Subunit Communications Crucial for the Functional
Integrity of the Yeast RNA Polymerase II Elongator (
-Toxin
Target (TOT)) Complex*
Frank
Frohloff,
Daniel
Jablonowski,
Lars
Fichtner, and
Raffael
Schaffrath
From the Institut für Genetik, Biologicum,
Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 10, D-06120 Halle (Saale), Germany
Received for publication, October 1, 2002, and in revised form, November 4, 2002
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ABSTRACT |
In response to the Kluyveromyces
lactis zymocin, the
-toxin target (TOT) function of the
Saccharomyces cerevisiae RNA polymerase II (pol II)
Elongator complex prevents sensitive strains from cell cycle
progression. Studying Elongator subunit communications, Tot1p
(Elp1p), the yeast homologue of human IKK-associated protein, was found to be essentially involved in maintaining the structural integrity of Elongator. Thus, the ability of Tot2p (Elp2p) to interact
with the HAT subunit Tot3p (Elp3p) of Elongator and with subunit Tot5p
(Elp5p) is dependent on Tot1p (Elp1p). Also, the association of
core-Elongator (Tot1-3p/Elp1-3p) with HAP (Elp4-6p/Tot5-7p), the
second three-subunit subcomplex of Elongator, was found to be sensitive
to loss of TOT1 (ELP1) gene function.
Structural integrity of the HAP complex itself requires the
ELP4/TOT7, ELP5/TOT5, and ELP6/TOT6 genes, and
elp6
/tot6
as well as
elp4
/tot7
cells can no longer promote
interaction between Tot5p (Elp5p) and Tot2p (Elp2p). The association
between Elongator and Tot4p (Kti12p), a factor that may modulate the
TOT activity of Elongator, requires Tot1-3p (Elp1-3p) and Tot5p
(Elp5p), indicating that this contact requires a preassembled
holo-Elongator complex. Tot4p also binds pol II hyperphosphorylated at
its C-terminal domain Ser5 raising the possibility that
Tot4p bridges the contact between Elongator and pol II.
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INTRODUCTION |
Microbial rivalry between Kluyveromyces lactis killer
strains and sensitive Saccharomyces cerevisiae cells relies
on secretion of zymocin, a heterotrimeric (

) protein toxin
complex that acts as a cell cycle blocker in G1 (1-3).
Zymocin docking involves interaction of its
-subunit, an
exo-chitinase, with S. cerevisiae cell wall chitin (4, 5),
and anti-zymotic activity resides within the
-subunit, also termed
-toxin (6, 7). In an effort to identify the intracellular
-toxin target
(TOT)1 process, seven
TOT genes were found to abrogate toxicity when mutated (8,
9). TOT1-3 and TOT5-7 are identical with
ELP1-6 coding for Elongator, an RNA polymerase II (pol II)-
associated histone acetyltransferase (HAT) complex (8-15). In
addition, loss of KTI11, KTI13, and
SIT4 results in tot phenotypes characteristic for
TOT mutants, implying that these genes may also be linked to Elongator
function (16-18). Tot4p (Kti12p) can be found promoter-associated, and
it contacts Elongator and pol II (8, 19, 20). Since both removal and
overproduction of Tot4p induce zymocin resistance, Tot4p is likely to
influence Elongator by modulating its TOT activity (16, 20).
Holo-Elongator contains the core complex, Elp1-3p (Tot1-3p), and HAP,
a second heterotrimer composed of Elp4-6p (HapI-3p/Tot5-7p) (8, 9,
13-15). Elp1p is the largest subunit within core-Elongator and
homologous to human IKK-associated protein, an I
B kinase
scaffold protein and a member of a five-subunit protein complex (21,
22). Elp2p is a WD40 protein homologous to murine STAT3-interacting
protein StIP1 (12, 23, 24), and Elp3p is the HAT subunit (11, 25) whose
activity requires the HAP complex (26). Consistent with a role in
transcription, Elongator facilitates pol II activity through chromatin
and stably associates with hyperphosphorylated pol II (II0) (10, 27). Its HAT activity is essential for Elongator to function as TOT and
HAT-minus scenarios yield zymocin resistance (8, 14). Together with the
finding that the TOT function can be dissociated from Elongator
by mutagenesis of its HAT gene without affecting other Elongator
properties (9), the HAT function of Elongator plays a key role in
mediating zymocicity. As judged from the observations that (i) pol
II-driven transcription is down-regulated in zymocin-treated cells (8,
9), (ii) pol II underassembly and general pol II defects elicit
zymocin-hypersensitivity (9, 28), (iii) interfering with pol II
C-terminal domain (CTD) modification alters the response of a cell to
zymocin (9), and (iv) the phospho-states of pol II are imbalanced in
zymocin-treated cells (29), zymocin may work by hijacking the TOT
function of Elongator to convert it into a global pol II inhibitor.
Genetic analyses have shown that deletion of any one of the
ELP/TOT genes phenocopies the full range of
elp/tot phenotypes induced by elp3
point mutations that drastically reduce the HAT activity of Elongator
(8, 12, 25). Thus, it has been speculated that the functional integrity of Elongator is compromised in these deletants, leading to
non-productive Elongator HAT scenarios (12, 25).
To study TOT/Elongator function further, we analyzed subunit
communications within the complex by co-immune precipitation (co-ip).
We found Elongator subunit Tot1p (Elp1p) to be essential for Tot2-Tot3
(Elp2-Elp3) and Tot2-Tot5 (Elp2-Elp5) protein-protein interactions.
Also, the association of core-Elongator (Tot1-3p/Elp1-3p) with the
HAP complex was dependent on Tot1p (Elp1p). The interaction between
Elongator and Tot4p (Kti12p) required Tot1-3p (Elp1-3p) and Tot5p
(Elp5p), suggesting that Tot4p contacts Elongator as a preassembled
holo-complex. Tot4p also bound pol II hyperphosphorylated at its CTD
Ser5 position, implying that it may bridge Elongator and
pol II.
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EXPERIMENTAL PROCEDURES |
Strains, Media, General DNA Techniques, and K. lactis Zymocin
Methods--
All yeast strains used are listed in Table
I. Routine yeast growth media, YPD (1%
yeast extract, 2% peptone, 2% dextrose) and synthetic dextrose (SD)
medium (0.67% yeast nitrogen base, 2% dextrose), were as
described by Sherman (30). Yeast DNA transformation utilized the
lithium acetate protocol of Gietz et al. (31). Construction
of TOT2 3'-end deletions involved PCR amplification of
TOT2 open reading frame fragments (promoter primer, 5'-CGA GCA TCG CGG AAA CGC GAT TTA AGA-3'; TOT2 deletion primers:
1, 5'-CTA AGC TGG CTC CTT TTG GTG CCT CCA TAC-3', and
2, 5'-CTA ATC TTC CAT ATT TCT TTC CCA AAG CGC-3') using plasmid template pFF10, a
multicopy YEplac195 derivative carrying wild-type TOT2 (8,
32). After subcloning these deletions into pCR2.1-TOPO (Invitrogen),
they were moved, together with wild-type, full-length TOT2,
into single-copy YCplac33 (32) using SalI/SacI.
The resultant plasmids (pTOT2
1-2) were tested with
wild-type TOT2 (pTOT2) for genetic
complementation of tot2
-associated defects by
transforming strain FFY3/4-dt-2d (Table I). Zymocin sensitivity
of individual S. cerevisiae mutants was tested using killer
eclipse or zymocin YPD plate assays (5, 33). Testing sensitivity to
-toxin expression involved transformation with pHMS14 and glucose to galactose shift assays as described (8).
Gene Disruptions, Epitope-Tagging, and Immunological
Techniques--
Individual TOT gene deletions were created
in vivo by one-step PCR-mediated gene replacement using
plasmid template tools as described (8, 9). (HA)6 and
(c-Myc)3 epitopes were fused to Tot (Elp) gene products
using PCR-based one step in vivo tagging (34) as described
previously (8). Similarly, chromosomally encoded truncation variants of
Tot2p were C-terminally (HA)6 epitope-tagged using PCR from
template plasmid pYM3 (34) and S2-TOT2 (8) and S3 primers
(S3-TOT2
1, 5'-CGT CCA GGG ATA AAA CTG TCA AAG TAT GGA GGC
ACC AAA AGG AGC CAG CTC GTA CGC TGC AGG TCG AC-3'; S3-TOT2
2, 5'-GTG TTT GTA GAG ACA GAA AAT GGG CGC TTT GGG
AAA GAA ATA TGG AAG ATC GTA CGC TGC AGG TCG AC-3'). Detection of tagged proteins involved anti-c-Myc antibody 9E10 (Roche Molecular
Biochemicals) and anti-HA antibody 3F10 (Roche Molecular Biochemicals)
as described (8). Antibody cross-linking to protein A-Sepharose,
preparation of protein extract, and co-ip were carried out as described
(35). RNA pol II form II0 was distinguished from IIA using antibodies H14, H5, and 8WG16 (Covance) as well as anti-CTD-P (36), a kind gift from Dr. David Bentley (University of Colorado Health Science Center, Denver, CO). Protein-protein cross-linking involved yeast grown
to A600 ~1.0 followed by treatment with 1%
(v/v) formaldehyde for 15 min.
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RESULTS |
To analyze subunit communication within TOT/Elongator, we
constructed strains in which one subunit gene was deleted and different subunits were c-Myc- and HA epitope-tagged. Co-ip was then done in an
effort to study protein-protein interaction. As illustrated in Fig.
1A, tot6
and
tot7
cells were found to lose the ability of Tot5p
(Elp5p) to associate with core-Elongator subunit Tot2p (Elp2p). Thus,
TOT6 (ELP6) and TOT7 (ELP4)
influence the structural integrity of the HAP complex, and deletion of
either gene causes it to lose its capability to interact with
core-Elongator. The fact that protein-protein interaction between the
two three-subunit entities also becomes distorted upon deletion of
TOT1 (ELP1) (Fig. 1A) can be regarded
as evidence that Tot1p (Elp1p) is crucial in mediating this
inter-complex communication. Consistently, the ability to co-ip Tot5p
(Elp5p) by the HAT subunit, Tot3p (Elp3p), was lost when Tot1p (Elp1p)
was removed (not shown). Together with the fact that Tot5-Tot3
(Elp5-Elp3) protein-protein interaction is not compromised by
TOT2 (ELP2) deletion (19), this indicates that
the HAP complex communicates with core-Elongator mainly by virtue of
Tot1p (Elp1p)-mediated contact(s). Tot4-Tot5 protein-protein interaction, however, does require TOT2 (ELP2)
and again TOT6 (ELP6) function (Fig.
1B). As for the contact between Tot4p and core-Elongator,
this may indicate that Tot2p (Elp2p) directly communicates with Tot4p,
and indeed, if Tot2p is missing or C-terminally truncated, Tot4p can no
longer associate with the HAT subunit Tot3p (Elp3p) of Elongator (see
below). Thus, both Tot4p and the HAP complex contact core-Elongator in
a different manner that is likely to happen in parallel and not to be
mutually exclusive.

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Fig. 1.
Dependence of Elongator
subunit interactions on TOT1, TOT2,
TOT6, and TOT7 gene function. As
shown in A, Tot2-Tot5 protein-protein interaction depends on
TOT1, TOT6, and TOT7. wt,
wild type. As shown in B, Tot4-Tot5 protein-protein
interaction depends on TOT2 and TOT6. Protein
extracts obtained from the indicated strains were subjected to co-ip
using the anti-c-Myc antibody 9E10. The immune precipitates were
probed with anti-HA antibody to detect Tot5p (A and
B) and with anti-c-Myc antibody to detect Tot2p
(A) and Tot4p (B). Protein positions are
indicated by arrows. MSM denotes molecular size
markers (BenchmarkTM protein ladder, Invitrogen) in kilodaltons.
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The yeast Elongator subunit Tot1p (Elp1p) has been shown to be related
to IKAP, a scaffold protein that associates with I
B kinase and that
is the largest subunit of the human Elongator complex (21, 24). To
assess its role in mediating the structural integrity of
Elongator, we required data concerning individual Elongator subunit
interactions in the presence and absence of TOT1
(ELP1). Thus, the ability of Elongator subunit Tot2p (Elp2p) to co-ip the HAT subunit Tot3p (Elp3p) of Elongator was fully dependent
on the presence of a functional TOT1 (ELP1) gene
(Fig. 2A). Similarly,
Elongator-associated factor Tot4p required Tot1p (Elp1p) to interact
with the HAP component Tot5p (Elp5p) (Fig. 2B). Together
with our previous findings that the interactions between Tot1p (Elp1p)
and Tot3p (Elp3p) as well as Tot5p (Elp5p) and Tot3p (Elp3p) are
insensitive to Tot2p (Elp2p) (19), our observations suggest that Tot1p
(Elp1p) may play a role as a scaffold to which Tot3p (Elp3p) and Tot2p
(Elp2p) assemble onto to form core-Elongator. Moreover, Tot1p (Elp1p)
can be considered to mediate the contact between core-Elongator and the
HAP complex since this inter-subcomplex communication is lost in the
absence of Tot1p (Elp1p). As for the capability of Tot3p (Elp3p) to
co-ip Tot4p, both Tot1p (Elp1p) and Tot2p (Elp2p) are required
(Fig. 3). Together with the
surprising observation that this also holds true for a functional
TOT5 (ELP5) gene (Fig. 3), these data strongly
indicate that the association of Tot4p with Elongator requires a
completely assembled six-subunit holo-complex.

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Fig. 2.
Elongator subunit Tot1p mediates Tot2-Tot3
and Tot4-Tot5 protein-protein interactions. Tot2-Tot3
(A) and Tot4-Tot5 (B) protein-protein
interactions require a functional TOT1 gene. Protein
extracts obtained from the indicated strains were subjected to co-ip
using the anti-c-Myc antibody 9E10. The immune precipitates were probed
with anti-HA antibody to detect Tot3p (A) and Tot5p
(B) and with anti-c-Myc antibody to detect Tot2p
(A) and Tot4p (B). Their positions are indicated
by arrows. MSM denotes molecular size markers
(see the legend for Fig. 1).
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Fig. 3.
The association of Tot4p and the HAT subunit
Tot3p of Elongator depends on TOT1,
TOT2, and TOT5. Protein extracts
obtained from the indicated strains were subjected to co-ip using the
anti-c-Myc antibody 9E10. The immune precipitates were probed with
anti-HA antibody to detect Tot4p and with anti-c-Myc antibody to detect
Tot3p. Protein positions are indicated by arrows.
MSM denotes molecular size markers (see the legend for Fig.
1). wt, wild type.
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Tot2p (Elp2p) contains eight WD40 domains and was hypothesized to serve
the structural integrity of Elongator (12). Intriguingly, however,
TOT2 (ELP2) deletion does not compromise
Tot1-Tot3 (Elp1-Elp3) and Tot3-Tot5 (Elp3-Elp5) protein-protein
interaction, indicating that communication between both of these
core-Elongator subunits and a representative HAP component is
insensitive to Tot2p (15, 19). As for the established role
TOT2 (ELP2) plays in the association of Tot4p
with Elongator (19), we tested episomal TOT2
(ELP2) deletion alleles coding for C-terminally truncated
variants (Fig. 4A,
pTOT2
1-2) for their abilities to mediate Tot4-Tot3
protein-protein interaction by co-ip. As illustrated in Fig.
4C, none of these truncations lacking up to one WD40 domain
was able to restore Tot4-Tot3 protein-protein interaction in a
tot2
(elp2
) background, whereas full-length
episomal TOT2 (ELP2) mediated association of
Tot4p with Tot3p (Elp3p) in a way indistinguishable from chromosomally encoded wild-type TOT2 (ELP2) (Fig.
4C). Anti-HA Western analysis of protein extracts obtained
from yeast strains expressing the truncations as HA-tagged proteins
revealed that the Tot2p (Elp2p) variants were synthesized (Fig.
4D), suggesting that their incapability in mediating co-ip
between Tot4p and Tot3p (Elp3p) in tot2
(elp2
) cells was not due to protein instability or lack
of protein synthesis. Thus, the function of Tot2p in mediating
association of Tot4p with Tot3p (and core-Elongator) resides in
its extreme C terminus. Consistently, the appropriate truncation
alleles conferred zymocin resistance in killer eclipse assays,
suggesting that this region plays a role in TOT function and K. lactis zymocicity, too (Fig. 4B). Taken together, these
data indicate that Tot2p (Elp2p) mediates at least in part the contact
between Tot4p and Elongator. However, for inter-complex communication
and interaction between Tot1p (Elp1p) and Tot3p (Elp3p), it appears to
be dispensable (15, 19). As for the zymocin mode of action, which is
abrogated in the absence of the C terminus of Tot2p, our findings
indicate that zymocin requires Tot4p to be associated with Elongator,
providing further evidence that Tot4p influences the TOT function of
Elongator (19, 20).

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Fig. 4.
Effect of progressive C-terminal Tot2p
truncations on zymocin sensitivity and Tot3-Tot4 protein-protein
interactions. In A, C-terminal deletions are shown on
the horizontal axis, and each plasmid-encoded allele is indicated
(e.g. pTOT2 1). Numbers denote amino
acid residues being deleted ( ). Black boxes indicate the
presence of eight (numbered) WD40 motifs. B,
killer eclipse assay of wild-type TOT2 (pTOT2),
empty vector (YCplac33), and the episomal TOT2 deletions
(pTOT2 1-2) in a zymocin-resistant tot2
background. Inability of the TOT2 truncations to complement
zymocin resistance is indicated by a symbol, whereas
complementation by wild-type pTOT2 is denoted with a + symbol. As shown in C, co-ip of Tot3p and Tot4p requires
TOT2 function. Protein extracts obtained from the indicated
strains were subjected to co-ip using the anti-c-Myc antibody 9E10. The
immunoprecipitates were probed with the anti-HA antibody to detect
Tot4p and with the anti-c-Myc antibody to detect Tot3p. Their positions
are indicated by arrows. D, synthesis of the
C-terminal Tot2p truncations. Protein extracts or immune precipitate
(ip) of the indicated strains were subjected to Western
analysis using the anti-HA antibody. The positions of wild-type Tot2p
and the two truncations are shown by arrows. MSM
denotes molecular size markers (see the legend for Fig. 1).
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Consistent with copurification of Elongator and pol II0 (10, 24),
c-Myc-tagged Tot2p (Elp2p) and, to a lesser extent, c-Myc-tagged Tot5p
(Elp5p), were able to associate with pol II form II0 using co-ip (Fig.
5A). Similarly, c-Myc-tagged
Tot4p interacted with pol II form II0 (Fig. 5A). Remarkably,
as judged from utilizing different anti-pol II antibodies, the
interaction between Tot4p and pol II was solely restricted to form II0
hyperphosphorylated at Ser5 within the CTD repeat (Fig.
5B). In contrast, neither pol II form IIA
(hypophosphorylated on its CTD) nor pol II form II0
(hyperphosphorylated at the Ser2 of CTD) were
co-precipitable with HA-tagged Tot4p (Fig. 5B). Also, lack
of interaction could not be overridden by subjecting yeast cells to
cross-linking prior to protein extraction and co-ip to amplify weaker
interactions (Fig. 5B). Thus, Tot4p interacts with pol II
form II0 hyperphosphorylated at the Ser5 of CTD, a
modification that is transcriptionally characteristic for a
post-initiation event and for pol II0 engaged in promoter clearance
and/or early transcript elongation (37-39).

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Fig. 5.
Elongator and Tot4p associate with pol II
hyperphosphorylated within the CTD repeat. A, co-ip
between pol II's largest subunit Rpb1p and Tot2p, Tot4p, and Tot5p.
Protein extracts obtained from the indicated strains were subjected to
co-ip using the anti-c-Myc antibody 9E10. The immunoprecipitates were
subjected to 10% SDS-PAGE and immunoprobed with 9E10 to detect Tot2p,
Tot4p, and Tot5p and with the anti-CTD-S5-P antibody H14 to detect pol
II form II0. The positions of Tot2p, Tot4p, and Tot5p and pol II's
largest subunit Rpb1p are indicated by arrows.
MSM denotes molecular size markers. wt, wild
type. B, co-ip between Tot4p and pol II form II0. Extracts
from TOT4-(HA)6-expressing
cells grown in the absence ( ) or presence (+) of formaldehyde to
induce cross-linking (cl) were subjected to co-ip using the
anti-HA antibody 3F10. The immunoprecipitates were subjected to 6%
SDS-PAGE and immunoprobed with anti-CTD (8WG16),
anti-CTD-S5-P (H14), anti-CTD-P (Bentley laboratory), and
anti-CTD-S2-P (H5) antibodies to detect both
hyperphosphorylated and hypophosphorylated pol II forms. ip,
immune precipitate.
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DISCUSSION |
Taken together, our findings can be hypothetically incorporated in
a working model (Fig. 6) in which the
assembly of holo-Elongator requires the association of preformed
core-Elongator with the HAP complex. This inter-complex communication
largely relies on Tot1p (Elp1p), which may serve as a scaffold protein.
Fully assembled holo-Elongator is capable to contact Tot4p (Fig. 6), an
Elongator-associated factor that is not a structural subunit but rather
transiently contacts Elongator, presumably to promote its interaction
with elongation-competent pol II0. Consistent with this, Tot4p
associates both with core-Elongator subunits and HAP components (8) as well as with pol II form II0 hyperphosphorylated at the
Ser5 of CTD. Since this modification occurs after
preinitiation complex formation (37-39) and since Tot4p is able to
occupy the promoter rather than the coding sequence of the
ADH1 gene in chromatin immune precipitations (19), Tot4p may
be recruited to and communicate with pol II form II0 engaged in
promoter escape and/or early transcript elongation (39). One
interpretation of such a scenario includes the conclusion that Tot4p
mediates the association of Elongator with pol II to yield
HAT-productive holo-enzymes ready for efficient transcript elongation.
Consistent with such a role for Tot4p as a loading factor, its removal
yields tot/elp phenotypes indistinguishable from
TOT/Elongator mutants (8, 9). Moreover, multicopy TOT4 induces zymocin resistance and intermediate tot phenotypes,
indicating that excess Tot4p levels affect Elongator function, too (8, 16, 20). According to the identification of a putative ATP/GTP binding
P-loop motif in the N terminus of Tot4p that is necessary for protein
function, Tot4p may be a G-protein (20). Together with the fact that a
deletion of KTI13 (ATS1) encoding a putative GTP
exchange factor (40) results in zymocin resistance and tot phenotype expression (18) that can be suppressed by multicopy TOT4 (16), excess Tot4p levels may bypass the requirement
for this GTP exchange factor under normal conditions (20). Thus, the
role of Tot4p in Elongator function is likely to be regulatory. Provided that Tot4p was a key component of a distinct complex that
associates both with Elongator and pol II, loss of TOT4
function would impair complex formation, suppress Elongator loading,
and ultimately result in a HAT-minus scenario and zymocin resistance. Alternatively, excess Tot4p levels due to multicopy TOT4
might perturb normal complex stoichiometry by separately titrating the other subunits and thereby reducing the amount of completely assembled Tot4p complex able to interact with Elongator and pol II. In
favor of this model, tandem affinity purification-tagged Tot4p has been recently reported to be a constituent of a protein complex that is
distinct from Elongator but whose identity awaits further analysis (15). If Tot4p was able to regulate TOT by loading Elongator onto pol
II (Fig. 6), one would assume that the underlying contact between Tot4p
and Elongator involved interaction with a preassembled complex rather
than individual Elongator subunits. Consistently, TOT4
deletion does not affect the structural integrity of Elongator (20),
and the capability of Tot4p to interact with Elongator requires
TOT1 (ELP1), TOT2 (ELP2),
TOT3 (ELP3), and TOT5
(ELP5) gene functions (Figs. 1-4) (Ref. 19). Thus,
disruption of TOT1 (ELP1) abolishes the ability
to co-ip Tot4p by Tot2p (Elp2p), tot3
(elp3
) cells lack Tot2-Tot4 protein-protein interaction, deletion of TOT2 (ELP2) no longer admits Tot4p to
associate with Tot3p (Elp3p), and tot5
(elp5
) cells do not allow Tot4p to interact with Tot3p
(Elp3p). These data suggest that association of Tot4p with Elongator
requires not only more than one subunit but a preassembled holo-Elongator complex. It will be interesting to determine
whether the inability of mutant Elongator to interact with Tot4p
prevented it from being associated with pol II0. If this holds true,
Tot4p may as well be envisaged to be instrumental in bridging Elongator and pol II, a scenario necessary for Tot4p to act as a putative Elongator loading factor. In contrast to a previous report (13), which
considers the HAP complex to be preferentially associated with pol
II-free rather than pol II-bound core-Elongator, the HAP component
Tot5p (Elp5p) was found to co-ip pol II (II0), albeit to a lesser
extent than the core-Elongator subunit Tot2p (Elp2p). Also, our model
(Fig. 6) predicts that a functionally productive Elongator HAT complex
requires the association of the six-subunit holo-Elongator with
elongating pol II. The recent demonstration that all three HAP subunits
Elp4-6p (Tot5-7p) are indeed required for Elongator HAT activity
in vivo supports the model and indicates an essential role
for Elp4-6p in either structural organization or substrate recognition
of holo-Elongator (26). Intriguingly, both Elp4p and Elp6p were
recently suggested to be ATPase homologues, implying putative roles,
for instance in ATP-dependent chromatin remodeling (41).
Consistent with results showing that the majority of all human
core-Elongator subunits are located in the cytoplasm (24, 27), yeast
core-Elongator has also been reported to localize primarily to the
cytoplasm (19, 42). Together with the finding that Elongator subunits
did not appear to occupy promoters or coding regions of genes in
chromatin immune precipitations, whereas other transcriptionally
relevant elongation factors clearly did, the likelihood that Elongator
is at all recruited to transcribed genes as part of the pol II
elongation apparatus has been questioned (42, 43). Nevertheless, these
data could not eliminate the possibility that Elongator may enter the
nucleus to become engaged in transcription. The finding that StIP1, the
murine homologue of yeast Elp2p (Tot2p) and interactor of STAT3, is
located in the cytoplasm and becomes translocated into the nucleus upon
IL-6 treatment (23) supports this notion and indicates that murine Elongator is subject to compartmentalization. Our data showing that the TOT function of Elongator depends on an nuclear
localization signal present in Elp1p (Tot1p) provide evidence that
yeast Elongator function may require nuclear localization
signal-mediated nuclear import (19) and may be regulated by subcellular
compartmentalization in a manner similar to other transcription factors
(44).

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Fig. 6.
Working model on how Tot4p (Kti12p) may
regulate the TOT function of Elongator. Holo-Elongator formation
requires the association of core-Elongator (Elp1-3p/Tot1-3p) with the
HAP complex (Elp4-6p/Tot5-7p) (1) in a manner largely
dependent on the putative scaffold protein, Elp1p/Tot1p. Individual
subunit interactions within the HAP complex cannot be derived from the
co-ip studies presented, although individual deletions
(tot5 , tot6 , and tot7 cells)
remove association with core-Elongator, and two-hybrid interaction has
been shown between Elp5p/Tot5p and Elp6p/Tot6p (45). Fully assembled
holo-Elongator enables interaction with Tot4p/Kti12p (2) in
a fashion that requires the functional integrity of the C terminus of
Elp2p/Tot2p. This contact may be necessary for piggy-backing Elongator
onto elongation-competent pol II form II0 (3). During this
transition, Tot4p/Kti12p becomes replaced by Elongator and can be
recycled (4). For simplicity, Tot4p/Kti12p is shown
monomeric. The possibility, however, that Tot4p/Kti12p contacts
holo-Elongator and pol II form II0 as part of a protein complex cannot
be eliminated for the time being.
|
|
 |
ACKNOWLEDGEMENTS |
We thanks Drs. D. Bentley and J. Svejstrup
for providing us with anti-CTD-P and anti-Elp3p antibodies.
 |
FOOTNOTES |
*
The project was supported by a Deutsche
Forschungsgemeinschaft (Scha 750/2) grant (to R. S.), a stipend by the
'Graduierten Förderung des Landes Sachsen-Anhalt' (to D. J.),
and a grant by the Martin-Luther Universität Halle-Wittenberg (to
L. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Tel.:
49-345-5526333; Fax: 49-345-5527151; E-mail:
schaffrath@genetik.unihalle.de.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M210060200
 |
ABBREVIATIONS |
The abbreviations used are:
TOT,
-toxin target;
pol II, RNA polymerase II;
HAT, histone
acetyltransferase;
HAP, histone acetyltransferase-associated protein(s);
CTD, C-terminal domain;
IIA, hypophosphorylated pol II;
II0, hyperphosphorylated pol II;
co-ip, co-immune precipitations;
HA, hemagglutinin;
IKK, I
B-related kinases.
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