexibility in transcriptional regulation. Thus, the TFIID complex consists of the TATA-binding protein (TBP), associated with accessory factors (TBP-associated factors and coactivators; Dynlacht et al.(1991)), which are necessary for activated transcription (Chen et al., 1994). Mediators (Kim et al., 1994) and SRBs (Koleske and Young, 1994; Koleske et al., 1992) are associated with the carboxyl-terminal tail of the largest subunit of RNAPII and may form a RNAPII holoenzyme complex, which is recruited en masse to promoters during activation. The SWI/SNF complex (Peterson et al., 1994) may be required for activators to overcome chromatin-mediated repression, perhaps by associations with activators (Cote et al., 1994; Kwon et al., 1994) and TBP (Imbalzano et al., 1994).
Finally, putative transcriptional adaptors are required for full function of certain acidic activators. In previous work we identified these cofactors by their biochemical (Berger et al., 1990) and genetic interaction (Berger et al., 1992) with the activation domain of herpes simplex virus VP16. Several genes were cloned, termed ADA2 (Berger et al., 1992), ADA3 (Piña et al., 1993), and GCN5 (Marcus et al., 1994), and mutations in them affected transcription in a manner suggestive of adaptor function. For example, mutations in ADA2 affected transcriptional activation in vivo and in vitro by certain acidic activators including VP16 and yeast GCN4, but did not affect activation by the acidic activator HAP4 (Berger et al., 1992). This functional specificity was reflected in physical interactions between ADA2 and activation domains derived from VP16 (Silverman et al., 1994) and GCN4, but not HAP4 (Barlev et al., 1995). In yeast nuclear extracts, ADA2 both interacted with the TBP component of TFIID and was required for VP16 activation domain interaction with TBP (Barlev et al., 1995). These data suggested that adaptors may be required for physical interaction between activation domains and the basal transcription apparatus.
ADA2, ADA3, and GCN5 interacted in vitro (Horiuchi et al., 1995; Marcus et al., 1994), and activation in vivo by ADA2 fused to the bacterial lexA DNA binding domain (lexA-ADA2) was dependent on GCN5 and ADA3 (Marcus et al., 1994; Silverman et al., 1994). Here we report a primary structure and functional study of the adaptors. We mapped regions of GCN5 necessary for interaction with ADA2. We also defined regions of ADA2 required for interaction with GCN5 and ADA3 in vitro and then analyzed the dependence of these ADA2 mutants on ADA3 for in vivo function. We found parallels in the behavior of these mutants in vitro and in vivo. Moreover, we detected physical interaction between ADA2, ADA3, and GCN5 in yeast extracts. These data indicate that the adaptors function as a complex in vivo.
The ada2 derivative of BJ2168 (mata ura3-52
leu2 trp1 prb1-1122 pep4-3 prc1-407) was
generated by transformation with pyADA2.KO as described (Berger et
al., 1992).
pRS316-lexA-ADA2 was constructed by PCR generation of ADA2 with NotI restriction sites and ligation into pRS316-lexA (Piña et al., 1993) digested with NotI.
GAL4-VP16 expression vectors were described (Berger et al., 1992). The ADA3 expression vector was described (Piña et al., 1993).
For in vitro translation, PCR fragments of ADA3 bearing BamHI restriction sites or ADA2 and GCN5 bearing BglII restriction sites were cloned into SP64 (Pharmacia Biotech Inc.). The three genes were cloned into a yeast expression vector and were functional as assayed for complementation of ada2 or ada3 disruption strains.
SP64-NotI was generated cloning a NotI linker in SP64 digested with SmaI. To generate the ADA2 deletion mutants for in vitro translation, the lexA-ADA2 deletion mutants were digested with NotI and cloned into SP64-NotI.
To generate the deletion mutants of GCN5 for in vitro translation, fragments between residues 1-440, 1-350, 1-250, 1-170, 95-440, 170-440, and 250-440 of GCN5 bearing a NotI restriction site at the 5` end and an EagI restriction site at the 3` end were amplified by PCR. These fragments were digested with EagI and inserted in SP64-NotI opened with NotI.
To generate the two halves of ADA3 for in vitro translation, fragments of ADA3 between residues 1-364 with BamHI restriction sites and 363-702 with a BglII restriction site at the 5` end and a BamHI restriction site at the 3` end were amplified by PCR. These fragments were digested with BamHI or BamHI/BglII and inserted in SP64 digested with BamHI.
pRS426-GAL1-ADA2 and pRS425-GAL1-GCN5 were constructed by PCR generation of ADA2 or GCN5 bearing BglII restriction sites and ligation into the BamHI site of pRS426-GAL1 or pRS425-GAL1 (Mumberg et al., 1994) pRS424-GAL1-HA-ADA3 was constructed in three steps. First, a linker with the sequence encoding the 12CA5 epitope derived from the influenza hemagglutinin protein (Kolodziej and Young, 1991) was inserted into the NotI site of SP64-NotI to generate SP64-HA (only the NotI site downstream of the linker was restored after the ligation); second, ADA3 was PCRed with a NotI restriction site at the 5` end and an EagI restriction site at the 3` end was cloned into the NotI site of SP64-HA to generate SP64-HA-ADA3; finally, SP64-HA-ADA3 was digested with EcoRI and PstI, and HA-ADA3 was cloned into pRS424-GAL1 digested with EcoRI and PstI.
Growth inhibition by
GAL4-VP16 was carried out in BWG1-7A ada2 cotransformed with the ADA2 deletion mutants and
GAL4-VP16 high expression plasmid and plated on SD minimal medium.
-Galactosidase assays (Rose et al., 1988) were carried
out in PSY-17A
ada2
ada3 or in PSY316
ada2 transformed with the plasmids described for each experiment.
-Galactosidase activity was determined as units/mg of protein.
The lacZ reporters used were pLGSD5 (Guarente et al., 1982) for GAL4-VP16 activation, and YEp21 (Brent and Ptashne, 1985) for lexA-ADA2 activation.
50 µl
of whole cell extract prepared as described (Berger et al.,
1992), except without reducing agents, were incubated with 0.2 µl
(to precipitate ADA2 and coprecipitate GCN5) or 2 µl (to
coprecipitate HA-ADA3) of -ADA2 antisera and a 40-µl slurry of
Gammabind Sepharose beads as described for the in vitro immunoprecipitation. Because ADA2 and GCN5 electrophorese
similarly to IgG, the samples used to immunoblot with
-ADA2
antisera were electrophoresed in non-reducing conditions, and the
samples used to immunoblot with
-GCN5 antisera were eluted with 1 M NaCl, 0.2% SDS. Input was 30% of the material used in the
immunoprecipitation reaction. The immunoblot probed with
-GCN5
showing the elutions from the protein G precipitate was exposed for 20
min, and all other immunoblots were exposed for 3 min.
Figure 1:
Schematic of ADA2 deletion
derivatives. Full-length ADA2 comprises amino acids 1-434.
1, deletion of the cys-rich region, a putative
zinc-binding domain;
2, deletion of a region of sequence
similarity to the myb oncogene (Lane et al., 1990);
3, deletion of a region with similarity with the D.
melanogaster PGM gene (Stoffers et al., 1989);
4, deletion of a region of strong negative charge;
5, deletion of a region rich in leucine residues.
6-
17 are a set of 200-base pair
overlapping deletions.
Several assays were used to
determine the effect of the deletions on the function of ADA2. We
showed previously that genetic deletion of ADA2 results in
defective growth on minimal media (Berger et al., 1992). Thus,
as an overall estimate of function, the ability of the ADA2 deletion mutants to complement growth of an ada2 disruption strain was tested. Each deletion mutant was
transformed, and each transformant was restreaked onto minimal media
along with wild type ADA2 or vector alone, as positive and
negative controls for growth ( Fig. 2and Table 1, column
1). Growth of the mutants divided into three groups. Some mutants grew
similar to (ADA2and ADA2
) or indistinguishable from (ADA2
, ADA2
, and ADA2
) wild type. Five mutants
complemented very poorly (ADA2
,
ADA2
,
ADA2
,
ADA2
, and
ADA2
). The remaining mutants gave
intermediate growth phenotypes (ADA2
,
ADA2
,
ADA2
,
ADA2
, and
ADA2
). In summary, the carboxyl end of
the protein was dispensable for growth. Two regions were crucial for
complementation, the amino terminus (
6 and
7) and a region
within the middle of the protein (
12,
13, and
14).
Immunoblot analysis demonstrated that the amount of protein of each
mutant was similar to wild type, except mutant ADA2
,
which was not detected (data not shown), even though the mutant protein
was stable in vitro (see below). The instability of
ADA2
may be significant with regard to the structure
and function of the adaptor complex, as will be discussed below.
Figure 2:
Growth complementation assay in the ada2 disruption strain. Each ADA2 deletion mutant was
transformed into PSY316 ada2, and each transformant was
restreaked onto minimal medium. The dividing line shown in the
figure separates two different experiments. Full-length ADA2 and vector alone were used as positive or negative controls in
each experiment.
In
cells containing wild type ADA2, overproduction of GAL4-VP16
results in a strong inhibition of growth and deletion of ADA2 relieves this toxicity (Berger et al., 1992). The growth
inhibition by GAL4-VP16 was the basis of the genetic selection used to
identify the adaptors ADA2 (Berger et al., 1992), ADA3 (Piña et al., 1993), and GCN5 (Marcus et al., 1994). This assay was used to
assess the effect of the deletion mutations on ADA2 function (Table 1, column 2). The results were largely consistent with the
growth complementation assay, since the mutations that allowed wild
type colonial growth in the first assay (1,
5,
15,
16, and
17) did not reduce the ability of ADA2 to
complement growth inhibition by GAL4-VP16. Similarly, mutations that
resulted in defective growth (
6,
7,
12,
13, and
14), were defective for growth inhibition by GAL4-VP16. The
intermediate group for complementation (mutations
2,
3,
4,
9, and
10) was somewhat more negative in the growth
inhibition assay, particularly
2,
3, and
4. Overall the
results were similar in the assays and pointed to two regions crucial
for ADA2 function, one at the amino terminus and the second in the
middle of the protein.
A third measure of function of the ADA2 mutants is their ability to substitute for wild type ADA2 in transcriptional activation. For example, a total deletion of ADA2 results in a 7-10-fold reduction in activation by
GAL4-VP16 (Berger et al., 1992). Each deletion mutant was
cotransformed into the ada2 disruption strain with a GAL4-VP16
low copy expression plasmid and a -galactosidase reporter plasmid.
The level of GAL4-VP16 activation dependent on each of the ADA2 deletion mutants was largely consistent with the results of growth
complementation and growth inhibition assays (Table 1, column 3),
since the amino terminus (
2,
3,
6, and
7) and a
middle region (
12,
13 and
14) were required for activity
and the carboxyl terminus (
15,
16, and
17) was
dispensable. However, this assay seemed to be more stringent than the
other two, since certain mutations were intermediate in the previous
assays, but negative for GAL4-VP16 activation (
2,
3,
9,
and
10).
In summary all of these assays suggest that regions of ADA2 required for function are within the amino terminus and the middle of the protein. The carboxyl terminus was dispensable in all assays.
To define sequence elements within ADA2
that are required for interaction with GCN5 in vitro, the
overlapping ADA2 deletion mutants (Fig. 1) were tested for their
ability to coimmunoprecipitate GCN5 (Fig. 3). As shown
previously (Marcus et al., 1994), wild type ADA2
co-immunoprecipitated GCN5. Deletion of the amino terminus of ADA2
(6 or
7) abolished interaction with GCN5, while deletions
downstream of
7 did not affect interaction with GCN5. This was
consistent with previous results showing that regions contained within
the first 166 amino acids (i.e. corresponding to the regions
deleted in ADA2
through ADA2
) were
sufficient to interact with GCN5, and the rest of the protein did not
form a complex with GCN5 (Candau et al., 1996).
Figure 3:
Effect of deletions in ADA2 on
coimmunoprecipitation of GCN5. GCN5 was cotranslated with each ADA2
deletion mutant. Cotranslated proteins were immunoprecipitated with
-ADA2 antisera, and
S-labeled proteins were
visualized by autoradiography after 8% SDS-PAGE. in, input; ppt, precipitate. ADA2 proteins and GCN5 are indicated on the left. The migration of ADA2 varies because of the internal
deletions.
To more
finely map the region of ADA2 required for interaction with GCN5, the
series of smaller deletion mutants, ADA2 through
ADA2
, was tested in the same assay (Fig. 3).
Only
1, the Cys-rich region, and
3, the region showing
similarity to PGM, eliminated interaction. This was consistent with the
results of the coarser deletions, since
1 and
3 lie within
the larger deletions
6 and
7, respectively. The other three
small deletions (
2,
4, and
5) did not affect
coprecipitation of GCN5. These results support growth complementation
and other assays (Table 1) and suggest that mutants containing
deletions in the amino terminus of ADA2 (ADA2
,
ADA2
, and ADA2
) fail to complement
growth because, at least in part, they are unable to interact with
GCN5. Mutant ADA2
did not fit this correlation, since
coprecipitation was reduced but the mutant was similar to wild type in vivo, which is discussed below.
To map the region of GCN5 responsible for interaction with ADA2, a series of amino- and carboxyl-terminal deletions were made in GCN5 (Fig. 4A). The protein was divided into five regions, based on sequence conservation with the putative human homologue of GCN5 (hGCN5). Since hGCN5 interacts with both the human and yeast ADA2 homologues (Candau et al., 1996), regions of strong sequence similarity between yGCN5 and hGCN5 may be involved in the GCN5/ADA2 interaction. The region between residues 1 and 95 in the yeast protein has low similarity to the equivalent human region, the regions between 95 and 170 and between 250 and 350 have strong similarity, and the region between 170 and 250 has very strong similarity. Finally, the bromodomain, between 350 and 440, not only has strong similarity between yeast and human GCN5 (Candau et al., 1996), it is conserved with other transcription factors (Georgakopoulos and Thireos, 1992). These yeast GCN5 mutants were then tested for their ability to interact with ADA2, using the coimmunoprecipitation assay (Fig. 4B). Neither the amino terminus (amino acids 1-250) or, as previously observed, the carboxyl-terminal bromo domain (amino acids 350-440) of GCN5 were required for the interaction with ADA2. All mutants containing the region between amino acids 250 and 350 interacted with ADA2, while all mutants deleted in this region did not coimmunoprecipitate with ADA2. Thus, the region between 250 and 350 is crucial for this interaction. In summary, the central region of GCN5 (amino acids 250-350) is necessary for the interaction with two separate regions within the amino terminus of ADA2.
Figure 4:
GCN5 deletion mutants and interaction with
ADA2 in vitro. A, schematic of GCN5 deletion
derivatives. Full-length GCN5 comprises amino acids 1-440.
Deleted versions of GCN5 are composed of residues 1-350,
1-250, 1-170, 95-440, 170-440, and
250-440, which are each shown relative to full-length GCN5. The
percentage of sequence similarity to human GCN5 (Candau et
al., 1996) is indicated above the schematic, and ability to bind
to ADA2 (from data in Fig. 4B) is indicated on the right. B, ADA2 immunoprecipitation of GCN5 deletion
mutants. ADA2 was cotranslated with each GCN5 deletion mutant.
Cotranslated proteins were immunoprecipitated with -ADA2 antisera,
and
S-labeled proteins were visualized by autoradiography
after 15% SDS-PAGE. in, input; ppt, precipitate. Side arrows indicate GCN5 peptides and
ADA2.
Figure 5:
Interaction between ADA2 and ADA3 in
vitro. A, effect of deletions in ADA2 on
coimmunoprecipitation of ADA3. ADA3 and each ADA2 deletion mutant were
cotranslated and immunoprecipitated with -ADA2 antisera.
S-Labeled proteins were visualized by autoradiography
after 8% SDS-PAGE. in, input; ppt, precipitate. Side arrows indicate ADA2 proteins and ADA3. B, ADA2
immunoprecipitation of ADA3 deletion mutants. ADA2 was cotranslated
with each half of ADA3 and immunoprecipitated with
-ADA2 antisera.
S-Labeled proteins were visualized by autoradiography
after 12% SDS-PAGE. in, input; ppt, precipitate. Side arrows indicate ADA3 peptides and
ADA2.
To map the region of ADA3 required for interaction with ADA2, the amino- and carboxyl-terminal halves of ADA3 were translated in vitro and tested for interaction with ADA2 (Fig. 5B). The carboxyl-terminal half of ADA3 was immunoprecipitated by ADA2, as previously observed (Horiuchi et al., 1995), while the amino-terminal half of ADA3 did not interact with ADA2. However, the amino terminus of ADA3 was immunoprecipitated by ADA2, in the presence of the carboxyl terminus of ADA3. This result indicates an intramolecular interaction between the two halves of ADA3 and provides a molecular explanation for the previous observation that the two halves of ADA3 complemented growth when cotransformed, but not when singly transformed (Horiuchi et al., 1995).
In summary, the carboxyl terminus of ADA3 interacts with amino acids 201-301 of ADA2.
Activity
of lexA-ADA2 decreased in an ada3 disruption strain, and
increased when ADA3 was overproduced (Silverman et al.,
1994). To understand the basis of the induction of
lexA-ADA2 by ADA3, we examined transcriptional activity and the amount
of lexA-ADA2 protein in the absence of ADA3, in the presence of
endogenous levels of ADA3 and in the presence of overproduced ADA3. The
lexA-ADA2 protein and activity were nearly undetectable in the ada3 disruption strain, were induced in ADA3
cells, and were induced further in the presence of high levels of
ADA3 (Fig. 6A). In contrast with these effects on
lexA-ADA2, lexA alone did not vary in activity or protein levels with
alterations in the amount of ADA3. Thus, activity measurements
correlated with the amounts of lexA-ADA2 protein, and ADA3 was
necessary for stability of lexA-ADA2.
Figure 6:
Analysis of the interaction between ADA2
and ADA3 in vivo. A, ADA3 induction of
lexA-ADA2 transcriptional activity and protein. Either
lexA-ADA2
(lanes 4-6) or lexA (lanes
1-3) was cotransformed with either a plasmid overexpressing
ADA3 (lanes 3 and 6) or vector alone (lanes
1, 2, 4, and 5). The yeast strains were
either
ada2
ada3 (lanes 1, 3, 4, and 6) or
ada2 (lanes 2 and 5). In all cases the reporter
plasmid contained one lexA binding site upstream of the bacterial lacZ gene. Equal amounts of whole cell extract were tested for
-galactosidase activity (shown at the bottom) and
immunoblotted with
-lexA antisera to determine the amount of lexA
or lexA-ADA2 protein. B, ADA3 induction of the protein level
of lexA-ADA2 deletion mutants. Immunoblot analysis using
-lexA
antisera of whole cell extract from
ada2
ada3 yeast transformed with lexA-ADA2 deletion mutants. Each lexA-ADA2 mutant was cotransformed with vector alone(-)
or vector that overproduced ADA3 (+).
Since both activity and amount
of lexA-ADA2 were dependent on the level of ADA3 protein, we tested the
effect of ADA3 on the lexA-ADA2 deletion mutants. The activity of each
lexA-ADA2 deletion mutant was measured in the absence of ADA3, in the
presence of endogenous levels of ADA3, or in cells overproducing ADA3 (Table 2). The mutants subdivided into three groups, depending
upon the extent of induction by ADA3. This was true whether the
activity of lexA-ADA2 in the presence of overproduced ADA3 was compared
to activity in the presence of endogenous levels of ADA3 or no ADA3 (Table 2), although the effect was more dramatic when
overproduced ADA3 was compared to no ADA3. The induction by ADA3 of
mutants lexA-ADA2 was indistinguishable from
wild type. Mutants lexA-ADA2
were inducible to
a lower extent than wild type. In contrast, mutants
lexA-ADA2
were not inducible by ADA3 at all,
suggesting that these deletions were in a region that interacted with
ADA3.
We then measured the protein level of each lexA-ADA2 mutant
fusion (lexA-ADA2). Equal amounts of protein
from extracts used for activity determination were used for immunoblot
analysis (Fig. 6B). The results paralleled the activity
assay. Mutants that were strongly induced for activity showed strong
induction of protein levels (lexA-ADA2
) and
were indistinguishable from wild type. Mutants that were moderately
inducible for activity showed moderate induction of protein levels
(lexA-ADA2
). The three mutants that were not
inducible for activity (lexA-ADA2
) were also
not inducible for the level of protein. Mutants lexA-ADA2
and lexA-ADA2
were stable even in the absence
of ADA3, unlike wild type lexA-ADA2 or any of the other mutants, and
did not respond to increased levels of ADA3. Mutant
lexA-ADA2
was not stable in the presence of any
amount of ADA3. The lack of protein for lexA-ADA2
was
not caused by an intrinsic instability in the protein, because the
mutant was able to be translated and was stable in vitro ( Fig. 3and 5A). Thus the effects of ADA3 on lexA-ADA2
activity and protein suggest an interaction within the
12,
13, and
14 regions of ADA2. Moreover, these three deletion
mutants, which were independent of ADA3 in vivo, did not
interact with ADA3 in vitro (Fig. 5A) and
failed to complement growth ( Fig. 2and Table 1). The
congruence of the in vivo assays with the in vitro assay suggests that these deletions define a functional
interaction with ADA3. Most important, the consistent results strongly
argue that the physical interaction observed in vitro is
relevant in vivo.
Figure 7:
Immunoprecipitation analysis of the
adaptor complex in vivo. ADA2 was immunoprecipitated using
-ADA2 antisera from whole cell yeast extracts prepared from either
wild type (transformed with plasmids overexpressing ADA2, HA-ADA3, and
GCN5) or
ada2 (transformed with plasmids overexpressing
HA-ADA3 and GCN5) strains. Immunoblots were performed using
-ADA2
antisera (upper panel),
-HA monoclonal antisera (middle panel), to detect ADA3, or
-GCN5 antisera (lower panel). Inputs were 30% of amounts used in
immunoprecipitation. Baculovirus-expressed ADA2 served as a size
marker.
Transcriptional activation mediated by certain acidic activators in yeast appears to require a family of cofactors, which have been termed adaptors (Berger et al., 1992). The function of the putative adaptors may be to promote interaction between activation domains and the basal transcription apparatus (Barlev et al., 1995). Several lines of evidence suggest that the adaptors may function in a complex (Horiuchi et al., 1995; Marcus et al., 1994; Piña et al., 1993; Silverman et al., 1994). In this study we present direct evidence that ADA2 interacts with ADA3 and GCN5 in vivo, using coimmunoprecipitation from extracts prepared from yeast overexpressing the three proteins. We have mapped regions of the adaptors required for the formation of the complex in vitro and have shown that these regions are important for function in vivo.
In
previous work we have identified human genes (hADA2 and hGCN5) whose products are similar in sequence and function to
yeast ADA2 and yeast GCN5 (Candau et al., 1996). The yeast and
human ADA2 proteins contain conserved putative domains, especially at
their amino termini. Also, as was observed for yeast ADA2 and GCN5
(Marcus et al., 1994), human ADA2 and GCN5 interacted in a
two-hybrid assay in vivo, and yeast ADA2 and human GCN5
interacted as well (Candau et al., 1996). These similarities
stimulated a primary structure and functional analysis of yeast ADA2,
to dissect the structural requirements for functional interactions
between ADA2 and GCN5 or ADA3. An extensive deletion mutagenesis was
done through ADA2, and selected regions of possible functional
significance were separately deleted. Analysis of these deletions in vivo indicated that two regions were necessary for ADA2
function, including growth complementation of the ada2 disruption strain and GAL4-VP16 activity. These regions are the
amino terminus (defined by 6 and
7) and the middle of the
protein (defined by
12,
13, and
14).
We tested whether these regions were important for interaction with two other putative adaptors GCN5 and ADA3. Analysis of the interaction between ADA2/GCN5 and ADA2/ADA3 in vitro showed distinct regions were required for ADA2 interaction with each of these proteins.
The region of ADA2 that forms a complex with GCN5 was previously defined by immunoprecipitation and glutathione S-transferase-ADA2 pull-down assay (Candau et al., 1996). The region of ADA2 between 1 and 166 was sufficient for interaction with GCN5 in the coimmunoprecipitation assay, and between 1 and 180 in a glutathione S-transferase-ADA2 interaction assay with purified recombinant GCN5. The region between 167 and 434 did not interact with GCN5 in either assay.
In this study we have used deletions within the amino
terminus of ADA2 to define important subregions for GCN5 interaction in vitro. Deletions that eliminated interaction with GCN5 in vitro (1,
3,
6, and
7) were all in the
amino terminus, and deletion of no other portion of ADA2 reduced the
interaction with GCN5 and thus was consistent with previous data. The
larger deletions (
6 and
7) overlap the smaller deletions
(
1 and
3), and thus it is not possible to determine from
these data whether there are additional small regions in the amino
terminus aside from those within
1 and
3 that contribute to
the interaction with GCN5. However, it is clear that regions within
both
1 and
3 are required for in vitro interaction
with GCN5, since deletion of either region causes the loss of GCN5
immunoprecipitation. ADA2
complemented growth in
vivo, while ADA2
was defective, suggesting
interaction of the PGM region (defined by
3) with GCN5 in
vivo. There may be additional determinants of GCN5 interaction in
the region carboxyl to the Cys-rich region, since
ADA2
, in which the Cys-rich region and additional
residues are deleted, is the single most defective mutant in the in
vivo assays. The region deleted in ADA2
may
contain determinants for GCN5 interaction that are necessary in
vitro but not in vivo. Alternatively, the interaction
between GCN5 and the mutant ADA2
may be stabilized in vivo by other putative adaptors in the complex, such as
ADA5 (Marcus et al., 1994), that are not present in the in
vitro assay.
The regions of ADA2 required for interaction with GCN5 are strongly conserved between the human and yeast ADA2 proteins (Candau et al., 1996). This is interesting in light of the previous observation that yeast ADA2 interacts with human GCN5 (Candau et al., 1996), suggesting conservation of the interacting sequences. There is also conservation of sequence within the Cys-rich region of ADA2 and Cys-rich regions in mammalian CBP and p300 (Arany et al., 1994), two putative adaptors for the cAMP response element-binding protein activator (Lundblad et al., 1995). Since the Cys-rich region is implicated in our experiments as being involved in interaction with GCN5, it is possible that the Cys-rich regions in CBP and p300 have a similar role, in interacting with human GCN5 or with other possible homologues of yeast GCN5. Previously it was found that a 133-amino acid region of CBP, including the ADA2-like Cys-rich region, was sufficient for TFIIB interaction using recombinant proteins (Kwok et al., 1994). We have tested interactions between ADA2 and endogenous TFIIB or TBP in yeast nuclear extracts and have observed interaction with TBP, but not TFIIB (Barlev et al., 1995). Thus, it is possible that the Cys-rich regions in ADA2 and CBP have different functions.
Our deletion analysis of GCN5 suggests that the region necessary for its interaction with ADA2 localizes to the middle of the protein, between amino acids 250 and 350, in a region of GCN5 that shares a high degree of sequence similarity with its human counterpart (Candau et al., 1996). Most interesting is the finding of another region of GCN5 (residues 170-250), having exceedingly high conservation (80% similarity) between human and yeast, that is not necessary for the interaction of GCN5 with ADA2 in vitro. It is possible that this region is responsible for interaction of GCN5 with another transcription factor conserved between human and yeast. We are currently investigating this possibility. This factor is not likely to be ADA3, since GCN5 and ADA3 do not interact directly in vitro (Horiuchi et al., 1995).
We have studied the interaction between ADA2 and ADA3 using
two assays: induction of lexA-ADA2 by ADA3 in vivo, and the
ability of ADA2 to immunoprecipitate ADA3 in vitro. The
results indicate that ADA3 interacts with ADA2 in the regions defined
by 12 and
13 (residues 201-301).
ADA2
, ADA2
, and ADA2
lost the ability to coimmunoprecipitate ADA3. The second half of
the region defined by
14 appears not to contact ADA3, since this
region is also deleted in ADA2
, which
immunoprecipitated ADA3. Our results in vivo are consistent
with these results. It has been shown that lexA-ADA2 activity is
dependent on ADA3, and we show here that lexA-ADA2 is unstable in the
absence of ADA3. Mutant lexA-ADA2
has no activity and
is unstable in the presence or absence of ADA3. Deletion of flanking
regions (
12 or
14) results in lexA fusion proteins that are
stable independent of ADA3 expression and whose activity is not
inducible by ADA3. Mutant lexA-ADA2
and all other mutants
are unstable in the absence of ADA3 and have protein that is induced in
amount and activity proportionately to increasing ADA3. Thus only
deletions
12,
13, and
14 result in lexA fusion proteins
that are not inducible by ADA3 in vivo, and this is the only
region required for in vitro interaction with ADA3. Thus,
overall, the consistency in ADA2 mutant behavior between the in
vitro immunoprecipitation and the in vivo induction
assays suggests that the interaction observed in vitro between
ADA2 and ADA3 also occurs in vivo. Also, we previously
observed a correlation between in vitro and in vivo assays in interactions between ADA2 and GCN5. The amino-terminal
166 amino acids of ADA2, but not the remainder of ADA2, interacted with
GCN5 in vitro, and lexA-ADA2(1-166) was stabilized by
GCN5 in vivo, but GCN5 had no effect on the remainder of ADA2
(Candau et al., 1996).
One manifestation of ADA3
interaction with, and stabilization of, ADA2 is that ADA3 appears to
induce the activity of lexA-ADA2. We have observed a similar
stabilizing effect of ADA3 on endogenous ADA2. ()Thus the
level of activity of lexA-ADA2 is proportionate to its concentration,
which is directly related to the concentration of ADA3. This also
suggests that the amount of lexA-ADA2 in the complex limits the overall
activity. This may also be true in the actual activation complex,
during interactions with activation domains.
Mutants
lexA-ADA2 and lexA-ADA2
were
constitutively stable in the presence or absence of ADA3;
lexA-ADA2
was unstable in the presence or absence of
ADA3; lexA-ADA2
and the remaining mutants were stable in
the presence of ADA3 and unstable in its absence. Our interpretation of
these data is that the regions flanking the interaction with ADA3 are
particularly sensitive to proteolysis, and that these regions may be
protected from degradation when ADA3 is present and interacting with
the region defined by
13. This hypothesis explains the
constitutive stability of lexA-ADA2
and
lexA-ADA2
, since each lacks one of the
proteolysis-sensitive regions, and also explains the constitutive
instability of lexA-ADA2
, since it has both
proteolysis-sensitive regions and cannot interact with ADA3.
Consistent with previous results (Horiuchi et al., 1995), we found that ADA2 interacted with the carboxyl terminus of ADA3, but not the amino terminus. However, when we simultaneously translated ADA2 and both halves of ADA3, ADA2 precipitated both the carboxyl terminus, as expected, and the amino terminus of ADA3. This result shows a physical interaction between the two halves of ADA3. Previous studies of the ability of the two halves of ADA3 to complement growth of a ada3 disruption strain showed that, although the two halves of ADA3 were not able to complement growth when transformed independently, they did complement when they were cotransformed (Horiuchi et al., 1995). Here we provide a molecular explanation for this observation, since the two halves of ADA3 apparently interacted with each other to reconstitute the full-length protein.
ADA2 was defective in all assays in vivo, but was able to
interact with both ADA3 and GCN5 in vitro. We have previously
shown that ADA2 interacts directly with GAL4-VP16 and indirectly with
TBP (Barlev et al., 1995). This mutant is likely to interact
with GAL4-VP16 because lexA-ADA2
is inactive (we would
expect a level of activity similar to wild type in the lexA fusion of
mutants unable to interact with activators). Thus, it is possible that
ADA2
has lost the ability to interact with TBP.
In summary, we have demonstrated physical interaction between the adaptor proteins in vivo. Interactions among the three adaptors, ADA2, GCN5, and ADA3, were mapped to define sequence elements necessary for their binding. The region of ADA2 necessary for interaction with ADA3 in vitro was required for functional interaction in vivo. These data indicate physiological relevance of the adaptor complex.