From the Laboratoire de Génétique
Moléculaire (GEMO), University of Namur (Facultés
Universitaires Notre-Dame de la Paix), 61 Rue de Bruxelles, 5000 Namur, Belgium and the § Haartman Institute & Biocentrum
Helsinki, University of Helsinki, 00014 Helsinki, Finland
Received for publication, November 7, 2002, and in revised form, December 27, 2002
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ABSTRACT |
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Here we report functional characterization
of the essential fission yeast Skp1 homologue. We have created a
conditional allele of skp1 (skp1-3f) mimicking
the mutation in the budding yeast skp1-3 allele. Although
budding yeast skp1-3 arrests at the G1/S transition, skp1-3f cells progress through S phase
and instead display two distinct phenotypes. A fraction of the
skp1-3f cells arrest in mitosis with high Cdc2 activity.
Other skp1-3f cells as well as the
skp1-deleted cells accumulate abnormal thick septa leading
to defects in cell separation. Subsequent identification of 16 fission
yeast F-box proteins led to identification of the product of
pof6 (for pombe F-box) as a
Skp1-associated protein. Interestingly, cells deleted for the essential
pof6 gene display a similar cell separation defect noted in
skp1 mutants, and Pof6 localizes to septa and cell tips.
Purification of Pof6 demonstrates association of Skp1, whereas the Pcu1
cullin was absent from the complex. These findings reveal an
essential non-Skp1-Cdc53/Cullin-F-box protein function for the
fission yeast Skp1 homologue and the F-box protein Pof6 in cell separation.
Degradation of targets critical for the G1/S
transition is mediated by the Skp1-Cdc53/Cullin-F-box protein
(SCF)1 ubiquitin ligase
complex. The prototype SCFCdc4 (where the superscript
denotes the identity of the variable F-box protein subunit) was defined
in budding yeast by both genetic analysis and in vitro
reconstitution. Mutants of cdc34, cdc4, cdc53, and skp1 have overlapping phenotypes and
are compromised in their ability to degrade the cyclin-dependent
kinase inhibitor Sic1 (1, 2). Degradation of Sic1 through
ubiquitination is required for the G1/S transition and is
mediated by the SCF components Cdc4, Cdc53, and Skp1 together with E1,
the E2 enzyme Cdc34p, ATP, and ubiquitin (3, 4). Phosphorylation
of Sic1 on a set of cyclin-dependent kinase consensus sites is a
prerequisite for its recognition by the ubiquitin ligase (5, 6), and phosphorylation appears to be a general requirement for the recognition by a SCF ubiquitin ligase (7).
In addition to its role in G1, budding yeast Skp1 also
belongs to at least three non-SCF type complexes. It is part of the centromere-binding CBF3 complex (8). On the budding yeast kinetochore, Skp1 is required for the activation of Ctf13 (9). Together with the
F-box protein Rcy1, Skp1 plays a role in the recycling of the SNARE
Snc1 (10, 11). Finally, Skp1 has also been reported to form a complex
called RAVE with Rav1 and Rav2 to promote assembly of the
vacuolar-ATPase holoenzyme (12, 13).
The 40-residue F-box domain is required (1) but not sufficient (14) for
the interaction between a given F-box protein and Skp1. F-box proteins
are implicated in the regulation of many cellular processes including
transcription, signal transduction, development, metabolism, and cell
cycle control (15-17) reflected by the abundance of F-box proteins in
budding yeast and mammalian cells (16, 18).
In the fission yeast Schizosaccharomyces pombe, four F-box
proteins have been characterized. The Cdc4-related Pop1 and Pop2/Sud1 (19-22) participate in the formation of SCF as either hetero- or homodimers (21-23). In pop mutants, both the short-lived
Cdc2-Cdc13 inhibitor Rum1 and the S-phase regulator Cdc18 were
stabilized (20, 21). Rum1 is the functional homologue of Sic1 (24), and
Cdc18 is the homologue of budding yeast Cdc6 (25). Both budding yeast
proteins are degraded through SCFCdc4 (26). pop
mutations also result in constitutive transcription of cig2
(27) encoding a B-type cyclin required for normal S-phase (28, 29).
Two other non-essential F-box proteins Pof10 and Pof3 have been
reported. Although the function of Pof10 remains to be characterized (30), Pof3 is required for genome integrity and control of telomere length (31).
In this study we have identified and characterized functions of the
fission yeast Skp1 homologue. We have also identified a novel essential
F-box protein Pof6 that associates with Skp1 in a non-SCF complex.
Interestingly, both Skp1 and Pof6 appear to be essential for cell
separation of fission yeast cells.
Yeast Strains and Techniques--
S. pombe strains
used: h+ ade6-210 ura4D18 leu1-32,
h Cloning of skp1 Genomic Insert and cDNA--
A small-scale
library of S. pombe genomic DNA was constructed and probed.
Southern blot analysis revealed that when genomic DNA is digested with
EcoRI-HindIII, a probe corresponding to the partial skp1 cDNA hybridizes a band of about 1.7 kb. Genomic DNA was digested with EcoRI and
HindIII, and the fragments ranging from 1.5 to 2 kb were
cloned in pRS306. The resulting library was probed with the same probe.
Positive clones were sequenced and shown to contain a 1.7-kb insert
harboring the skp1 gene. A full-length cDNA of
skp1 was cloned by PCR on an S. pombe
cDNA library (a kind gift of M. Minet and F. Lacroute) with
the following primers:
5'-AGC-GGC-CGC-GTC-GAC-GAA-TTC-ATG-TCC-AAA-ATC-AAA-C-3' and
5'-TGC-GGC-CGC-GGA-TCC-CTC- GAG-CTA-TCT-GTC-TTC-GG-3'.
Deletion of skp1 and pof6--
700 bp upstream (PCR1) and 400 bp
downstream (PCR2) regions of the skp1 open reading
frame were amplified using the following primers:
5'-CCG-GAA-TTC-TAG-TAA-CTC-CAC-TAA-CA and
5'-CCC-AAG-CTT-TGT-TGA-TGT-ATG-TAA-GTA-ATG-GAT-3' for PCR1
and 5'-CCC-AAG-CTT-TTT-GTG-GCT-TTT-TGT-GTA-TA-3' and
5'-CCG-CTC-GAG-TAA-GCA-ATT-CGG-GAT-AGT-AAG-3' for
PCR2. The EcoRI-HindIII-digested PCR1 fragment
and the HindIII-XhoI-digested PCR2 fragments were
cloned together in a pSK vector. A HindIII fragment
containing the S. pombe ura4 gene was inserted between the
PCR1 and PCR2 fragments to give the pSK-skp1
A diploid strain was transformed with an
EcoRI-XhoI fragment of the
pSK-skp1 Construction and Integration of skp1 Mutants--
A two-step PCR
approach was used to generate the skp1-3f and
skp1-4f alleles using the following primers:
external oligos, 5'-CCG-GAA-TTC-TAG-TAA-CTC-CAC-TAA-CA-3'
and 5'-CCG-CTC-GAG-TAA-GCA-ATT-CGG-GAT-AGT-AAG-3'; mutated
oligos for skp1-3f,
5'-TCG-GAA-AAC-ATT-CAA-CAA-CCC-TAA-TGA-3' and
5'-TCA-TTA-GGG-TTG-TTG-AAT-GTT-TTC-CGA-3'; mutated oligos for
skp1-4f, 5'-GGA-TAT-CAA-ACC-TTC-GCT-TGA-TAC-TGG-TT-3' and 5'-AAC-CAG-TAT-CAA-GCG-AAG-GTT-TGA-TAT-CC-3'. The
EcoRI-XhoI-digested fragments of the final PCR
products were cloned in pSK digested in
EcoRI-XhoI and sequenced. Subsequently, the
kanR cassette from pFA6a-kanMX (33) was introduced
downstream of the mutant alleles. The
EcoRI-HindIII linear fragments containing the
skp1-3f and skp1-4f mutants, respectively, were
used to transform the strain. Integration was confirmed by Southern
blotting and sequencing.
Western Blotting and H1 Kinase Assays--
For Western blots,
boiled extracts were prepared as described (32) and blots were
probed with anti-Cdc2, anti-Y15, anti-tubulin, anti-Cdt1, or anti-GFP
(all gifts of P. Nurse). The anti-Psh1 is a kind gift of D. Wolf
(34). For the H1 kinase, native extracts were prepared, and 1 mg of
proteins was subjected to immunoprecipitation with anti-Cdc2 antibodies
(a kind gift of P. Nurse) for 40 min, and protein-A-Sepharose was added
for a further 20 min. After 4 washes in HB buffer, kinase buffer
(1 mg/ml Calbiochem H1 histone, 200 µM
[ Fluorescence in Vivo Imaging--
GFP fusions were constructed
under the control of the nmt1 promoter in diploids and visualized in
living cells. Cells were cultured in minimal media supplemented or not
with 5 µM thiamine. Cells were observed using a
100×/1.30 Zeiss oil immersion objective lens. Images were obtained on
a cooled CCD camera with an exposure time of 2 s. Images
were processed with Adobe Photoshop 5.0.
Nitrogen Starvation--
Cells were grown in rich media
overnight. They were harvested and resuspended in minimal media
supplemented with NH4 and amino acids.
When reaching late log phase, cells were filtered, washed, and
resuspended in minimal media lacking NH4. They were grown
in these conditions for 18 h. NH4 was then added back
and temperature raised at the same time.
Spore Germination Assay--
Diploid cells were induced to
sporulate in minimal media lacking NH4. When 99% of asci
were formed, they were digested 16 h in glusulase at 25 °C and
washed. Spores were inoculated in minimal medium, and samples were
examined after 14 and 24 h, fixed and stained with
DAPI.
Tandem Affinity Purification (TAP)--
The pof6 open
reading frame has been tagged with the TAP cassette using the PCR
approach and vectors described by Tasto et al. (45). The TAP
cassette (hereafter referred to as "TAP fusion" or "Pof6-TAP")
consists of a calmoduline-binding domain followed by a peptide
corresponding to the consensus sequence of TEV protease and two
repeats of the IgG-binding domain of protein A. This enables two
purifications to be performed in a row, first taking advantage of the
affinity of IgG for protein A beads and second, after elution from the
beads with TEV, using calmodulin beads. This method has been widely
used in yeast as described by Gavin et al. (35).
The tagged protein can be detected with an IgG-peroxydase antibody
(Sigma) due to the high affinity of IgG for protein A. Hereafter, we
refer to this antibody as "anti-TAP."
Skp1 Is the Essential Fission Yeast Skp1 Homologue--
During a
two-hybrid screen with the Mcs2 cyclin (36), one of the cDNA clones
isolated was found to encode for a fission yeast protein with strong
similarity to Skp1. Subsequently a 1.7-kb genomic fragment containing
an apparent full-length open reading frame was isolated and predicted
to encode a protein with 50% identity to budding yeast Skp1. As the
name skp1 was already in use in fission yeast (37), we have
first entered the new gene into the fission yeast protein data base
(PombeDB) and GenBankTM (accession number AF071066)
as shp1 for Skp1 homologue
pombe, although several other names have been used since,
including psh1 (23, 34) and skp1 (31). However,
for simplicity we decided to use the name skp1 in this
study. The lack of sequences with similarity to skp1 in the
fission yeast genome suggests that as its budding yeast counterpart,
skp1 is unique. Alignment of Skp1 with homologues from
various species reveals high conservation throughout the length of the
protein with the highest similarity in the C terminus (Fig.
1A), especially in the
residues identified as being involved in binding F-box proteins in the
published SCF structure (14, 38).
skp1 is an essential gene as determined by the formation of
two viable spores from sporulation of diploid S. pombe cells
in which one copy of skp1 was deleted (Fig. 1B).
On plates, skp1-deleted spores germinate, and a fraction of
these cells divide once (data not shown). The terminal phenotype of
skp1-deleted cells was further studied by a spore
germination assay. To this end, spores from a diploid heterozygous
for skp1 (skp1/skp1::ura4)
were cultured in medium lacking uracil allowing only the
skp1::ura4 spores to germinate. Analysis of these
spores 24 h after germination (when cell number did not increase
anymore) by DAPI staining revealed slightly elongated cells (Fig.
1C). Unexpectedly, a significant fraction (25%) of the
skp1-deleted cells arrested with two nuclei and a septum
(Fig. 1C, arrows).
A Temperature-sensitive (ts) skp1 Allele Can Pass S-phase and
Arrests with a Pleiotropic Phenotype--
The identification of both a
G1/S and a mitotic function for the budding yeast
SKP1 was based on two functionally distinct classes of ts
mutants. skp1-11 (1) and skp1-3 (8) arrests at
the G1/S transition. By contrast, skp1-12 (1)
and skp1-4 (8) cells display either a severe defect
(skp1-12) or total inability (skp1-4) to
progress through mitosis and accumulate as large budded elongated cells
with a G2 DNA content. skp1-3 and the
skp1-4 alleles contain single amino acid changes in highly conserved residues (arrows in Fig. 1A).
skp1-4 contains a serine substitution at Leu-146
forming part of helix 6, and skp1-3 contains an asparagine
substitution at Ile-172 directly contacting the F-box in the human
Skp1-Skp2 structure (14). This prompted us to test whether mimicking
these mutations in fission yeast skp1 would generate
ts alleles. The resulting alleles were named skp1-3f (I139N) and skp1-4f (L113S; Fig. 1A). When
analyzed for temperature sensitivity skp1-4f cells did not
differ from wild type cells (Fig. 1D). By contrast, the
skp1-3f allele was unable to grow at 37 °C (Fig.
1D) and was chosen for further analysis. At the permissive
temperature (25 °C), exponentially growing skp1-3f cells
were indistinguishable from wild type cells, but after 4 h at the
restrictive temperature (37 °C), skp1-3f cells arrested cell cycle progression with a 2C DNA content as shown by FACS (Fig. 1E). Cytological analysis revealed that a fraction of
the arrested cells are elongated, resulting in a slight drift of the FACS 2C peak. Beside this phenotype, cells with multiple or very thick
septum were also observed (Fig. 1E,
calcofluor).
The 2C content of skp1-3f-arrested cells was unexpected
considering the G1 arrest observed with the identical
budding yeast mutant (8). To analyze G1/S
progression in more detail, cells were synchronized in G1
by nitrogen starvation. Simultaneously, with release from the
G1 block cells were transferred to 37 °C, and
progression through S-phase was monitored by FACS analysis demonstrating comparable progression of control and skp1-3f
cells through S-phase (Fig. 1F).
To further characterize the arrest point in skp1-3f, a time
course experiment monitoring Cdc2 activity and Tyr-15 phosphorylation was performed. Initiation of mitosis is triggered by activation of Cdc2
due to dephosphorylation of Tyr-15 by the Cdc25 phosphatase (39).
Western blotting analysis of boiled extracts of skp1-3f cells indicated a marked decrease in Tyr(P)-15 following a shift to the restrictive temperature (Fig. 1G,
phospho-Tyr-15), whereas Cdc2 levels remained constant. This
was correlated with a strong increase in Cdc2 activity (Cdc2 H1
kinase). These characteristics of the arrested skp1-3f
cells were in marked contrast to G2-arrested cdc25-22 cells, where phosphorylation of Cdc2 Tyr-15 is
increased and Cdc2 activity is decreased as expected (Fig.
1G, cdc25-22; at 6 h the mutant leaks
through the block).
Skp1 Is Required for Septum Processing and Cell
Separation--
The appearance of aberrant septa in both the
skp1-deleted cells as well as in skp1-3f cells
at the restrictive temperature was intriguing and suggested a role for
Skp1 in septum processing and cell separation. However, demonstration
of this was complicated by the fact that the majority of cells arrest
earlier in mitosis, which precedes cytokinesis by 10-15 min. To our
knowledge, the only efficient way to block fission yeast cells in
mitosis is to use the nda3-311 allele encoding a
cold-sensitive allele of beta-tubulin (40). Therefore a strain was
generated combining nda3-311 (restrictive temperature
20 °C) and skp1-3f (restrictive temperature 37 °C).
This would potentially allow a successive restriction of first
nda3-311 and then skp1-3f functions leading to
an increase in the number of cells with the septum defect. To this end,
control nda3-311 cells and double mutant skp1-3f nda3-311 cells were synchronized for 6 h at 20 °C
followed by a rapid shift (in a PCR machine) to 36 °C after which
fraction of cells with septa was monitored (Fig.
2A). The septation index in
the nda3-311 control cells peaked at 75% at 10 min and had decreased to 47% at 20 min. In the double-mutant skp1-3f
nda3-311 cells, the septation index rose similarly to the
controls, but no decrease was noted at 20 or 30 min (Fig.
2A), consistent with impaired septum processing.
A second approach to increasing the number of cells displaying aberrant
septa involved a "block-release-block" experiment. This was
initiated by incubating skp1-3f and control cultures at
36 °C for 4 h in the presence of 15 mM hydroxyurea
(HU). This led to an early S arrest in control cells (Fig.
2B, 240 min, control) with a 1C DNA content,
whereas the skp1-3f cells displayed a predominantly 2C DNA
content (Fig. 2B, 240 min, skp1-3f), suggesting
they were blocked after S-phase, which is consistent with our previous
results (Fig. 1).
Subsequently, control cells (blocked at G1/S) and
skp1-3f cells (mostly blocked in mitosis) were shifted to
25 °C (the permissive temperature for skp1-3f) with
simultaneous removal of HU and incubated in fresh medium at 25 °C
for 20 min. Cells were then re-shifted to 36 °C and followed by FACS
analysis. In the skp1-3f cells a 4C DNA peak appeared at 30 min and increased until the last time point (Fig. 2B),
whereas the 1C wild type culture completed S-phase and subsequently
continued a normal cycle.
Cytological analysis of the 4C skp1-3f cells revealed
accumulation of pronounced thick septa (Fig. 2C). Based on
this, it is likely that the 4C DNA content of these cells is due to a
2C content in both daughter nuclei still attached by the thick septum, indicating that both nuclei have undergone replication. The possibility that the peak would be due to re-replication of unseptated cells was
ruled out by unchanged levels of Cdc18 levels throughout the experiment
(data not shown). The completion of DNA synthesis in the two daughter
nuclei in the septated cells is consistent with the results above (Fig.
1), demonstrating that Skp1 is not required for replication.
The results described above indicate that skp1-3f arrest
with a pleiotropic phenotype with elongated cells and with the majority of cells arresting in mitosis with high Cdc2 activity, but with other
cells arresting just prior to cell separation with a septum defect.
These pleiotropic phenotypes are likely to reflect the various
functions Skp1 mediates in distinct SCF complexes with various F-box
proteins. For example, elongation of skp1-3f cells at
37 °C is likely to reflect inactivation of SCFPof3 as
pof3 deletion has a similar phenotype (31).
A number of F-box proteins can be identified by similarity searches
from the fission yeast genome. We have also performed two-hybrid
screening with Skp1, leading to the isolation of several F-box proteins
partly overlapping with those identified through similarity
searches.2 One of the F-box
proteins identified in this screen was Pof6.
Pof6 Is an Essential F-box Protein Required for Septum
Processing--
As described above, Pof6 was identified through a
two-hybrid interaction with Skp1 (Fig.
3A). The predicted Pof6
protein encodes a 872-amino acid protein with the F-box region between
amino acids 33 and 75 (Fig. 3B). Pof6 also has a CAAX-motif
at its C terminus, suggesting that Pof6 may be modified through
prenylation (41). Pof6 does not have any closely related sequences in
fission yeast, and it is most closely related to Yarrowia
lipolytica Sls2p (28% identity between amino acids 45 and 872 in
Pof6 (42)) and budding yeast Rcy1p proteins (24% identity between
amino acids 150 and 872 in Pof6 (10)). Sls2p has been implicated in
secretion and Rcy1p is involved in recycling. In addition,
significant sequence similarity was detected between Pof6 and the
exocyst subunit Sec10 involved in exocytosis (43). Using reiterations
on PSI-BLAST this similarity was noted between amino acids 147 and 853 of Pof6 (13% identity, 30% similarity with fission yeast
Sec10). As septum processing and cell separation require exocyst
function (44), Pof6 was chosen for further studies as a candidate F-box
protein mediating the septum processing phenotype noted in
skp1 mutants.
Pof6 protein was found to be constantly expressed during the cell cycle
(Fig. 3C). pof6 is an essential gene as
determined by the formation of two viable spores from sporulation of
diploid S. pombe cells in which one copy of pof6
was deleted (Fig. 3D). Germinated spores form highly
branched structures corresponding to unseparated cells (Fig.
3E), and in some cases, a thick septum between two cells was
observed (Fig. 3F). This phenotype is highly reminiscent of
what we observed in skp1-3f except that no branching was
noted with skp1-3f, which is likely due to the fact that
cells lacking Skp1 cannot proceed through several cell cycles because Skp1 is also required for other essential steps (see Fig.
1).
Pof6 Interacts with Skp1 in a Non-SCF Complex and Localizes at the
Septum--
To test if the Pof6 F-box protein is part of an SCF, a TAP
fusion protein was expressed from the endogenous pof6 locus.
After tandem affinity purification (45), the eluate was separated on
lithium dodecyl sulfate-PAGE and silver-stained revealing
several bands (Fig. 4A). The
19-kDa band represents Skp1 as demonstrated by Western blot analysis
(Fig. 4A). The identity of several other bands is unknown
and will be subjected to further study. However, interestingly, no
bands were detected at the expected size of Pcu1-myc (112 kDa, the only
essential cullin in S. pombe (21)) or Pip1 (13 kDa, the Rbx1
homologue). Furthermore, the absence of Pcu1 from the Pof6 complex
could also be demonstrated by Western blot analysis, showing that
although the characteristic doublet representing Pcu1-myc (46) was
clearly detected in the extract used for the purification, it did not
co-purify with Pof6. Separate co-immunoprecipitation analysis confirmed
this result (data not shown).
To localize Pof6 in the cell, three GFP-Pof6 constructs corresponding
to wild type, a mutant lacking the F-box, or a mutant lacking the CAAX
box have been integrated in diploid cells at the pof6 locus
under the control of the thiamine-regulated nmt1 promoter. Fig.
4B shows anti-GFP Western blots performed on extracts from
these strains grown in the presence or absence of thiamine. The diploid
strains were subsequently sporulated, and tetrads were analyzed
indicating that only the disruption of the F-box of Pof6 was
detrimental to fission yeast survival based on the 2:2 segregation
(Fig. 4C). Germinated spores on the plate have a phenotype
identical to the pof6 deletion (not shown).
To analyze the subcellular localization of GFP-Pof6, GFP-Pof6-
The localization of Pof6 to the septum is consistent with a function in
septum processing and cell separation. It is interesting to note that
this subcellular localization is similar to that noted for the Sec8p
exocyst component (44).
From these studies we conclude that Pof6 and Skp1 are physically
associated in a non-SCF complex. Both Skp1 and Pof6 are required for
normal septum processing and cell separation, a phenomenon that has not
been characterized in much detail. However, recently the Sec8 subunit
of the exocyst complex was also found to be required for septum
processing and cell separation (44), and several other exocyst proteins
in addition to Sec8p were found at the septa. Thus it is interesting to
speculate that Pof6 and Skp1 functions in cell separation would be
related to exocyst function. In this regard it is very interesting to
note that the apparent homologues of Pof6 in Y. lipolytica (Sls2p) and budding yeast (Rcy1p) have been
implicated in transport and recycling, respectively (10, 11, 42).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
ade6-216 ura4D18 leu1-32,
h+ leu1-32 (Stratagene),
h+ cdc25-22 ade6-216 leu1-32 (lab
stock), and h+ nda3-311 leu1-32
his2 (Lab stock). Classical S. pombe techniques were
performed as described (32).
.
, and transformants were selected on plates
lacking uracil and checked by Southern blot analysis. Deletion of
pof6 was performed as described by Bähler
et al. (33) using the following oligonucleotides:
5'-GTA-GTC-GTC-ACT-ATT-TTA-TGA-AGT-ATG-CGG-AAT-ACC-TGT-GAT-CAT-AAT-ATC-ATG-TTC-GCT-AGA-ACC-AAA-TAG-ATT-TCA-TGA-CGG-ATC-CCC-GGG-TTA-ATT-AA-3' and
5'-GCC-TAA- AGC-AGA-AAG-CAA-GAT-GCA-TTT-TTA-AAG-TGT-ATG-GTA-ATA- ACA-AAA-AAA-GGA-TAT-GAA-CAA-AAA-ATA-AAA-AAT-ATG-AAT- TCG-AGC-TCG-TTT-AAA-C-3'.
-32P]ATP 40 µCi/ml) was added for 25 min at
30 °C. Proteins were separated by 12% SDS-PAGE and the gel exposed
to x-ray film for a few hours.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Skp1 is an essential protein
required for mitotic progression. A, multiple alignment
of C-terminal regions of indicated Skp1 homologues including Skp1 using
ClustalX (47). Hs, Homo sapiens;
Dd, Dictyostelium discoideum; At,
Arabidopsis thaliana; Sp, S. pombe;
An, Aspergillus nidulans; Sc,
Saccharomyces cerevisiae; Ce,
Caenorhabditis elegans. Arrows indicate
locations of the single mutations identified in S. cerevisiae skp1-4 (L146S) and skp1-3
(I172N) (8) and mimicking mutations made into skp1 here to
generate skp1-3f (I139N) and skp1-4f (L113S).
B, photograph of colonies growing following
dissection of spores from tetrads of a diploid heterozygous for
skp1 (skp1/skp1::ura4).
The 2:2 segregation of growing versus non-growing spores
indicates the gene is essential. C, spores deleted for
skp1 were germinated in minimal media lacking uracil for
24 h. Aliquots of fixed cells were stained with DAPI.
Arrows indicate septa noted in 25% of the cells. The
asterisk indicates an ungerminated wild type spore.
(Bar = 10 µm). D, photographs
of plates on which the indicated skp1 mutant strains were
streaked followed by incubation at indicated temperatures.
E, flow cytometry analysis of the skp1-3f strain
at 25 °C or after 6 h at 37 °C. The bottom
micrograph shows skp1-3f cells grown for 6 h in
liquid culture at 37 °C and stained with calcofluor.
(Bar = 10 µm). F, flow cytometry analysis
of wild type and skp1-3f strains at indicated times
following a release from synchronization in G1 by 18 h
nitrogen starvation. The cultures were shifted to 37 °C at time of
release as shown in the schematic outline of the experiment.
G, Western blotting analysis to determine levels of Cdc2
( -Cdc2, a kind gift of P. Nurse) (48) and Cdc2 Tyr(P)-15
(Biolabs) at indicated time points in the skp1-3f and
cdc25-22 strains cultured in minimal media following a
shift from 25 °C to 37 °C. Cdc2 activity was also assayed for
histone H1 after immunoprecipitation from native extracts of the
skp1-3f and cdc25-22 strains following a shift
from 25 to 37 °C at indicated times.
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Fig. 2.
The skp1-3 ts
mutant is impaired in cell separation.
A, nda3-311 or nda3-311 skp1-3f
cells were cultured for 6 h at the restrictive temperature of
nda3-311 (20 °C) to synchronize cells in metaphase.
Following a rapid shift to 36 °C, the percentage of septated cells
was calculated (septation index) at indicated time points.
B, a block-release-block experiment with control and
skp1-3f cells as shown schematically on the
left. At time 0, 15 mM HU was added to control
and skp1-3f cells subsequently cultured at 36 °C for
4 h. HU was washed and cells were shifted to 25 °C for 20 min.
Subsequently, they were shifted back to 36 °C. Aliquots of cells
were collected during HU incubation at 60, 120, 180, and 240 min, and
following the release and re-shift back to 36 °C at 30, 60, 90, 120, 150, and 180 min and analyzed by flow cytometry. The 1C, 2C, and 4C DNA
contents are indicated at the bottom. C,
micrographs of calcofluor-stained skp1-3f or control cells
from the last time point in B. (Bar = 10 µm).
View larger version (29K):
[in a new window]
Fig. 3.
Pof6 is an essential F-box protein essential
for cell separation. A, two-hybrid analysis of
Gal4-BD-Skp1 and Gal4-AD-Pof6 using the b-gal gene as
reporter (Clontech Matchmaker) with indicated
controls. B, a schematic of Pof6 showing the position of
both the F-box and the CAAX-box as well as the area of similarity with
Sec10 (amino acids 147-852). C, to analyze Pof6 levels
during cell cycle progression a cdc25-22 pof6-TAP strain
was synchronized in G2 by growing cells at 37 °C for 3h
30'. Following a shift to the permissive temperature (25 °C) samples
were collected every 20 min for Western blotting analysis with control
anti-Cdt1 (a G1 marker, gift of P. Nurse) or anti-TAP
antibodies. D, photograph of colonies growing
following dissection of spores from tetrads of a diploid heterozygous
for pof6 (pof6/pof6::kan+).
The 2:2 segregation of growing versus non-growing spores
indicates the gene is essential. E, phase contrast
micrographs of spores from the diploid
pof6/pof6::kan+ germinated in minimal
media in the presence of kanamycine for 4 days. F,
micrographs of spores from the diploid
pof6/pof6::kan+ germinated in minimal
media in the presence of kanamycine for 24 h showing thick septa
in both phase contrast or following calcofluor staining as
indicated. (Bar = 10 µm).
View larger version (50K):
[in a new window]
Fig. 4.
Pof6 belongs to a non-SCF complex and
localizes at the septum. A, TAP purification was
performed on a pof6-TAP pcu1-myc strain exactly as described
(45). The eluate (Pof6-TAP) and an eluate from the control
strain pcu1-myc (TAP Ctr) were separated by
lithium dodecyl sulfate-PAGE and the gel was silver-stained. The gel
was blotted and probed with anti-Psh1 (left panel) (= Skp1,
a gift of D. Wolf) or anti-myc (right panel). For each
Western blot, the original lysate used for the purification
(Extr.) was also separated and probed as indicated.
B, anti-GFP Western blot analysis of diploid strains
expressing either GFP-Pof6 (wt) or mutants lacking the F-box
( -F-box) or the CAAX box (
-CAAX box) under
control of the nmt1 promoter. Cells were grown in presence
or absence of thiamine to repress or de-repress the promoter,
respectively. C, GFP-Pof6 (wt), GFP-Pof6-
F-box
(
-F-box), or GFP-Pof6-
CAAX-box (
-CAAX
box) strains from B were sporulated, and tetrads were
dissected as indicated. The 2:2 segregation obtained with the
-F-box
mutant indicates F-box is essential for growth. D,
fluorescence micrographs of GFP-Pof6 (wt), GFP-Pof6-
F-box
(
-F-box), or GFP-Pof6-
CAAX-box (
-CAAX
box) strains grown in the presence of thiamine (low expression)
and observed under fluorescence. Arrows point to GFP-Pof6
localization to the septum and the arrowhead to cell tips.
(Bar = 10 µm).
F-box,
or GFP-Pof6-
CAAX, the strains were grown in the presence of thiamine
(low expression, see Fig. 4B). The wild type GFP-Pof6 displays a nuclear enrichment but is excluded from a nuclear structure that could correspond to the nucleolus. GFP-Pof6 is also localized at
cell tips and on both sides of the septum in septated cells (Fig.
4D, wt). Localization of the GFP-Pof6-
F-box
and GFP-Pof6-
CAAX were similar to wild type (Fig. 4D,
-F-box,
CAAX) except that in the CAAX box
mutant, localization at the tips was less pronounced. This indicates
that the F-box, and by extension association with Skp1, is not required
for proper localization of Pof6, though it is essential for Pof6
function. It also shows that the CAAX box is not required for the
essential function of Pof6.
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ACKNOWLEDGEMENTS |
---|
We thank P. Nurse for reagents and comments on results. We thank M. Minet, F. Lacroute, J. Bähler, K. Gould, D. Wolf, and K. Tanaka for providing reagents. We thank A. Borgne and A. Decottignies for advice and help, and H. Browning and S. Yamaguchi for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This study was supported by grants from Fonds National de la Recherche Scientifique (Convention Fonds de la Recherche Fondamentale et Collective 2.4504.00/1999), Academy of Finland, Biocentrum Helsinki, Finnish Cancer Organization, Finnish Cancer Institute, and Sigrid Juselius Foundation.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF071066.
¶ These authors contributed equally to this work.
A FNRS Postdoctoral Researcher.
** To whom correspondence should be addressed: Cancer Research UK, 44 Lincoln's Inn Field, London WC2A 3PX, United Kingdom. Tel.: 44-(0)20-7269-3235; Fax: 44-(0)20-7269-3610; E-mail: D.Hermand@cancer.org.uk.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M211358200
2 L. Tafforeau, S. Bamps, J. Vandenhaute, and D. Hermand, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SCF, Skp1-Cdc53/Cullin-F-box protein; E1, ubiquitin-activating enzyme; E2, ubiquitin conjugating protein; SNARE, soluble NSF attachment protein receptors; GFP, green fluorescence protein; TAP, tandem affinity purification; DAPI, 4',6-diamidino-2-phenylindole; ts, temperature-sensitive; FACS, fluorescence-activated cell sorter; HU, hydroxyurea; wt, wild type.
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