Báo cáo khoa học: Phosphorylation of the Saccharomyces cerevisiae Grx4p glutaredoxin by the Bud32p kinase unveils a novel signaling pathway involving Sch9p, a yeast member of the Akt / PKB subfamily pot

Phosphorylation of the Saccharomyces cerevisiae Grx4p
glutaredoxin by the Bud32p kinase unveils a novel
signaling pathway involving Sch9p, a yeast member
of the Akt / PKB subfamily
Caterina Peggion1,*, Raffaele Lopreiato1,*, Elena Casanova1, Maria Ruzzene1,2, Sonia Facchin1,
Lorenzo A. Pinna1,2, Giovanna Carignani1 and Geppo Sartori1
`
1 Dipartimento di Chimica Biologica dell’Universita di Padova, Italy
2 Venetian Institute of Molecular Medicine (VIMM), Padova, Italy

Keywords
Bud32p kinase; EKC ⁄ KEOPS complex;
Grx4p glutaredoxin; Sch9p kinase
Correspondence
G. Sartori, Dipartimento di Chimica Biologica
`
dell’Universita di Padova, viale G. Colombo,
3-35121 Padova, Italy
Fax: +39 049 8073310
Tel: +39 049 8276141
E-mail:
*These authors contributed equally to this
work
(Received 31 July 2008, revised
26 September 2008, accepted
1 October 2008)
doi:10.1111/j.1742-4658.2008.06721.x

The Saccharomyces cerevisiae atypical protein kinase Bud32p is a member
of the nuclear endopeptidase-like, kinase, chromatin-associated ⁄ kinase,
endopeptidase-like and other protein of small size (EKC ⁄ KEOPS) complex,

known to be involved in the control of transcription and telomere homeostasis. Complex subunits (Pcc1p, Pcc2p, Cgi121p, Kae1p) represent,
however, a small subset of the proteins able to interact with Bud32p,
suggesting that this protein may be endowed with additional roles unrelated to its participation in the EKC ⁄ KEOPS complex. In this context, we
investigated the relationships between Bud32p and the nuclear glutaredoxin
Grx4p, showing that it is actually a physiological substrate of the kinase
and that Bud32p contributes to the full functionality of Grx4p in vivo. We
also show that this regulatory system is influenced by the phosphorylation
of Bud32p at Ser258, which is specifically mediated by the Sch9p kinase
[yeast homolog of mammalian protein kinase B (Akt ⁄ PKB)]. Notably,
Ser258 phosphorylation of Bud32p does not alter the catalytic activity of
the protein kinase per se, but positively regulates its ability to interact with
Grx4p and thus to phosphorylate it. Interestingly, this novel signaling
pathway represents a function of Bud32p that is independent from its role
in the EKC ⁄ KEOPS complex, as the known functions of the complex in
the regulation of transcription and telomere homeostasis are unaffected
when the cascade is impaired. A similar relationship has already been
observed in humans between Akt ⁄ PKB and p53-related protein kinase
(Bud32p homolog), and could indicate that this pathway is conserved
throughout evolution.

The Bud32p protein of Saccharomyces cerevisiae
belongs to the piD261 family of atypical Ser ⁄ Thr protein kinases, which has representatives in virtually all
eukaryotic and archaeal organisms. Unlike the majority of eukaryotic protein kinases, the protein preferen-

tially recognizes acidic substrates [1–3]. Several
different approaches have shown that Bud32p is able
to interact with many yeast proteins [4–6]. Particularly
remarkable is its tight association with the still uncharacterized Kae1p, as the two proteins make up a single

Abbreviations

Akt ⁄ PKB, protein kinase B; EKC ⁄ KEOPS, endopeptidase-like, kinase, chromatin-associated ⁄ kinase, endopeptidase-like and other protein of
small size; HA, hemagglutinin; PRPK, p53-related protein kinase; pSer, phosphorylated Ser; Ni-NTA, Ni2+-nitrilotriacetate–agarose.

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Role of Bud32p in a new signaling pathway in yeast

C. Peggion et al.

polypeptide in some archaeans, and their human
homologs are also able to interact [4]. Two recent
papers have highlighted the importance of this association, by describing a novel and highly conserved protein complex named endopeptidase-like, kinase,
chromatin-associated ⁄ kinase, endopeptidase-like and
other protein of small size (EKC ⁄ KEOPS) [7,8], composed of Bud32p, Kae1p and three additional small
proteins, lacking a known biochemical signature
(Pcc1p, Pcc2p, Cgi121p). The EKC ⁄ KEOPS complex
is essential for yeast viability, and is functionally
related to telomere homeostasis and transcription control, as its mutations cause transcriptional impairment
in the expression of specific gene groups, as well as
relevant shortening of telomeres. Although the molecular mechanism of EKC ⁄ KEOPS activity remains
elusive, it has been proposed that the complex might
promote the accessibility to chromatin, at telomeres as
well as elsewhere on the genome, and regulate the
recruitment of specific factors to their site of action
[7,8]. Although the kinase activity of Bud32p is relevant for the functions of the EKC ⁄ KEOPS complex in
yeast, it is actually unknown whether Bud32p-dependent phosphorylation of other subunits of the complex
could directly regulate its activity.

In addition to the components of the EKC ⁄ KEOPS
complex, many other proteins have been identified as
Bud32p interactors [4–6], suggesting that this protein
kinase may have additional roles by specifically phosphorylating other substrates. Among these Bud32p
interactors, our attention has been drawn to the glutaredoxin Grx4p, which is an in vitro substrate of the
protein kinase, being readily phosphorylated by recombinant, purified Bud32p at Ser134 [4], suggesting that
Grx4p may be one of the physiological substrates of
Bud32p in yeast cells.
Grx4p belongs to the subfamily of yeast monothiolic
glutaredoxins, together with Grx3p and Grx5p [9,10].
Whereas the function of Grx5p in mitochondrial Fe–S
cluster assembly has been extensively investigated, the
role of the nuclear glutaredoxins Grx3p and Grx4p is
less well characterized. The single deletion of either
GRX3 or GRX4 leads to weak growth defects, but the
double deletion strongly affects cellular growth and the
response to oxidative stress. As the two proteins display relevant sequence similarity, they might have
overlapping or redundant functions. Accordingly, it
has been shown that both Grx3p and Grx4p are
involved in the transcriptional modulation of the iron
regulon, by controlling the nucleo-cytoplasmic shuffle
of the transcriptional activator Aft1p [11–13].
In this work, we demonstrate that Grx4p is a physiological substrate of Bud32p in yeast cells, and show
5920

that this relationship is influenced by the phosphorylation state of Bud32p. In fact, Bud32, as well as its
human homolog p53-related protein kinase (PRPK)
[14,15], displays a highly conserved C-terminal
sequence, rich in basic amino acids, that fulfils the consensus recognized by protein kinase B (Akt ⁄ PKB)
(RxxRxS ⁄ THy) [16]. Interestingly, the activity of

PRPK on its known substrate (Ser15-p53) mainly (but
not exclusively) depends on the phosphorylation of its
Ser250 residue by Akt ⁄ PKB [17]. This prompted us to
investigate whether the activity of Bud32p could also
be modulated by phosphorylation of its Ser258 residue,
possibly mediated by Sch9p, which is considered to be
a yeast homolog of mammalian Akt ⁄ PKB. Sch9p is an
AGC kinase [18] involved in a number of cellular processes, including the response to nutrient-mediated
stimuli and the regulation of replicative and chronological lifespan [19–23]. Recently, Sch9p has been identified as a transcriptional activator that is recruited, only
in stress conditions, to the chromatin of genes induced
by osmotic stress [24]. Sch9p is also regulated by TOR
complex 1, which phosphorylates several amino acids
situated in its C-terminal sequence [25]. Recent data
have implicated the PAS kinase Rim15p and the Rps6p
protein as substrates of yeast Sch9p [25–28].
Here, we identify a novel phosphorylation cascade
implicating Sch9p, Bud32p and Grx4p, which apparently does not affect the telomeric and transcriptional
activities of the EKC ⁄ KEOPS complex, suggesting an
additional function for Bud32p in yeast cells.

Results and Discussion
Grx4p is an in vivo substrate of Bud32p
The characterization of Bud32p as a protein kinase
was achieved by using a recombinant form of the
enzyme, purified from Escherichia coli [1]. Bud32p was
shown to be able to autophosphorylate and to phosphorylate in vitro the Ser ⁄ Thr residues of acidic substrates, such as casein. As is the case with many other
protein kinases, autophosphorylation of Bud32p on its
activation loop correlated with an increased activity on
substrates, whereas all the mutant forms unable to
autophosphorylate were also inactive on substrates [1–

3]. A subsequent search for Bud32p-associated proteins
in yeast identified Grx4p as a Bud32p interactor, and
the observation that recombinant Bud32p was able to
phosphorylate recombinant Grx4p in vitro [4] suggested that Grx4p might be an in vivo substrate of the
protein kinase.
To verify this assumption, we constructed several
yeast strains in which Bud32p (wild-type or mutant) is

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C. Peggion et al.

expressed from its chromosomal location in fusion
with the hemagglutinin (HA) epitope at the C-terminus. The two mutations analyzed here substitute
respectively Asp161 and Lys52, two amino acids that
are essential for the catalytic activity of the recombinant protein [2]. The two bud32 mutants are characterized by a slow growth phenotype that is, however, less
stringent than that exhibited by cells in which BUD32
is deleted (Fig. 1A).
First, we checked the activity of Bud32p upon
immunoprecipitation in an in vitro assay on recombinant, purified Grx4p. Preliminary experiments, performed in the conditions used in the biochemical
characterization of recombinant Bud32 (i.e. in the
presence of Mn2+ as bivalent ion) [1–4], showed that
the kinase activity of immunoprecipitated Bud32p was
extremely low. Conversely, the substitution of Mn2+
with Mg2+ significantly improved the enzymatic activ-

A

B

Fig. 1. (A) The kinase activity of Bud32p is relevant for its in vivo
functionality. The wild-type [W303 and BUD32-HA (WT)], the bud32
mutants K52A-HA (K52A), D161A-HA (D161A) and the BUD32
deleted strain (bud32D) were grown until stationary phase in YPD
medium, and diluted to 3 · 107 cellsỈmL)1; 10-fold serial dilutions
were then spotted onto solid YPD medium. Growth was observed
after 2 days at 28 °C. (B) Immunoprecipitated yeast endogenous
Bud32p autophosphorylates and phosphorylates recombinant Grx4p
in vitro. HA-tagged Bud32p, wild-type or catalytically inactive, was
immunoprecipitated from lysates (500 lg of total protein) of strains
BUD32-HA (WT), D161A-HA (D161A) and K52A-HA (K52A) grown
in complete glucose medium (YPD) until exponential phase. Immunocomplexes were subjected to an in vitro phosphorylation reaction
in the presence of [33P]ATP[cP] and 500 ng of recombinant, purified
Grx4p. After SDS ⁄ PAGE, proteins were blotted onto a filter that
was autoradiographed (left panel) and then revealed with specific
antibodies for Bud32p or Grx4p (right panel).

Role of Bud32p in a new signaling pathway in yeast

ity of native Bud32p. This behavior could be related to
subtle differences between the structures of the recombinant and the native protein kinase (e.g. protein folding and ⁄ or post-translational modifications). Even in
the presence of Mg2+, the catalytic efficiency detected
for the native kinase remains low, the results, however,
being consistent with those already reported for the
recombinant protein [4]. It is worth noting that very
recent data [29] have indicated that the activity of
Bud32p is inhibited by its functional partner Kae1p,
suggesting that the protein(s) coprecipitating with
Bud32p might reduce its activity also in our in vitro

assays. Altogether, native Bud32p displays specific
kinase activity on recombinant Grx4p: in fact, the
wild-type protein, which undergoes autophosphorylation, is able to phosphorylate Grx4p, whereas the
mutants D161A and K52A almost completely fail to
autophosphorylate and exhibit a significantly lower
activity on Grx4p (Fig. 1B).
The partial phosphorylation of Grx4p still observed
in the case of the two mutants (corresponding to
30–40% of that of the wild-type) could be explained
either by the intervention of another (contaminant)
kinase, copurified with Bud32p, or, alternatively, by
residual activity of the mutant proteins, which would
still be able to catalyze the kinase reaction in the presence of an excess of exogenous Grx4p substrate.
Together, these results demonstrate that native Bud32p
is able to phosphorylate Grx4p, further suggesting that
the same relationship may exist in yeast cells.
In a further approach, we investigated whether
endogenous Grx4p was phosphorylated by Bud32p.
Native, myc-tagged Grx4p was immunoprecipitated
from yeast, and the precipitate was analyzed for the
presence of coprecipitated Bud32p. The ability of
Bud32p to interact in vivo with Grx4p has already been
reported, although coimmunoprecipitation of the two
proteins depends on the experimental conditions [4,7].
In our present experiments, a weak but specific signal
was detected when the Grx4p immunoprecipitates were
analyzed for the presence of wild-type Bud32p.
Interestingly, this signal was clearly stabilized in the
presence of a catalytic mutant of Bud32p, such as
D161A (Fig. 2A). This observation is consistent with

Grx4p being an in vivo substrate of Bud32p, based on
the assumption that the transient interaction between
the enzyme and its substrate is strengthened if the
course of the catalytic reaction is hampered. In accordance with this, the low amount of wild-type Bud32p
present in the precipitate is still active on native
Grx4p when subjected to in vitro phosphorylation,
whereas the reaction is impaired in the case of
coimmunoprecipitation of Grx4p with D161A mutant

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C. Peggion et al.

A

C

B

D

Fig. 2. Bud32p coimmunoprecipitates with and phosphorylates native Grx4p. Myc-tagged Grx4p was immunoprecipitated from 500 lg of
total protein from the following strains: GRX4-myc ⁄ BUD32-HA (WT), GRX4-myc ⁄ bud32-D161A-HA (D161A), BUD32-HA (no tag) and
GRX4-myc ⁄ bud32D (bud32D), grown as in Fig. 1. Immunocomplexes were either directly subjected to SDS ⁄ PAGE and immunoblotting (A, D)
or subjected to an in vitro phosphorylation reaction in the presence of [33P]ATP[cP] and then to SDS ⁄ PAGE and western blotting (B, C). The

band corresponding to Grx4p in (C) is indicated (*). In (A), the starting amounts of Bud32p present in the cell lysates were equivalent, as
revealed by antibody against HA (Input). In (B), the amounts of Bud32p and Grx4p present in the kinase reaction after immunoprecipitation
are revealed by antibody against HA or Myc, respectively.

(Fig. 2B). To be sure that the observed Grx4p phosphorylation was catalyzed by coimmunoprecipitated
Bud32p, we performed a similar experiment in a
bud32D ⁄ GRX4-myc mutant strain; the result, shown in
Fig. 2C, clearly indicates that Bud32p is responsible
for the radioactivity incorporated into Grx4p. In this
assay, the phosphorylation reaction occurs by addition
of the [33P]ATP[cP] directly to the resin containing the
native, immunoprecipitated Grx4p and proteins associated with it. Any phosphotransferase activity on
Grx4p therefore requires the presence in the immunoprecipitate of (at least) one protein kinase. Our results
confirmed the presence of Bud32p in the Grx4p immunoprecipitate, supporting the idea that Bud32p is
responsible (or coresponsible) for the kinase activity
observed on Grx4p. Accordingly, in the case of the
bud32D ⁄ GRX4-myc mutant strain, in which Bud32p is
lacking (Fig. 2C), the phosphorylation of Grx4p completely disappeared. However, the possibility cannot be
excluded that another, unidentified kinase may act on
Grx4p, but in this case it should be associated with
Bud32p rather than with Grx4p, as no activity was
detected when the immunoprecipitation was performed
in the absence of Bud32p. Finally, we showed
(Fig. 2D) that the phosphorylation state of Grx4p, as
revealed by antibody against phosphorylated serines
(pSer), was much lower when Grx4p was immuno5922

precipitated from the bud32D mutant strain, as compared to the wild-type. However, the detectable
presence of pSer on Grx4p, even in the absence of
Bud32p, indicates that the glutaredoxin is phosphorylated also by other kinases in yeast cells. Taken

together, these results demonstrate that Bud32p participates in the in vivo phosphorylation status of Grx4p.
Phosphorylation of Ser134 of Grx4p by
Bud32p contributes to the functionality
of the glutaredoxin in yeast cells
As indicated by previous in vitro data, phosphorylation
of Grx4p by wild-type Bud32p occurs mainly at
Ser134 and, more weakly, at Ser133, two residues
embedded in a highly acidic stretch of the protein [4].
This sequence is situated in the linker region between
the thioredoxin-like and the glutaredoxin domains of
Grx4p [10], and its modification would be likely to
influence, directly or indirectly, the activity of the
enzyme. To evaluate the contribution of either Ser134
or Ser133 phosphorylation to the biological competence of Grx4p in yeast cells, we created a series of
unphosphorylatable mutants (S134A, S133A, S133A ⁄
S134A), as well as the phospho-mimic S134D, in order
to compare their behavior with that of the wild-type
sequence. S. cerevisiae, however, possesses another

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C. Peggion et al.

Role of Bud32p in a new signaling pathway in yeast

nuclear monothiolic glutaredoxin, Grx3p, which is very
similar to Grx4p; the two proteins cooperate and show
interchangeable roles, e.g. in the transcriptional regulation of iron-dependent genes [11–13]. Therefore, to
specifically investigate in vivo the effect of mutating

Grx4p, we created the double null strain grx3D ⁄ grx4D.
Surprisingly, we noticed that, unlike what was
observed with other commonly used yeast strains (such
as BY4742 and CML128), cells containing the double
mutations are nonviable in the W303 genetic background (Fig. S1), indicating that in the W303 strain
the functions of nuclear monothiolic glutaredoxins are
essential. This may reflect the subtle differences existing between yeast laboratory strains, in particular with
regard to the responses to environmental changes or
stresses involving these oxidoreductases.
The different GRX4 coding sequences were inserted
in galactose-inducible yeast plasmids (see Experimental
procedures), which were used to transform heterozygous diploid grx3D ⁄ grx4D cells. After sporulation
and tetrad dissection, we isolated a complete set of

haploid grx3D ⁄ grx4D strains containing wild-type or
mutant GRX4 plasmids. We then compared their
growth in glucose medium, where the plasmidic alleles
are weakly expressed, and observed (Fig. 3A, left
panel) that, in these conditions, wild-type GRX4 was
able to fully restore yeast growth, similarly to the
bona fide positive control (wild-type W303 cells carrying the empty plasmid). Although the substitution of
Ser133 by Ala (S133A) did not affect the in vivo functionality of Grx4p, the mutation of Ser134 (S134A)
slightly affected its function, this impairment, however,
being completely restored by the phospho-mimic substitution by Asp (S134D). Accordingly, the double
mutation of Ser133 and Ser134 (SS-AA) showed the
same effect as the single S134A mutation, confirming
that Ser134 of Grx4p has a major role in vivo with
respect to Ser133. In addition, we checked the effects
of Grx4p overexpression, by growing the yeast strains
in galactose medium, in which the expression of plasmid-carried genes is strongly induced (Fig. 3A, right

panel). We observed that overexpression of wild-type

A

Fig. 3. Ser134 phosphorylation of Grx4p
by Bud32p contributes to its functionality
in vivo. (A) The wild-type W303 strain
carrying the empty plasmid and the mutant
strain grx4D ⁄ grx3D, carrying the plasmids
coding for either wild-type or mutant Grx4p
(S134A, S133A, SS-AA, S134D) were grown
until stationary phase in SD selective medium and diluted at 3 · 107 cellsỈmL)1. Tenfold serial dilutions were spotted either onto
solid SD (Glucose) or SG (Galactose) plates.
Growth was observed after 3 days at 28 °C.
See text for details. (B) Total protein lysates
(500 lg) of yeast cells expressing wild-type,
HA-tagged Bud32p (Bud32–HA) were used
to immunoprecipitate Bud32p as in Fig. 1.
Immune complexes were subjected to an
in vitro phosphorylation reaction in the presence of [33P]ATP[cP] and 25 ng of recombinant wild-type Grx4p (WT) or 50 ng of the
Grx4p double mutant S133A ⁄ S134A (SS-AA). After SDS ⁄ PAGE and blotting, filters
were autoradiographed (upper panels), and
then visualized with specific antibodies
against Bud32p or Grx4p (lower panels). The
radiolabeled bands (*) are produced by an
unidentified contaminant of the purified
Grx4p proteins. The strong signals in
western blots (**) are from the IgG light
chains released by the resin used for
Bud32–HA precipitation.

B

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C. Peggion et al.

Grx4p was toxic to yeast cells, whereas overexpression
of either the single S134A mutant or the double
S133A ⁄ S134A mutant was less detrimental, indicating
that these substitutions somehow impaired the activity
of the glutaredoxin, rendering it less toxic to the cell.
Remarkably, the phospho-mimic substitution S134D
was able to restore the toxicity of the glutaredoxin,
supporting the relevance of Ser134 phosphorylation
for the biological properties of Grx4p.
Taken together, our data indicate that under normal
growth conditions, Ser134 phosphorylation may be
almost dispensable for Grx4p functionality, although it
could be relevant in the regulation of specific pathway(s) upon environmental changes, allowing yeast
cells to respond appropriately to these stimuli.
Finally, to evaluate the specific contribution of
Bud32p to Grx4p phosphorylation at Ser134-Ser133,
we used a mutant version of recombinant, His-tagged
Grx4p (S133A ⁄ S134A) as substrate for an in vitro

phosphorylation reaction by native Bud32p immunoprecitated from yeast cells. Despite several purification
attempts, the recovery of wild-type and mutant Grx4p
was low, and the phosphorylation assays were run with
quantities of substrate consistently lower than in other
experiments (such as the one shown in Fig. 1). However, as shown in Fig. 3B, the results demonstrated
that Bud32p was able to phosphorylate the minimal
amount of wild-type Grx4p present in the reaction
(left, upper panel), whereas the phosphorylation of
mutant Grx4p completely failed to do so (right, upper
panel), despite the presence of a higher amount of
recombinant protein, as revealed by the western blot
(lower panels). Accordingly, a parallel experiment, performed with the same Grx4p substrates and an aliquot
of recombinant Bud32p, showed that the recombinant
protein was also unable to phosphorylate the mutant
Grx4p (not shown), thus confirming that the phosphorylation of Grx4p by Bud32p specifically involves
Ser134.
Activity of Bud32p on Grx4p is regulated through
phosphorylation by Sch9p
As recently demonstrated [17], the kinase activity of
PRPK (the human homolog of Bud32p) on its known
substrate Ser15-p53 is positively regulated in vitro and
in vivo by phosphorylation of Ser250, which is specifically mediated by Akt ⁄ PKB. The observation that
Bud32p at the homologous residue (Ser258) also
displays the consensus sequence for Akt ⁄ PKB (RxxRxS ⁄ THy), and the existence in yeast of a functional
homolog of Akt ⁄ PKB, the protein kinase Sch9p,
prompted us to look for the presence in yeast of a
5924

similar enzyme–substrate relationship and to determine
whether the activity of Bud32p could be modulated by

Sch9p.
Synthetic genetic interaction between BUD32
and SCH9
In order to find whether a functional relationship
existed between Sch9p and Bud32p, we took advantage
of a genetic approach, easily carried out in yeast, looking for a possible genetic interaction between sch9 and
bud32 mutants. Figure 4A shows that the combination
of the SCH9 and BUD32 deletions is nonviable, and
that deletion of SCH9, when combined with a bud32
catalytically inactive mutant (D161A), affects the
growth of yeast cells more severely than each of the
two single mutations (Fig. 4B). These results supported
the hypothesis that Sch9p and Bud32p are functionally
related, and prompted us to examine their relationship
in depth.
In vivo phosphorylation of Ser258 of Bud32p is
strongly reduced in an sch9D mutant strain
Phosphorylation of Bud32p at Ser258 can be identified
by the use of antibodies (anti-pSer258) that recognize
the phosphorylated target site for Akt ⁄ PKB present at
the C-terminus of the protein [17]. We first established
that wild-type, His-tagged Bud32p, when overexpressed in yeast and purified by Ni2+–nitrilotriacetic acid
agarose (NiNTA), is recognized by the antibodies,
indicating that the protein is phosphorylated in vivo at
Ser258. The antibodies are specific, as a similarly overexpressed and purified Bud32p mutant, in which the
Ser258 residue is replaced with Ala (S258A), is recognized very weakly (Fig. 5A).
Next, to ascertain whether phosphorylation of
Ser258 of Bud32p is due to Sch9p, we immunoprecipitated endogenous HA-tagged Bud32p (expressed from
its chromosomal location) from a wild-type strain
(BUD32-HA) and from a mutant strain in which

SCH9 had been deleted (sch9D ⁄ BUD32-HA). Figure 5B shows that Bud32p was recognized by the antibodies against pSer258 when immunoprecipitated from
the wild-type strain, but not (or very poorly) when
immunoprecipitated from the sch9D mutant, supporting the idea that Sch9p is implicated in the phosphorylation of Bud32p at Ser258.
In order to investigate the impact of Ser258 substitution in vivo, we compared the growth of the S258A
mutant with that of the wild-type strain and of
other bud32 mutants (D161A, K52A) and deletion
mutants (bud32D), and observed that cells were almost

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Role of Bud32p in a new signaling pathway in yeast

A

B

Fig. 4. Genetic interaction between BUD32 and SCH9. (A) The double deletion of BUD32 and SCH9 is lethal for yeast. Heterozygotic diploid
bud32D ⁄ sch9D cells were transformed with a centromeric plasmid (pFL38) carrying the wild-type BUD32 sequence and the URA3 marker
[counterselectable on 5-fluoroorotic acid (5-FOA)-containing medium). After tetrad dissection, haploid spores were recovered and genotypes
were determined. Yeast cells containing the plasmidic URA3 marker and coming from some complete tetrads were plated on 5-FOA
medium. Only cells containing the double deletion bud32D ⁄ sch9D cannot lose the plasmid and cannot grow on 5-FOA plates. Two representative complete tetrads are shown. DB, DS, DD are for bud32D, sch9D, and bud32D ⁄ sch9D, respectively. (B) The slow-growth phenotype of
the bud32-D161A mutant is exacerbated by the additional deletion of SCH9. Wild-type BUD32-HA (WT) and mutant strains D161A-HA
(D161A), sch9D ⁄ BUD32-HA (sch9D) and sch9D ⁄ D161A-HA (sch9D ⁄ D161A) were grown until stationary phase in YPD medium, and diluted
to 3 · 107 cellsỈmL)1; 10-fold serial dilutions were then spotted onto solid YPD medium. Growth was observed after 3 days at 28 °C.

A

B

Fig. 5. Bud32p is phosphorylated at Ser258 in the wild-type but not in an sch9D mutant strain. (A) Antibodies against pSer258 (anti-pSer258)
recognize ectopically expressed wild-type Bud32p and not the S258A mutant. The W303 wild-type strain, transformed with galactose-inducible vectors carrying the BUD32 sequence (WT or S258A mutant) fused to a His epitope, was grown in YPGal until exponential phase, when
Bud32–His (WT or S258A) was purified with the NiNTA resin from the cell lysate. The resin was subjected to SDS ⁄ PAGE and immunoblotting. (B) Endogenous HA-tagged Bud32p is phosphorylated at Ser258 when immunoprecipitated from the wild-type but not from an
sch9D mutant strain. The wild-type BUD32-HA strain (WT) and the sch9D ⁄ BUD32-HA mutant (sch9D), both expressing BUD32-HA from its
chromosomal location, were grown in YPD medium (until exponential phase). Native Bud32p was immunoprecipitated from cell lysate with
the anti-HA resin, and processed by SDS ⁄ PAGE and immunoblotting.

unaffected when compared to catalytically inactive or
null mutants (not shown). Although we cannot completely rule out the possibility that Sch9p phosphorylates Bud32p also at other Ser ⁄ Thr residues (which

would be embedded in sequences different from the
consensus recognized by Akt ⁄ PKB), we suppose that
Ser258 phosphorylation could affect cell growth only in
specific situations, and not in the (normal) conditions

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here tested. In this case, the S258A substitution should
not affect the main properties of the kinase, but may
operate as a regulatory site for Bud32p, e.g. by allowing ⁄ promoting its association with specific partners (or
substrates).

However, the absence of a growth phenotype for
the S258A mutant does not explain the strong
genetic interaction observed between BUD32 and
SCH9 (see above and Fig. 4); we must, then, infer
that the two genes, besides being interrelated via
phosphorylation of the Bud32p Ser258 residue by
Sch9p, have overlapping functions in a still unidentified pathway.

Sch9p interacts with Bud32p and phosphorylates
it in vitro at Ser258
The results described above are consistent with a role
for Sch9p in the phosphorylation of Bud32p at Ser258,
but do not prove that Bud32p is a direct substrate of
Sch9p. To clarify this point, we checked the ability of
Sch9p to interact with Bud32p and phosphorylate it
in vitro.
First, a pull-down experiment was performed by
incubating recombinant, purified Bud32-His, previously bound to the NiNTA resin, with a yeast cellular
lysate in which Sch9p was HA-tagged. The results
shown in Fig. 7A indicate that the two proteins are
able to interact. In fact, a western blot analysis
revealed the presence of Sch9p associated with the
NiNTA resin only where recombinant Bud32p had
been previously bound to the resin. The different
bands recognized by the antibodies against HA are
noteworthy, as they probably highlight different phosphorylation states of Sch9p, as already demonstrated
[21].
Next, we investigated whether Bud32p is a substrate
of Sch9p. The HA-tagged Sch9p was immunoprecipitated by the use of the anti-HA affinity matrix:
Fig. 7Ba confirms the immunoprecipitation of Sch9p

(lane 1); as controls, the anti-HA matrix was incubated
either with a cellular lysate of the untagged strain, or
with no lysate (lanes 2 and 3). The resin was subjected
to an in vitro phosphorylation reaction in the presence
of recombinant, purified Bud32-His. In the autoradiograph (Fig. 7Bb), it can be seen that, when immunoprecipitated Sch9p was present (lane 1), radioactivity
incorporation was greatly increased (2.5-fold) with
respect to the background levels (lanes 2 and 3), which
are due to autophosphorylation of recombinant
Bud32-His. Subsequent detection of the filter with antibodies against pSer258 (Fig. 7Bc) confirmed that this
phosphorylation takes place at Ser258.

Conditions that regulate Sch9p abundance
influence Bud32p phosphorylation at Ser258
The abundance of Sch9p, which is involved in the control of numerous nutrient-sensitive processes, is in fact
modulated by nutrients [21]. To verify whether the
onset of conditions that modify the amount of Sch9p
has an effect on the phosphorylation of Bud32p at
Ser258, we compared the level of Bud32p phosphorylation in wild-type cells grown on a fermentable carbon
source (glucose) to that in cells grown on glycerol. To
this end, we used a yeast strain expressing Bud32p and
Sch9p fused to different epitopes (SCH9-myc ⁄ BUD32HA), and first verified the abundance of Sch9p:
Fig. 6A shows that the amount of Sch9p in the cell
lysates (as revealed by western blot) was higher in the
case of cells grown in glucose than in the case of cells
grown in glycerol. Accordingly, phosphorylation at
Ser258 of Bud32p, immunoprecipitated from the same
lysates, is more extensive in cells grown in glucose than
in cells grown in glycerol (Fig. 6B). These results
suggest that Bud32p might be a physiological target of
Sch9p, representing one of the effectors of this protein

kinase known to be involved in multiple cellular
processes.

A

B

Fig. 6. Phosphorylation at Ser258 of Bud32p is related to Sch9p abundance. (A) Sch9p levels in cell lysates. Equal amounts (20 lg of total
protein) of cell lysates obtained from strain SCH9-myc ⁄ BUD32-HA, grown in YPD (glucose) or in YP with glycerol (Glycerol) until exponential
phase, were subjected to SDS ⁄ PAGE and immunoblotting to visualize Sch9p. (B) Phosphorylation state of immunoprecipitated Bud32p. HAtagged Bud32p was immunoprecipitated from the same lysates used in (A) (500 lg of total protein), using the anti-HA resin. Bound proteins
were eluted with SDS ⁄ PAGE loading buffer, electrophoresed, and immunoblotted with the indicated antibodies.

5926

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C. Peggion et al.

Role of Bud32p in a new signaling pathway in yeast

A

B

Fig. 7. Sch9p is able to interact with Bud32p and phosphorylate it in vitro. (A) Interaction between Sch9p and Bud32p by pull-down assay.
Five micrograms of recombinant, purified Bud32–His (lane 1), bound to the NiNTA resin, were incubated with 500 lg of a cellular lysate of
strain SCH9-HA. The resin was washed with a buffer containing imidazole, and subjected to SDS ⁄ PAGE and immunoblotting to detect
Sch9p or Bud32p. As a reference, the same amount of resin, with no Bud32–His bound, was incubated with the same lysate, and then
treated as described and loaded in lane 2. Western blot analysis revealed that Sch9p was retained by the resin only in lane 1. (B) Immunoprecipitated Sch9p phosphorylates recombinant Bud32p. HA-tagged Sch9p was immunoprecipitated with the anti-HA resin from 500 lg (total

proteins) of a lysate of strain SCH9-HA (lane 1). As a reference, the anti-HA resin was incubated with a lysate of wild-type strain W303, in
which SCH9 has no tag (lane 2), or with no lysate (lane 3). The immunoprecipitation of Sch9p only in lane 1 is revealed in (a). The resins
were subjected to a phosphorylation reaction in the presence of [33P]ATP[cP] (b) or cold ATP (c) and 100 ng of recombinant His-tagged
Bud32p [quantified by antibodies against His in panel (d)]. After SDS ⁄ PAGE and immunoblotting, Bud32p phosphorylation was detected as
radioactivity incorporation (b) and by anti-pSer258 (c).

Taken together, the in vivo and in vitro results on
Bud32p phosphorylation by Sch9p indicate that
Bud32p is one of the downstream targets of Sch9p,
whose substrates are still largely unknown, with few
exceptions, e.g. the Rps6p ribosomal protein, indicated
in a recent report as a probable Sch9p substrate, at
least in vitro [25]. Our data confirm that Sch9p recognizes the same target sequence as human Akt ⁄ PKB,
further supporting the assumption of the similarity
between the two protein kinases. This raises the question of whether phosphorylation of Bud32p at Ser258
represents a way to regulate its activity.
Ser258 phosphorylation of Bud32p promotes
recognition and phosphorylation of Grx4p
We next compared the two forms of Bud32p (phosphorylated, or not, at Ser258) for their ability to
phosphorylate recombinant, purified Grx4p. The HAtagged Bud32p was immunoprecipitated from the wildtype strain BUD32-HA (Fig. 8A, lane 1) and from the
sch9D mutant strain sch9D ⁄ BUD32-HA (lane 3), as
well as from a mutant in which wild-type SCH9 is
present but Ser258 of Bud32p is replaced by Ala
(S258A-HA) (lane 2); the precipitates were then subjected to an in vitro phosphorylation reaction in the

presence of recombinant Grx4p. Figure 8A shows that,
when Bud32p was not Ser258-phosphorylated, i.e. in
the sch9D strain, or when it carried the S258A mutation, phosphorylation of Grx4p was reduced by up to
40% of the wild-type activity, similarly to what was
seen with the K52A and D161A mutants. The observation that Grx4p phosphorylation was comparable in

both mutant strains (bud32-S258A and sch9D, lanes 2
and 3, respectively) rules out the possibility of Grx4p
being a direct substrate of Sch9p.
Nevertheless, unlike with catalytic-defective mutants,
autophosphorylation of immunoprecipitated Bud32p
was not affected with respect to the wild-type, strongly
suggesting that Ser258 modification of Bud32p does
not alter the catalytic activity of the protein kinase
per se. We further confirmed such evidence by checking in vitro the enzymatic activity of native Bud32p on
the model substrate casein, and observed that both
forms of Bud32p, phosphorylated or not, had similar
catalytic properties, as they were able to phosphorylate
casein (data not shown). These results may therefore
indicate that Ser258 modification could modulate the
ability of Bud32p to recognize the Grx4p substrate.
To confirm this hypothesis, we performed a pulldown experiment in which His-tagged wild-type
Bud32p and mutant S258A were bound to the NiNTA

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Role of Bud32p in a new signaling pathway in yeast

C. Peggion et al.

A

B

Fig. 8. Ser258 phosphorylation of Bud32p influences its interaction with Grx4p. (A) Yeast endogenous Bud32p phosphorylates the Grx4p
substrate only if activated by Ser258 phosphorylation. Native, HA-tagged Bud32p was immunoprecipitated from the wild-type BUD32-HA
strain (lane 1), the S258A-HA mutant strain (lane 2) and the sch9 null strain (sch9D ⁄ BUD32-HA, lane 3), and subjected to an in vitro phosphorylation reaction in the presence of recombinant Grx4p. (B) Grx4p coprecipitates with ectopically expressed wild-type Bud32p and not
with the S258A mutant. The GRX4-myc ⁄ BUD32-HA strain was transformed with an empty centromeric plasmid (lane 1) and with the same
plasmid carrying the wild-type (lane 2) or the S258A mutant (lane 3) forms of the BUD32 sequence, both fused to the His epitope. Cells
were grown until exponential phase in galactose medium to induce the ectopic expression of the plasmid-borne genes, and aliquots of the
lysates containing equal amounts of Grx4p (as revealed by anti-myc detection, shown in Input) were incubated with the NiNTA resin to bind
His-tagged Bud32p. The resin was finally subjected to SDS ⁄ PAGE and western blotting.

resin and analyzed for their ability to coprecipitate
Grx4p. Yeast strains expressing native, myc-tagged
Grx4p were transformed with a plasmid bearing wildtype BUD32, or the bud32-S258A mutant, both fused
to a His-tag. After growth in galactose medium (to
induce the expression of the plasmid inserted genes),
cells were lysed and treated with the NiNTA resin in
order to isolate His-tagged Bud32p together with the
associated proteins. In this experiment, we analyzed
the ability of ectopic Bud32p-His, phosporylated or
not at Ser258, to compete with endogenous Bud32p
for binding to native Grx4p. Figure 8B shows that
wild-type His (lane 2) efficiently substituted for the
endogenous kinase in the association with Grx4p,
whereas a similar amount of the S258A mutant
(lane 3) failed to bind Grx4p, as shown by a signal
comparable to the background level (lane 1). The
(unexpected) high signal of the Grx4p background
indicates that NiNTA resin may be somehow able to
aspecifically bind myc-tagged Grx4p. Taken together,
these results indicate that phosphorylation of Bud32p

at Ser258 positively regulates the ability of the protein
kinase to associate with Grx4p and therefore to phosphorylate it, whereas this modification does not affect
the catalytic activity of the protein kinase per se.
Phosphorylation of Bud32p at Ser258 is unrelated
to its functions within the EKC/KEOPS complex
The observed phosphorylation of Bud32p at Ser258,
besides having an effect on Grx4p, might also influence
in yeast cells the activity of the whole EKC ⁄ KEOPS
complex, of which Bud32p is a crucial component.
5928

Notably, Bud32p-Ser258 modification might impact on
both functions (transcription control and telomere
homeostasis), in which the EKC ⁄ KEOPS complex is
involved [7,8].
We therefore investigated whether phosphorylation
of Bud32p-Ser258 is linked to these processes, first by
analyzing telomere length in several wild-type and
mutant strains. As shown in Fig. 9A, catalytically inactive or null bud32 mutations (K52A; D161A; bud32D)
led to telomeres that were shortened in comparison to
the wild-type, whereas the telomere length of the
S258A mutant was unaffected, being almost identical
to that of the wild-type (W303 or BUD32-HA).
Remarkably, deletion of either SCH9 or GRX4 did not
impair telomere elongation. We have then examined
the effects of Ser258 phosphorylation of Bud32p on
the transcriptional activity of the EKC ⁄ KEOPS complex by analyzing the activation rate of the galactoseinducible gene GAL1, known to be regulated by the
complex. By using real-time RT-PCR and northern
blot analyses (a representative northern blot is shown
in Fig. 9B), we compared the levels of GAL1 mRNA

in wild-type and bud32 mutant strains upon transcription induction, observing (as expected) a reduction of
mRNA levels in kinase-dead or null mutants, but no
difference between the wild-type and the S258A
mutant strains, in accordance with the effects observed
on telomere elongation. The results presented here thus
indicate that the phosphorylation cascade involving
Sch9p, Bud32p and Grx4p is apparently not relevant
to the telomeric or to the transcriptional function
of the Bud32p-associated EKC ⁄ KEOPS complex.
Accordingly, Grx4p has never been isolated as a

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C. Peggion et al.

A

Role of Bud32p in a new signaling pathway in yeast

cellular role of Bud32p (via Grx4p phosphorylation),
which would be unrelated to the functions of Bud32p
as a component of EKC ⁄ KEOPS, but could be
involved in responses to environmental stimuli or
endogenous stresses. Whether Bud32p could achieve
the two tasks while being simultaneously associated
with the EKC ⁄ KEOPS complex and the Grx4p substrate or, alternatively, whether only a cellular fraction
of Bud32p, not included in EKC ⁄ KEOPS, could associate with the glutaredoxin, will be a matter for future
investigation.

Conclusions
B

Fig. 9. Ser258 phosphorylation of Bud32p does not influence its
functions in telomere elongation and transcriptional regulation.
(A) Telomere length analysis in wild-type and in mutant strains
impaired in the Sch9–Bud32–Grx4 cascade. Genomic DNA of the
indicated strains (grown in rich medium until exponential phase)
was purified and digested with XhoI, producing telomeric terminal
DNA fragments of about 1 kb, which were separated on 1.2%
agarose, tranferred onto a nitrocellulose membrane, and checked
with a 33P-labeled probe specific for telomeric TG1–3 repeats.
(B) Transcriptional activation of GAL1 is unaffected by Ser258
mutation. Yeast strains were grown in noninducing raffinose medium to exponential phase, and then incubated for 30 min in galactose medium to activate the GAL regulon. Total mRNAs were
extracted and subjected to standard northern blot analysis. GAL1
mRNA, and ACT1 mRNA (considered as a loading control), were
detected by the use of specific radiolabeled probes (see Experimental procedures).

component of EKC ⁄ KEOPS, indicating that the phosphorylation of Grx4p by Bud32p is independent from
the known activities of the complex and must therefore
be involved in different functions and unrelated pathways. Furthermore, we noticed that growth of bud32
mutants lacking the highly conserved C-terminal tail
was not affected, similarly to what has been observed
in the case of the single S258A substitution, indicating
that these mutations do not impair the main biological
properties of the kinase. Moreover, recent data [29]
from the 3D structure of an archeal Bud32 homolog
(Mj1130p) indicate that the Bud32p C-terminal tail is
located far from the catalytic site, suggesting that its
alteration should not be detrimental to the overall

structure. Finally, our data are consistent with a

In this article, we describe a novel S. cerevisiae signaling pathway that implicates Bud32p and Sch9p (yeast
homologs of mammalian PRPK and Akt ⁄ PKB, respectively) in modulating the phosphorylation state of
Grx4p in yeast cells, with possible implications for the
regulation of its activity. Notably, we show that Sch9p
phosphorylates, both in vitro and in vivo, Ser258 of
Bud32p, and that this modification does not affect the
catalytic properties of the enzyme, but promotes its
ability to associate with its substrate Grx4p and, consequently, to phosphorylate it. This event appears to
be physiologically regulated by the cellular levels of
Sch9p, suggesting that nutrient-mediated stimuli,
detected by Sch9p, may also be important in controlling Bud32p phosphorylation state and finely tuning its
functions. These results highlight for the first time
Bud32p as a substrate of Sch9p and probably one of
the effectors of this crucial protein kinase. Interestingly, this pathway is unrelated to the known functions
of the Bud32p-associated EKC ⁄ KEOPS complex in
both telomere metabolism and transcription, suggesting that Bud32p participates in multiple pathways in
yeast cells.
An important aspect of these data concerns the evolutionary conservation of this regulatory system. It has
been demonstrated that in human cells, phosphorylation at Ser250 of PRPK (Bud32p homolog) by
Akt ⁄ PKB positively regulates in vivo the activity of
PRPK on its physiological substrate Ser15-p53 [17].
However, it remains to be established whether Ser250
modification directly influences the PRPK catalytic
properties or, alternatively, promotes the association
with its substrate p53. On the basis of the results
described here, we are tempted to speculate that
in eukaryotic cells, phosphorylation by the Akt ⁄ PKB
kinases on Bud32-like proteins would create adhesive

surfaces, which promote the association with specific
targets, without major effects on the catalytic activity
of the enzyme.

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Role of Bud32p in a new signaling pathway in yeast

C. Peggion et al.

Experimental procedures
Yeast and bacterial strains
The yeast strains used in this study (Table S1) are derived
from the W303 strain [30]. E. coli InvaF¢ [endA1, recA1,
hsdR17 (r)K, m)K), supE44, k), thi-1, gyrA, relA1, F80lacZaDM15D (lacZYA-argF), deoR+, F¢] was used for DNA
manipulation, and E. coli BL21 (DE3) [F), ompT, hsdSb
(rb), mb)) gal dcm (DE3)] was used as the host for expression of heterologous proteins, as described in [1] and [4].

Media
YP (1% yeast extract, 1% bactopeptone) contained 2%
dextrose (YPD) or 2% galactose (YPGal) or 3% glycerol
(YPGly) as carbon source; SD (0.67% yeast nitrogen base
without amino acids, 2% dextrose or galactose) contained
auxotrophic requirements as needed. The bacterial medium
was LB (1% Bactotryptone, 0.5% yeast extract, 0.5% NaCl
and, when requested, 100 lgỈmL)1 ampicillin). Solid media
were obtained by the addition of 2% agar. Media components were from Difco (Becton Dickinson and Company,

Franklin Lakes, NJ, USA), and auxotrophic requirements
were from Sigma (St Louis, MO, USA). Overexpression of
genes from the pYeDP plasmid [31] was obtained by pregrowing the transformed cells in SD (galactose). Then,
the cultures were diluted to D600 nm = 0.2 and grown in
YPGal until mid-log phase. Cells were routinely incubated
at 28 °C.

Construction of mutant yeast strains
Standard DNA manipulation was performed as described
in [32]. Gene deletions and C-terminal epitope-tagging of
wild-type and mutant alleles were performed using the
PCR-based one-step in vivo strategies described, respectively, in [33] and [34]. The mutagenized strains have been
verified by PCR analysis and, when necessary, direct DNA
sequencing of the manipulated genomic regions.
The wild-type, along with the D161A and K52A mutant
alleles of BUD32 inserted in the pFL38 plasmid under the
control of the BUD32 promoter, were already available [4].
The S258A mutant form of BUD32 was obtained using the
QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene,
La Jolla, CA, USA), in combination with the S258A-S and
S258A-AS primers (Table S2), on the pFL38 plasmid bearing the wild-type BUD32 allele. All the BUD32 mutagenized sequences were isolated from the pFL38 plasmid by
BamHI ⁄ PstI digestion and cloned in the YIplac211 integrative plasmid [35], modified by deletion of the unique EcoRI
restriction site. Substitution at the appropriate genomic
locus of the wild-type BUD32 gene with the mutagenized
bud32 alleles was performed as described in [36] and verified
by DNA sequencing.

5930

The pYeDP–BUD32–His recombinant plasmid was

obtained as follows. The BUD32 coding sequence was amplified by PCR from yeast wild-type genomic DNA using the
BUD-S and BUD-AS primers (Table S2). The forward
primer (BUD-S) introduces the restriction site BamHI
(underlined) two nucleotides upstream of the ATG codon,
and the reverse primer (BUD-AS) introduces a sequence coding for six His residues (lower-case) in-frame with the 3¢-end
of the gene, immediately followed by a stop codon and
(underlined) the restriction site recognized by the KpnI
enzyme. The resulting amplification product was cloned in
the pYeDP-1 ⁄ 8.2 vector at the BamHI ⁄ KpnI sites [31]. In this
construct, the expression of the cloned gene is under the control of the CYC1–GAL1-10 promoter, allowing galactoseinducible overproduction of the Bud32–His6 fusion protein.
pYeDP–bud32-S258A–His was obtained using pYeDP–
BUD32–His as template DNA and the QuikChangeTM
Site-Directed Mutagenesis Kit, as previously described.
The same strategy was used to construct the pYeDP–
GRX4 recombinant plasmid. Amplification of the GRX4
coding sequence from genomic DNA was performed by
PCR using the GRX4-S and GRX4-AS primers (Table S2),
carrying, respectively, the restriction site for BamHI (underlined) two nucleotides upstream of the ATG codon, and
the restriction site for KpnI (underlined) two nucleotides
downstream of the TAA stop codon. After digestion with
these two enzymes, the PCR product was ligated in the
pYeDP-1 ⁄ 8.2 vector digested with the same enzymes. Starting from this recombinant plasmid, we obtained the S134A,
S133A, S133A ⁄ S134A and S134D mutagenized forms of
GRX4 as described previously, using, respectively, the
S134A-S ⁄ S134A-AS, S133A-S ⁄ S133A-AS, S133A-S134AS ⁄ S133A-S134A-AS and S134D-S ⁄ S134D-AS pairs of
primers (Table S2). Transformation of yeast cells with
recombinant plasmids was performed as described in [37].

Purification of wild-type and mutant
S133A ⁄ S134A Grx4 proteins from E. coli cells

The pET–GRX4 plasmid [4] was used to overexpress the
His-tagged wild-type form of Grx4 in E. coli. To produce
the S133A ⁄ S134A double mutant form of Grx4 in this
expression system, we mutagenized the sequence of the
pET–GRX4 plasmid, following the same strategy used to
create this mutant in the pYeDP-1 ⁄ 8.2 vector (see above).
Purification of both wild-type and mutant, His-tagged forms
of Grx4 from E. coli was performed as described in [4].

Northern blot analysis
Northern blot analyses were performed as described in [32].
ACT1-specific (as internal control) and GAL1-specific
probes were amplified by PCR from genomic yeast DNA
using, respectively, the ACT-S ⁄ ACT-AS and GAL-S ⁄ GALAS pairs of primers listed in Table S2. About 25 ng of the

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C. Peggion et al.

PCR products were radiolabeled using DECAprime II
(Applied Biosystems ⁄ Ambion, Austin, TX, USA) with
[32P]dCTP[aP] and used to probe 10 lg of total yeast
RNAs blotted on a nitrocellulose membrane. The membrane was scanned directly using the Cyclone apparatus
(Perkin Elmer, Waltham, MA, USA).

Preparation of cell extracts, immunoprecipitation
and immunoblotting
Immunoblots, as well all the assays performed with immunoprecipitated proteins, are representative of at least three
independent experiments.

Approximately 109 yeast cells, grown until mid-log
phase, were harvested by centrifugation at 3068 g for
5 min and washed with ice-cold water. Cells were resuspended in IP buffer [50 mm Tris ⁄ HCl, pH 8.0, 150 mm
NaCl, 0.1% NP-40, 10 mm NaF, 1 mm Na3VO4, 1 mm
phenylmethanesulfonyl fluoride, 50 mm b-glycerophosphate, 0.2% complete protease inhibitor cocktail (Roche
Diagnostics Ltd, Burgess Hill, UK), 1 mm EDTA, 1 mm
EGTA], at a concentration of 3 · 107 cells per 10 lL, in
the presence of an identical volume of glass beads (diameter 0.5 mm), and lysed by vortexing for 30 s at
6000 r.p.m. in a MagnaLyser apparatus (Roche Diagnostics). For the interaction between endogenous Bud32-HA
(or ectopically expressed Bud32–His6) and Grx4–myc, cells
were lysed in a modified IP buffer (20% glycerol instead
of 0.1% NP-40). The soluble fraction was obtained by
centrifugation (20 min at 10 000 g at 4 °C), and the protein concentration was determined by the Bradford
method. Lysate proteins (0.5 mg) were incubated with the
specific affinity matrix at 4 °C for 2 h, or overnight.
Immunocomplexes were washed with 50 mm Tris ⁄ HCl
(pH 8.0), 150 mm NaCl (500 mm in the case of phosphorylation assays), and 200 lm phenylmethanesulfonyl fluoride. Bound proteins were eluted by heating the beads for
5 min at 95 °C in SDS ⁄ PAGE loading buffer. Samples
were subjected to 11% SDS ⁄ PAGE, blotted on a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA,
USA), and processed with the indicated antibodies;
detection was obtained by enhanced chemiluminescence
(Amersham ⁄ GE Healthcare Ltd, Chalfont St Giles, UK),
and the signal was quantified on a Kodak Image
Station 440CF and analyzed with Kodak 1d image software. Monoclonal antibodies against phosphoserine (cat.
no. P3430) (anti-pSer), polyhistidine (cat. no. H1029)
(anti-His) and HA (cat. no. H9658) were from Sigma, and
monoclonal antibody against c-myc (anti-c-myc) (clone
9E10) was from Roche. For immunoprecipitation of HA
or c-myc-tagged proteins from yeast extracts, we used,
respectively, the anti-HA and the anti-c-myc affinity

matrix (Covance, Richmond, CA, USA). The phosphospecific antibody that recognizes the pSer258 residue of
Bud32 (anti-pSer258) is described elsewhere [17].

Role of Bud32p in a new signaling pathway in yeast

Bud32–His6 pull-down assay
To identify the interaction between Bud32 and Sch9, a
pull-down analysis was carried out using the NiNTA
affinity matrix (Ni2+–nitrilotriacetic acid agarose) (Qiagen,
Valencia, CA, USA) preloaded with 5 lg of recombinant,
purified Bud32–His6, prepared as described elsewhere [1].
Subsequently, 20 mL of NiNTA ⁄ Bud32–His6 resin were
incubated for 4 h at 4 °C with approximately 500 lg of
cell lysate obtained from the SCH9-HA3 strain (see
Table S1). In this protocol, yeast cells were lysed (as
previously described) with IPI buffer (IP buffer, 20 mm
imidazole). The resin was washed twice with IPI buffer,
and bound proteins were eluted with SDS ⁄ PAGE loading
buffer and analyzed by immunoblotting, using antibodies
as indicated. As a negative control, the same cellular
lysate was incubated with the NiNTA matrix without any
bound protein, and the sample was subjected to the same
protocol.

Immune complex protein kinase assays
The protein kinase activity of endogenous wild-type
Bud32–HA and of its mutant forms, immunoprecipitated
from a cellular lysate (as described in a previous section),
was routinely assayed by incubating about 50 ng of Bud32–
HA, bound to anti-HA sepharose beads (Covance), at

37 °C for 30 min in 20 lL of a medium containing 50 mm
Tris ⁄ HCl (pH 7.5), 10 mm MgCl2, and 25 lm [33P]ATP[cP]
(Amersham ⁄ GE Healthcare; specific radioactivity 2000–
3000 c.p.m.Ỉpmol)1) and 500 ng of purified, recombinant
Grx4–His (prepared as described in [4]) as phosphorylatable
substrate. All experiments were conducted in the presence
of 1 lm K25, a specific inhibitor of the casein kinase 2 protein. Phosphorylation assay on the S133A ⁄ S134A mutant
form of recombinant, His-tagged Grx4 (see Supporting
information) was performed in the same way, but with a
lower ( 25 ng) amount of recombinant substrate.
Phosphorylation of recombinant Bud32–His6 (100 ng) by
endogenous Sch9 was performed by incubation of Sch9–
HA3, immunoprecipitated from yeast cells as previously
described, in 20 lL of a medium containing 50 mm
Tris ⁄ HCl (pH 7.5), 12 mm MgCl2, and 10 lm [33P]ATP[cP]
( 1500 c.p.m.Ỉpmol)1) for 20 min at 30 °C.
The reaction was stopped by addition of gel electrophoresis loading buffer, and samples were subjected to 11%
SDS ⁄ PAGE. Proteins were blotted onto a nitrocellulose
membrane (Biorad), and the membranes were directly
scanned on the Cyclone apparatus (Packard) and detected
by immunoblotting using the appropriate antibodies.

Telomere length measurement
Telomere length was measured by Southern blotting as
described in [39]. Yeast cells were grown in YPDA medium

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5931

Role of Bud32p in a new signaling pathway in yeast

C. Peggion et al.

until exponential phase, when genomic DNAs were
extracted. XhoI-digested genomic DNA fragments were separated by gel electrophoresis in 1.2% agarose, transferred
to a Hybond-N+ membrane (Amersham Biosciences), and
probed with a radiolabeled Y¢-TG1–3 DNA fragment.

Acknowledgements
This work was supported by grants from: Ministero
`
Italiano dell’Universita e della Ricerca (MIUR), Progetto PRIN 2005, and University of Padova, Progetto
di Ateneo 2005.

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Supporting information
The following supplementary material is available:
Fig. S1. Activity of the Grx3 ⁄ Grx4 nuclear monothiolic
glutaredoxins is essential in the W303 yeast strain.
Table S1. Yeast strains [38].
Table S2. Oligonucleotides.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
material supplied by the authors. Any queries (other
than missing material) should be directed to the corresponding author for the article.

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