Skp1 and the F-box Protein Pof6 Are Essential for Cell Separation in Fission Yeast*

Damien HermandDagger §||**, Sophie BampsDagger DaggerDagger, Lionel TafforeauDagger , Jean VandenhauteDagger , and Tomi P. Mäkelä§

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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.

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ABSTRACT
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Yeast Strains and Techniques-- S. pombe strains used: h+ ade6-210 ura4D18 leu1-32, h- 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).

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-skp1Delta .

A diploid strain was transformed with an EcoRI-XhoI fragment of the pSK-skp1Delta , 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'.

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 [gamma -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.

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."

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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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).


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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 (alpha -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.

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.


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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).

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.


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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).

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).


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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 (Delta -F-box) or the CAAX box (Delta -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-Delta F-box (Delta -F-box), or GFP-Pof6-Delta CAAX-box (Delta -CAAX box) strains from B were sporulated, and tetrads were dissected as indicated. The 2:2 segregation obtained with the Delta -F-box mutant indicates F-box is essential for growth. D, fluorescence micrographs of GFP-Pof6 (wt), GFP-Pof6-Delta F-box (Delta -F-box), or GFP-Pof6-Delta CAAX-box (Delta -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).

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-Delta F-box, or GFP-Pof6-Delta 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-Delta F-box and GFP-Pof6-Delta CAAX were similar to wild type (Fig. 4D, Delta -F-box, Delta 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.

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).

    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.

Dagger Dagger A FNRS Research Fellow.

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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