Background
The ubiquitin/proteasome-dependent proteolysis system
has been implicated in a wide variety of cellular
regulatory mechanisms, including transcription, signal
transduction, and cell cycle control (reviewed in [ 1 ] ).
The system employs a cascade of enzymatic reactions that
lead to the covalent attachment of a chain of multiple
ubiquitins to substrate proteins [ 2 ] . In many cases,
modification by ubiquityl moieties targets proteins to the
proteasome, ultimately resulting in their degradation. The
ubiquitylation reaction involves a minimum of three
enzymes: An E1, which mediates the ATP-dependent activation
of ubiquitin, and an E2, or ubiquitin conjugating enzyme
(UBC), which, together with an E3 ubiquitin ligase,
transfers ubiquitin to the target protein. E3 enzymes are
of particular interest, as they mediate the substrate
specificity of ubiquitylation reactions.
Studies in budding yeast identified SCF Cdc4p, an E3
ubiquitin ligase complex that mediates the ubiquitylation
of the CDK inhibitor Sic1p [ 3 4 ] . SCF Cdc4pconsists of
at least four proteins: the cullin Cdc53p, the RING domain
protein Hrt1p/Rbx1p/Roc1p, the adapter protein Skp1p, and
Cdc4p (reviewed in [ 1 ] ). Cdc4p contains two sequence
motifs, which are conserved in a wide variety of so-called
F-box proteins: C-terminal WD-repeats that are involved in
binding the substrate Sic1p in a phosphorylation-dependent
manner, and a central F-box [ 5 ] that interacts with Skp1p
[ 6 7 ] . Cdc53p in turn binds to Skp1p and
Hrt1p/Rbx1p/Roc1p, which bridges Cdc53p with the
ubiquitin-conjugating enzyme Cdc34p/Ubc3p [ 6 8 9 10 11 12
] .
In vitro reconstitution demonstrated
that SCF Cdc4p, Cdc34p, E1, ubiquitin, and ATP are
sufficient to mediate Sic1p polyubiquitylation [ 6 7 ]
.
Components of the SCF system are widely conserved in
eukaryotes [ 1 3 ] . In human cells, for example, SCF
Skp2mediates destruction of the CDK inhibitor p27 [ 13 14 ]
, while SCF β-TRCPtargets IκB [ 15 16 17 ] . All of these
SCF complexes share homologues of the core components
CDC53/CUL1, SKP1, and HRT1/RBX1/ROC1, which associate with
different F-box proteins.
Several lines of evidence suggest that the SCF pathway
is also conserved in the fission yeast
Schizosaccharomyces pombe . Pcu1p, a
Cdc53p/CUL1-related protein, was shown to associate with
two different F-box/WD repeat proteins, Pop1p and Pop2p,
when overexpressed [ 18 ] . In addition, genetic studies
demonstrated that both of these F-box proteins are
simultaneously required for efficient destruction of Rum1p
and the replication initiator Cdc18p [ 18 19 20 ] . Rum1p
is a Sic1p-analogous CDK inhibitor, which accumulates in
G1, but is degraded as cells enter S phase [ 21 22 ] .
Failure to degrade Rum1p is the major phenotypic defect of
pop1 and
pop2 deletion strains, which leads to
disturbance of normal cell cycle progression, resulting in
polyploidy [ 18 19 20 23 ] . Based on the genetic data and
the biochemical observation that Pop1p and Pop2p interact
when overexpressed, a putative SCF Pop1p-Pop2pcomplex was
proposed, which would contain the heterooligomerizing F-box
proteins Pop1p and Pop2p bound to SCF core components [ 18
20 ] .
Whether this unusual heterooligomeric SCF
Pop1p-Pop2pcomplex exists
in vivo and whether it mediates Rum1p
ubiquitylation, remained unproven, as not all fission yeast
SCF core subunits were identified. In addition, based on
overexpression, distinct SCF Pop1pand SCF Pop2pcomplexes
were proposed to target unknown substrates, but no
biochemical evidence of their activity was provided [ 18 ]
. To address these questions, we cloned two additional
subunits of SCF Popand performed a detailed
characterization of its activity
in vitro and
in vivo . Our results indicate that
heterooligomeric SCF Pop1p-Pop2pmediates Rum1p
ubiquitylation whereas distinct SCF Pop1pand SCF
Pop2pcomplexes target unknown nuclear and cytoplasmic
substrates, thereby generating combinatorial diversity of
SCF function in fission yeast.
Results
Composition of SCF Pop1p-Pop2p
Based on the composition of known SCF complexes, we
identified in the
S. pombe genome database
psh1 (pombe skp1 homologue) and
pip1 (pop interacting protein 1),
two genes encoding proteins with strong similarity to
human SKP1 and HRT1/RBX1/ROC1, respectively (data not
shown). Consistent with these proteins being components
of the putative SCF Pop1p-Pop2pcomplex, they all
co-purified with Pop1p, Pop2p, and Pcu1p when
overexpressed pairwise (data not shown).
Co-immunoprecipitation experiments using affinity
purified rabbit antisera confirmed these interactions at
the level of the endogenous proteins. While each of the
five antisera co-precipitated at least one of the other
subunits, Pip1p, Pop1p, and Pop2p antisera
co-precipitated all five proteins from wild-type cell
lysate (Fig. 1A). Size fractionation of total cell
lysates prior to immunoprecipitation revealed co-elution
of Pip1p with Pop1p, Pop2p, Pcu1p, and Psh1p in a high
molecular weight complex of approximately 500 kDa, which
we refer to as SCF Pop1p-Pop2p(Fig. 1B). The composition
of the core complex Pip1p/Pcu1p/Psh1p did not undergo
major variations during the cell cycle (Fig. 1C). We have
carefully reexamined potential cell cycle variations of
Pcu1p neddylation apparent in the IP/immunoblotting
experiment in Fig. 1C. These variations were not seen
when samples were denatured in SDS immediately following
extract preparation (data not shown), suggesting that
they arise from varying degrees of deneddylation
presumably occuring during the immunoprecipitation step.
In addition, in a separate experiment, Pop1p-Pop2p
heterooligomerization was largely constant during the
cell cycle (Fig. 1C). These findings indicated that cell
cycle-dependent substrate degradation is unlikely to be
controlled at the level of SCF Pop1p-Pop2pcomplex
formation.
SCF Pop1p-Pop2pmediates polyubiquitylation of Rum1p
in vitro
Genetic experiments suggested that degradation of
Rum1p depends on the Pop1p and Pop2p F-box proteins [ 18
20 ] , but also requires phosphorylation of Rum1p on
serine 58 and threonine 62 by cyclin-dependent kinase
(CDK) [ 21 ] . Thus, phosphorylated Rum1p may be a
substrate for SCF Pop1p-Pop2p-mediated
polyubiquitylation. To test this, we first confirmed that
Rum1p is an
in vitro substrate for the Cdc2p
kinase in association with the cyclin Cig1p [ 21 ] as
judged by a mobility shift on SDS gels (Fig. 2A).
Bacterially expressed Rum1p purified to apparent
homogeneity was also efficiently phosphorylated by
Cdc2p/Cig1p complexes (Fig. 2A).
To determine whether phosphorylated Rum1p interacts
with Pop1p and Pop2p, protein lysate was prepared from
cells co-overexpressing epitope-tagged combinations of
Pop1p and Pop2p. Upon affinity purification on Ni-NTA
resin, HA-Pop1p/His-Myc-Pop2p complexes were incubated
with bacterially expressed, phosphorylated Rum1p. In this
reaction, Pop1p-Pop2p complexes specifically bound
phosphorylated Rum1p (Fig. 2B, lane 1). Consistent with
the genetic data [ 20 ] , His-Myc-Pop1p and His-Myc-Pop2p
individually purified upon overexpression in
pop1 pop2 double mutants exhibited
no Rum1p binding above background (Fig. 2B, lanes 2 and
3).
Given our ability to prepare immunopurified SCF
Pop1p-Pop2pthat bound phosphorylated Rum1p, we sought to
reconstitute Rum1p polyubiquitylation
in vitro. In addition, we required
an ubiquitin activating enzyme (E1) and an ubiquitin
conjugating enzyme (UBC). While human E1 is highly
similar to its fission yeast counterpart (data not
shown), inspection of the
S. pombe genome revealed fourteen
potential UBCs, none of which is an obvious homologue of
human UBC3 or budding yeast Cdc34p, since all lack the
characteristic C-terminal extension (data not shown). We
therefore purified recombinant human E1 and UBC3 (CDC34)
upon expression in bacteria (Fig. 2C).
In the presence of human E1, UBC3, ubiquitin, and ATP,
SCF Pop1p-Pop2pcomplexes immunopurified with Pip1p
antibodies converted a small portion of phosphorylated
Rum1p into high molecular weight species (Fig. 2D). This
conversion was dependent on the addition of E1, ATP (data
not shown), and wild-type UBC3 (Fig. 2D, lanes 2,4,6).
The activity of SCF Pop1p/Pop2pwas augmented when Pip1p
complexes where purified from
csn5 mutants (Fig. 2D, lane 5).
This mutant accumulates Pcu1p exclusively in a form
carrying the stimulatory Nedd8p modification, due to a
defect in COP9/signalosome-mediated cullin deneddylation
[ 24 25 ] . Replacing wild-type ubiquitin by a mutant
lacking all lysine residues prevented the formation of
high molecular weight products (Fig. 2D, lane 7),
indicating that they represent polyubiquitylated Rum1p
species generated in the reaction. Similar
polyubiquitylated reaction products were detected upon
incubation of phosphorylated Rum1p with Pcu1p
immunocomplexes, further suggesting that the activity is
mediated by SCF Pop1p-Pop2p(data not shown). Moreover,
Rum1p ubiquitylation was not obtained with Pip1p
complexes prepared from cell lysate of
pop2 deletion strains, proving the
F-box protein dependency of this reaction (Fig. 2D, lane
3). In addition, the reaction was specific for human
UBC3, as no ubiquitylation was obtained with fission
yeast Ubc1p, Ubc7p, Ubc11p, or Ubc13p (Fig. 2, lanes
10,11). Taken together these results strongly suggest
that SCF Pop1p-Pop2pmediates the polyubiquitylation of
CDK phosphorylated Rum1p
in vitro .
Differential subcellular localization of SCF
Pop1p-Pop2psubunits
The co-purification of the five identified SCF
Pop1p-Pop2psubunits and their
in vitro activity toward Rum1p
suggested that they coexist in a common subcellular
compartment. The nuclear localization of the only known
substrates, Cdc18p [ 26 ] and Rum1p (D.A.W.,
unpublished), indicated that a substantial portion of SCF
Pop1p-Pop2pis enriched in the nucleus. To test this
assumption, all five SCF Pop1p-Pop2psubunits were
expressed as fusion proteins with green fluorescent
protein (GFP) at low levels from an inducible pRep81
plasmid. While Pip1p, Psh1p, Pcu1p, and Pop2p were
present in both the cytoplasm and the nucleus,
surprisingly, GFP-Pop1p was largely restricted to the
nucleus (Fig. 3A). These localization patterns were
consistently observed in each single cell of an
asynchronous population, excluding major variations
during the cell cycle.
To rule out the possibility that overexpression or
N-terminal fusion to GFP affects their localization,
Pop1p and Pop2p were modified with 13 C-terminal Myc
epitope-tags at the endogenous genomic locus.
Immunoblotting proved the expression of correctly sized
proteins and, in addition, showed that endogenous Pop1p
is approximately twofold more abundant in
S. pombe cells than Pop2p (Fig.
3B). Indirect immunofluorescence staining with Myc
antibodies confirmed that Pop1p is predominantly
localized to cell nuclei, whereas Pop2p is expressed in
both the cytoplasm and the nucleus (Fig. 3C).
To confirm these localization patterns, cells derived
from the epitope-tagged strains were biochemically
fractionated into cytoplasmic and nuclear components. The
efficiency of enrichment of nuclear and cytoplasmic
components was estimated by analyzing fractions with
antibodies recognizing the nuclear marker PCNA and
cytoplasmic tubulin (Fig. 3D). Although both fractions
showed some contamination, Pop1p was detected mostly in
nuclear fractions, while Pop2p was apparent in both
nuclear and cytoplasmic fractions (Fig. 3D). Thus, all
five SCF Pop1p-Pop2psubunits appear to coexist in the
nucleus, although all but Pop1p are also present in the
cytoplasm.
Since Pop1p and Pop2p interact with each other [ 20 ]
, we asked whether their localization patterns depended
on the presence of the respective interaction partner.
For this, we created a
pop2 deletion strain carrying Pop1p
modified with 13 Myc epitope tags at the endogenous
genomic locus (
pop1-13myc Δpop2 strain ). In
addition, we created the reciprocal
pop2-13myc Δpop1 strain containing
epitope-tagged endogenous Pop2p in a
pop1 deletion background. The
distinct localization patterns of Pop1p-13Myc and
Pop2p-13Myc were fully maintained in these strains (Fig.
3E). This observation was confirmed by overexpressing
GFP-tagged Pop1p or Pop2p in
pop1 pop2 double deletion mutants
(data not shown). These data indicate that Pop1p and
Pop2p assume their subcellular localization pattern
independent of each other, indicating the possibility of
distinct nuclear and cytoplasmic homooligomeric SCF
Pop1pand SCF Pop2pcomplexes.
Differential F-box requirements of Pop1p and
Pop2p
As shown above and in previous genetic work [ 18 20 ]
, SCF Pop1p-Pop2p-dependent Rum1p degradation requires
two different proteins with highly conserved F-boxes
(Fig. 4A). To better understand the apparent dual F-box
requirement for SCF Pop1p-Pop2pfunction, we generated
mutants of Pop1p and Pop2p lacking F-boxes (Pop1p-ΔF,
Pop2p-ΔF; Fig. 4B). In addition, we prepared a set of
mutants, in which the F-boxes of Pop1p and Pop2p were
swapped (Pop1p-2F, Pop2p-1F; Fig. 4B). The mutants were
tested for their ability to suppress polyploidy and Rum1p
accumulation in the respective
pop mutant strains.
As described previously [ 20 ] , wild-type Pop1p
mildly overexpressed from a pRep81 plasmid fully
complemented the polyploidization phenotype of
pop1 mutants as determined by flow
cytometric measurement of the cellular DNA content (Fig
4C). In addition, Rum1p accumulation in
pop1 mutants was efficiently
reversed by wild-type Pop1p (Fig. 4D). In contrast, Pop1p
lacking its F-box (Pop1p-ΔF) or Pop1p, in which the F-box
was replaced by the F-box of Pop2p (Pop1p-2F) were
largely inactive in both assays (Fig. 4C,4D). Thus, as
with many other F-box proteins, the F-box of Pop1p is
essential for its
in vivo functions.
In contrast, wild-type Pop2p, the corresponding F-box
mutant, and Pop2p containing the Pop1p F-box were equally
effective in preventing Rum1p accumulation (Fig. 4D). The
same wild-type and mutant proteins also reversed the mild
polyploidy phenotype of
pop2 disruptants (Fig. 4C). Thus,
in contrast to Pop1p, Pop2p does not seem, to require its
F-box to mediate Rum1p degradation
in vivo .
To further substantiate this conclusion, we examined
Rum1p protein stability in wild-type and
pop mutant strains expressing
F-box-deleted Pop proteins from the weak pRep81 promoter.
Since the sensitivity of our Rum1p sera was insufficient
to detect the low levels present in wild-type cells (see
Fig. 4D, lane 9), these experiments were conducted in a
background where endogenous Rum1p was modified with 13
C-terminal c-Myc epitope tags. Rum1p half-life was
increased from ~20 minutes in wild-type to greater than
100 minutes in
pop1 or
pop2 mutants (Fig. 5). While F-box
deleted Pop2p expressed from plasmids reduced Rum1p
half-life to ~20 minutes in
pop2 mutants, F-box-deleted Pop1p
was completely defective in rescuing the Rum1p
proteolysis defect of
pop1 mutants (Fig. 5). Instead,
expression of Pop1p-ΔF in
pop1 mutants led to even greater
stabilization of Rum1p, potentially due to dominant
negative interference with the residual activity of Pop2p
and/or other F-box proteins.
F-box independent interaction of Pop1p and
Pop2p
The failure of the Pop2p F-box to replace the F-box of
Pop1p as well as the finding that it is not essential for
Rum1p degradation could be explained, if it was not
critically involved in SCF Pop1p-Pop2pprotein
interactions. We therefore tested the possibility that
Pop2p can be tethered to the SCF core complex
independently of its F-box via an interaction with Pop1p.
Consistent with this hypothesis, co-immunoprecipitation
experiments of overexpressed proteins revealed that the
Pop1p-Pop2p interaction occurs independently of the
F-boxes of both Pop1p and Pop2p (Fig. 6A).
We had previously mapped the domain of Pop2p that
interacts with Pop1p to an N-terminal fragment consisting
of the first 241 amino acids and lacking the F-box ( [ 20
] , Fig. 6B, lane 8). In co-immunoprecipitation
experiments with overexpressed proteins, this fragment
also bound to an N-terminal piece containing the first
402 residues of Pop1p (Fig. 6B, lane 12). Thus, the
Pop1p-Pop2p interaction is mediated by their N-terminal
domains. A further truncation mutant mapped the Pop2p
binding domain to a region between residues 228 and 402
of Pop1p (Fig. 6B, lanes 9,10).
Individual SCF Pop1pand SCF Pop2pcomplexes bearing
ubiquitin ligase activity
The apparent dispensibility of the F-box of Pop2p for
Rum1p degradation and binding to Pop1p raised the
question of why Pop2p does contain an F-box. Based on the
subcellular localization data, we considered the
possibility that the F-box of Pop2p may mediate the
assembly of a cytoplasmically localized SCF Pop2pcomplex,
independent of Pop1p. To demonstrate this, we again used
the strain in which endogenous Pop2p was modified by 13
Myc epitope tags in a
pop1 deletion background. The same
experiments were carried out with the reverse stain,
which contained Pop1p-13Myc in a
pop2 background. As a reference for
SCF Popcomplex formation, we used strains carrying 13Myc
epitope-tagged Pop1p or Pop2p integrated into the genome
of wild-type cells.
Pip1p immunoprecipitates were prepared from lysates of
these four strains and appropriate controls, and
co-purification of SCF components was determined by
immunoblotting. These experiments showed that both F-box
proteins, in the absence of their respective
heterooligomerization partner, could individually bind to
Pip1p in complexes that also contained Psh1p and Pcu1p
(Fig. 7A). These findings indicate the existence of
distinct SCF Pop1pand SCF Pop2pcomplexes
in vivo .
To further substantiate this conclusion, we used gel
filtration to compare the elution profiles of Pop1p and
Pop2p in the presence or absence of their respective
dimerization partners. If recruitment of Pop2p into a
high molecular weight SCF complex required
heterooligomerization with Pop1p, its elution profile
would be expected to shift to a smaller size in the
absence of Pop1p. Consistent with the results presented
in Fig. 1C, Pop2p, together with SCF core subunits,
eluted in fractions corresponding to 400 - 600 kDa,
irrespective of whether Pop1p was present or not (Fig.
7B). In the reverse experiment, the elution profile of
Pop1p was found to be independent of the presence of
Pop2p (Fig. 7B).
While these data support the existence of distinct SCF
Pop1pand SCF Pop2pcomplexes, individual binding of Pop1p
and Pop2p to SCF core components in the absence of their
heterodimerizing F-box protein partners does not rule out
the possibility that these complexes represent inactive
intermediates formed during the normal assembly of
functional SCF Pop1p-Pop2pcomplexes. To exclude this
possibility, we asked whether distinct SCF Pop1pand SCF
Pop2pcomplexes bear ubiquitin ligase activity
in vitro . To this end, we
performed
in vitro ubiquitylation assays.
Since the substrates of putative SCF Pop1pand SCF
Pop2pubiquitin ligases are unknown, we adopted a
substrate-independent assay originally described by
Lyapina
et al . [ 27 ] . For this
experiment, we again used the strains harboring
genomically integrated Myc epitope-tagged Pop1p or Pop2p
in a background deficient in the respective
heterooligomerization partner (
Δpop2 pop1-13myc ,
Δpop1 pop2-13myc strains). Pop1p
and Pop2p complexes were immunopurified with Myc
antibodies and employed in
in vitro ubiquitylation assays upon
addition of E1, UBC3, ubiquitin, and ATP. High molecular
weight products generated in the reaction were detected
by immunoblotting with ubiquitin antibodies. As
references, we used strains expressing Myc-tagged Pop1p
or Pop2p in a wild-type background. The experiment
demonstrated that Pop1p and Pop2p each associate with
polyubiquitylation activity even in the absence of their
respective heterooligomerizing F-box proteins (Fig. 7C).
Thus Pop1p and Pop2p appear to assemble into distinct SCF
complexes bearing ubiquitin ligase activity
in vitro .
Discussion
Molecular architecture of SCF Popcomplexes
The phenomenon of F-box protein oligomerization is not
unique to SCF Pop. Although the crystal structure of the
SKP1-SKP2 complex, derived from bacterially expressed
proteins, revealed a single SKP2 monomer bound to SKP1 [
28 ] , biochemical studies showed that budding yeast
Cdc4p, a close homologue of Pop1p\Pop2p, forms
homooligomers when expressed in insect cells (Correll and
Deshaies, personal communication). Similarly, β-TRCP1 and
2, which target IκBα for degradation as homooligomers,
form heterooligomers that each bind SCF core subunits,
although no biochemical activity for this
heterooligomeric complex was demonstrated [ 29 ] .
Finally, Pop1p and Pop2p homooligomerize, at least when
overexpressed [ 18 ] , indicating that both F-box
proteins may also be present as homooligomers in
individual SCF Pop1pand SCF Pop2p, respectively.
A surprising finding of this study was that the F-box
of Pop2p is dispensable for Rum1p degradation and ploidy
control, while the F-box of Pop1p is essential for both
functions (Fig. 4Dand 5). The Pop2p F-box is unlikely to
be a degenerate, non-functional, and hence dispensable,
motif, as it carries all signature residues of the F-box
(Fig. 4A). In addition, Pop2p, in the absence of Pop1p,
assembles into a complex containing all SCF core subunits
identified here (Fig. 7A). At present, we cannot exclude
that residues outside the F-box mediate binding of F-box
deleted Pop2p to Psh1p and other core subunits.
Consistent with this idea, biochemical studies based on
the crystal structure of the human SKP1-SKP2 complex
revealed cooperation of the SKP2 F-box with an adjacent
region in binding of SKP1 [ 28 ] . Consistent with this
finding, certain truncation mutants of the budding yeast
F-box proteins Grr1p and Cdc4p interact poorly with
Skp1p, despite the retainment of their F-boxes [ 30 31 ]
. On the other hand, the F-box of Pop1p is essential for
Rum1p degradation (Fig. 5), arguing that residues outside
the F-box are insufficient to mediate recruitment of Pop
proteins into SCF complexes.
Based on the non-essential function of the Pop2p
F-box, we propose a molecular architecture of SCF
Pop1p-Pop2p, in which Pop2p is tethered to the core
subunits through interaction with Pop1p. Although we have
no direct biochemical evidence to confirm this
proposition, which would require Pop2p point mutants
deficient in Pop1p binding, our data show that severely
truncated N-terminal fragments of Pop1p and Pop2p lacking
both F-boxes and WD repeat domains are sufficient to
mediate their interaction (Fig. 6B). Similarly,
dimerization of β-TRCP proteins is mediated by N-terminal
"D-domains" lacking binding of SKP1 and other SCF core
subunits [ 29 ] . According to our model, the F-box of
Pop2p would be essential only for the Pop1p-independent
activities of Pop2p, for which we provide tentative
evidence by demonstrating the
in vivo assembly of SCF
Pop2pcomplexes (Fig. 7A). These complexes bear ubiquitin
ligase activity in a substrate-independent
in vitro assay (Fig. 7C). To what
extent this assay reflects the
in vivo activity of SCF Pop2pwill
become testable, once the putative substrates of SCF
Pop2pare identified.
Subcellular compartmentalization of SCF Popas a
potential mechanism for substrate selection
Another surprising observation of this study was that
Pop1p is primarily localized to the nucleus, whereas
Pop2p is present in both the cytoplasm and the nucleus
(Fig. 3). While, nuclear localization was expected, since
both known substrates of SCF Pop1p-Pop2p, Cdc18p and
Rum1p, are primarily nuclear proteins, the cytoplasmic
localization of Pop2p suggests an activity of SCF
Pop2pdirected toward unknown cytoplasmic substrates. In
support of this notion, as with human SCF subunits [ 32 ]
, fission yeast SCF core subunits are also present in the
cytoplasm, as shown here for overexpressed GFP-fusions
(Fig. 3A), and for endogenous Pcu1p by immunostaining in
a previous report [ 24 ] .
By analogy, additional nuclear substrates of SCF
Pop1pmay exist. For example, Pop1p is involved in the
control of the RNA levels of the cyclin Cig2p [ 33 ] .
While it is unclear whether this effect is mediated at
the level of transcription or mRNA stability, the budding
yeast F-box protein Met30p was recently shown to regulate
the ubiquitylation and activity, but not degradation, of
the transcription factor Met4p [ 34 ] . In addition,
pop1 mutants display an increased
rate of chromosome loss, a phenotype that is not easily
explained by accumulation of Rum1p and Cdc18p [ 23 ] .
Finally,
pop1 mutants are sensitive to UV
irradiation, whereas
pop2 mutants are not (D. Griffiths
and D.A.W., unpublished observation). It is therefore
likely that other substrates of Pop1p and Pop2p, in
addition to their common substrates Cdc18p and Rum1p, do
exist.
The idea developed above that F-box protein
compartmentalization contributes to substrate selection
was recently confirmed directly for Cdc4p-mediated
degradation of Far1p in budding yeast [ 35 ] . Both,
Cdc4p and Far1p are nuclear proteins in vegetative cells,
owing to the presence of nuclear localization signals
(NLS). Fusion of Cdc4p with a nuclear export signal
(NES-Cdc4p) prevented its nuclear localization and its
ability to direct the degradation of nuclear Far1p. When
Far1p also was targeted to the cytoplasm by disrupting
its NLS, NES-Cdc4p degraded ΔNLS-Far1p in the cytoplasm [
35 ] .
A putative NLS is also present in the N-terminus of
Pop1p located between the F-box and the WD-repeat domain
( 393PEKIKRC). An N-terminal Pop1p fragment containing
this motif, when fused to the WD-repeat region of Pop2p,
is targeted exclusively to the nucleus (R.L. &
D.A.W., unpublished observation). Like wild-type Pop2p,
the reverse Pop2p-N/Pop1p-C chimera localizes to both the
nucleus and the cytoplasm, again indicating that Pop1p,
but not Pop2p, has a functional NLS in its N-terminus
(R.L. & D.A.W., unpublished observation). It is
unclear, at present, what regulates Pop2p distribution.
One possibility is that Pop2p is co-imported in a complex
with other SCF subunits that is preformed in the
cytoplasm. Since Pop2p distribution is independent of
Pop1p (Fig. 3E), SCF core subunits are the most likely
candidates for such a function. In line with this
suggestion, it was previously demonstrated that
HRT1/ROC1/RBX1 promotes nuclear accumulation of CUL1 [ 32
] .
Conclusion
Our data suggest homo- and heterooligomerization of the
F-box proteins Pop1p and Pop2p as a mechanism for
generating combinatorial diversity of SCF function in
fission yeast. A heterooligomeric SCF Pop1p-Pop2pcomplex
mediates polyubiquitylation of phosphorylated Rum1p. In
addition, compartmentalization of homooligomeric SCF
Pop1pand SCF Pop2pcomplexes may direct the ubiquitylation
of unknown nuclear and cytoplasmic substrates.
Methods
Plasmids and yeast strains
S. pombe genes for
psh1 and
pip1 were identified in the Sanger
Centre
S. pombe sequence database based on
their homology to the respective human and budding yeast
proteins. Complementary DNAs were amplified by PCR,
subcloned into pREp81.6xHis-Myc, pRep3.6His-Myc, or
pRep4.HA, and sequenced. Deletion strains and
epitope-tagged stains were constructed by one-step gene
replacement using PCR-generated fragments containing
kanamycin or
ura4 cassettes [ 36 ] . Growth
media, flow cytometry, and all other relevant
S. pombe techniques were described
previously [ 37 ] .
To generate the
pop1::ura4 pop2-13myc-kan strain,
an
h +
pop1::ura4 ura4-d18 leul-32
pRep81.pop1 strain was crossed with a
h -
leul-32 ura4-d18
pop2-13myc-kan strain, followed by selection of
spores on G418/ura -EMM plates. The pRep81.
pop1 plasmid required to complement
the sterility of the
pop1 deletion strain was lost by
growth in non-selective media (YES) for several
generations. The resulting strain was verified by PCR and
immunoblotting. The
pop2::ura4 pop1-13myc-kan strain
was generated in an analogous fashion.
Antibodies
Rabbit antisera were raised at Josman LCC (Napa, CA)
against bacterially expressed MBP-Psh1p, GST-Pcu1p,
GST-Pip1p, and GST-Pop1p. Sera were affinity purified on
affinity matrices containing immobilized GST-Psh1p,
MBP-Pcu1p, MBP-Pip1p, and MBP-Pop1p. Column eluates were
concentrated to ~1mg/ml and tittered by immunoblotting.
Rabbit antisera against Pop2p and Rum1p were described
before [ 20 ] . Monoclonal Myc and HA antibodies were
purified from 9E10 and 12CA5 tissue culture supernatants
by binding to protein A.
Immunoprecipitation and immunoblotting
Protein lysates for immunoblotting were prepared by
bead lysis in a Fastprep device (Bio 101) in the presence
of proteinase inhibitors, followed by boiling in SDS
sample buffer. Cell lysates for small scale
immunoprecipitations were prepared by disrupting cells in
immunoprecipitation buffer (20 mM Tris/HC1, pH7.4; 150 mM
NaC1; 0.5% Triton X-100, 10 ug/ml leupeptin, 10 ug/ml
pepstatin, 17 ug/ml aprotinin, 1 mM PMSF). Lysates were
cleared and precipitated with the respective antisera.
Immunocomplexes were collected by binding to protein A or
G beads, washed and analyzed by immunoblotting as
described [ 37 ] .
Large scale lysates for gel filtration and subsequent
immunoprecipitation were obtained by bead beater lysis.
Approximately 5 mg of total cell lysates was separated by
gel filtration on a 16/60 S300 column (Amersham Pharmacia
Biotech), and 1 ml fractions were immunoprecipitated with
Pip1p antibodies. Precipitates were fractionated by SDS
PAGE and assayed by immunoblotting with the respective
antisera.
Indirect immunofluorescence
Indirect immunofluorescence staining was performed
exactly as described [ 25 ] .
In vitroubiquitylation assay
For ubiquitylation reactions, Pip1p complexes were
immunoprecipitated from 100 - 200 ug total cell lysates
prepared as described above. Precipitates were washed
four times in 20 mM Tris/HCI, pH 7.4; 150 mM NaCl; 0.5%
Triton X-100, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 5
μg/ml aprotinin, 1 mM PMSF, and equilibrated in 20 mM
HEPES, pH 7.4, 100 mM potassium acetate, 1 mM DTT. A
cocktail was added that contained 8 μM ubiquitin, ATP
regenerating system (2 mM HEPES at pH 7.4, 1 mM ATP, 30
mM creatine phosphate, 1 mM magnesium acetate, 0.15 mg/ml
creatine kinase), reaction buffer (4 mM magnesium
acetate, 1mM DTT), 500 nM bacterially expressed 6 ×
His-UBCs, 100 nM 6 × His-tagged human E1, 0.5 μM
ubiquitin aldehyde in a volume of 15 ul. The reaction was
started by addition of phosphorylated Rum1p. After 90 min
at 30°C, the reaction was terminated by the addition 5 ×
SDS sample buffer. Samples were separated on 11 %
SDS-polyacrylamide gels and analyzed by
autoradiography.
Substrate-independent
in vitro ubiquitylation activity
was determined by immunoprecipitating Pop1p-13Myc or
Pop2p-13Myc with Myc antibodies. The same cocktail as
described above was added to the precipitates. Reaction
products were determined by immunoblotting with ubiquitin
antibodies (Zymed).
Subcellular fractionation
Cells grown in YES were harvested and washed in buffer
S (1.4 M sorbitol, 40 mM HEPES (pH = 7.2), 0.5 mM MgCl
2 ). Cells were resuspended in buffer
S, 1 mM PMSF, 10 mM β-mercaptoethanol and incubated for
10 min at 30°C. Cells were pelleted, resuspended in 4
pellet volumes of buffer S, 1 mM PMSF, Zymolyase (100
ug/ml) and incubated at 30°C for 40 min. Cells were
diluted in buffer S and pelleted by centrifugation and
resuspended in buffer F (18 % Ficoll 400, w/v; 20 mM
HEPES (pH = 7.2), 0.5 mM MgCl
2 and protease inhibitors. Cells were
lysed by homogenization using a teflon pestel fitted into
a microfuge tube. Cell lysis was monitored by microscopy.
Unlysed cells were pelleted by gentle centrifugation. The
lysate was placed on top of buffer GF (7% Ficoll 400,
w/v, 20% glycerol, 20 mM HEPES (pH = 7.2), 0.5 mM MgCl
2 ). Nuclei were pelleted by spinning
at 7000 rpm in a microfuge. The cytoplasmic fraction was
removed and mixed with SDS sample buffer. The nuclear
pellet was resolved in an equal volume of SDS sample
buffer. Fractions were analyzed by immunoblotting as
described in Fig. 3.
Abbreviations
SCF: SKP1/CUL1/F-box protein
UBC: ubiquitin-conjugating enzyme
HA: hemagglutinin
NLS: nuclear localization signal
NES: nuclear export signal
Authors' contributions
All experiments were performed by VS. with the following
exceptions: Building on reagents prepared by VS., CP.
performed the experiments shown in Figs. 2B, 4A, and 7. IS.
performed the experiments shown in Fig. 4D, 5, and 6A. ER.
performed the immunostaining experiments shown in Fig.
3Cand 3E. RL. performed the experiment shown in Fig. 3Aand
contributed data not shown. KA. produced recombinant UBCs
used in Fig. 2D. CZ. prepared the
csn5 mutant used in Fig. 2D,
contributed to the preparation of SCF Popantisera used in
Figs. 1, 2D, and 7, and assisted with study design. DAW.
performed the experiment in Fig. 6B, conceived the study,
drafted the manuscript, and participated in study design
and coordination. All authors read and approved the final
manuscript.
