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Molecular and Cellular Biology, March 2006, p. 1579-1588, Vol. 26, No. 5
0270-7306/06/$08.00+0 doi:10.1128/MCB.26.5.1579-1588.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Life Sciences, Ben Gurion University of the Negev, Beersheba 84105, Israel
Received 7 June 2005/ Returned for modification 10 July 2005/ Accepted 18 November 2005
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ABSTRACT |
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ddi1 cells arrest when full-length UFO1 is overexpressed. These results imply a role for the UIMs in turnover of SCFUfo1 complexes that is dependent on Ddi1, a novel activity for an UbL-UbA protein. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The F-box protein Ufo1 was identified during a study on the regulation of the activity of the mating switch endonuclease, Ho (22). Transcription of UFO1 is induced in response to DNA damage (20), and deletion of UFO1 has been observed to affect genome stability (40). Ufo1 is an unusual F-box protein in that besides the F-box and protein-protein interaction domains present in other F-box proteins, it has four copies of the ubiquitin-interacting motif (UIM). The UIM is a short N-terminal acidic box, followed by alternating long and short hydrophobic residues that terminates in an invariant signature sequence A-X-X-X-S (15). The UIM was originally identified as the ubiquitin chain-binding domain of subunit Rpn10 of the proteasome 19S RP (35); however, all other UIM proteins are components of the intracellular vesicular trafficking system (15). A comprehensive search of the protein databases indicates that it is only Ufo1 and its fungal orthologs that have UIMs. The UIM of the intracellular trafficking protein Epsin promotes ubiquitylation of glutathione S-transferase (GST) in the context of a GST-UIMEpsin fusion protein (34). Furthermore, during self-ubiquitylation of Hgs1, an adaptor protein that targets the EGF receptor for degradation, the Hgs1 UIM plays an essential role in recruitment of the E3, Nedd4 (25).
Here we report that deletion of the Ufo1 UIMs creates a dominant-negative phenotype of inactivation of the SCF pathway of protein degradation. The cells arrest with long buds resembling skp1-3 mutants at the restrictive temperature, corresponding to a G1/S arrest. Furthermore, we show that there is a specific interaction between Ufo1 and the UbL-UbA protein Ddi1. This interaction requires the presence of the UIMs of Ufo1 and is important for the turnover of SCFUfo1 complexes and cell cycle progression.
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MATERIALS AND METHODS |
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rad23 dsk2 ddi1 mutant strains were purchased from Euroscarf. The wild type and ufo1 mutant are Research Genetics BY4730 and #142, respectively. The G1/S skp1-3 mutant is described elsewhere (26). Temperature-sensitive (ts) cdc34-2 and cdc53-1 were obtained from M. Tyers and are in a W303 background with the genotypes MATa can1-100 ade2-1 his3-11,15 leu2,3,112 trp1-1 ura3-1 GAL+ cdc34-2 and MATa ura3-1 can1-100 GAL+ leu2-3,112 trp1-1 his3-11,15 ade2-1 cdc53-1, respectively. FY56 and its isogenic rsp5 mutant, Fw1808 (MATa his4-912P5 lys2-128 ura3-52 rsp5-1 [L7335]), were obtained from R. Haguenauer-Tsapis. The atg7 mutant was generated by one-step gene replacement in Euroscarf BY4741 by using the primers ATG7-URAF (ATAACTAAAGTTCATTATATTTCAACAAATATAAGATAATCAAGAATAAAATGTCGAAAGCTA CATATAAGG) and ATG7-URAR (CCGCAAGTTCTGCCTTTGGCTTTCCACAGTCCTCAAAATTATATAACGCT TTAGTTTTGCTGGCCGCATCTTC) to introduce URA3 at the genomic ATG7 locus. Cells expressing hemagglutinin (HA)-tagged Cln2 were prepared by integrating plasmid pRD114a from R. Deshaies at the LEU2 locus. SIC1 was deleted in the cdc53 ts strain above by using the primers SicHIS3F (AAATCAATCAACCAAACCTCTACGGAATTTTGACCCTTGAAGCAGGGACTATTACACGAAAATGACAGAGCAGAAAGCCC) and SicHIS3R (ATGTAGAATAAGTAAGTAAATAAAATATAATCGTTCCAGAAACTTTTTTTTTTCATTTCTCTACATAAGAACACCTTTGG) to substitute HIS3 for the genomic SIC1 gene.
Plasmids.
Construction of pGAL-GFP-UFO1 is described in reference 21; construction of of pTET-HO-LACZ is described in reference 22. pGFP-UFO1UIM was cloned in plasmid pYCPGAL (41) by using the primers Ufo1BamF (CGGGATCCATGGAGCGGCCTGGCTTGGTATTGC) and Ufo1SalR (CCGACAGGATCCCCCGCTTAAGCTTGATTCCG) to amplify genomic UFO1. The K134A K141A mutations were introduced by primer overlap extension by using the external primers described above and the mutagenic primers NLSHindF (CACGCAAGGCAGAAGAAATTTAAAGCTTTGATTTATATTCC) and NLSHindR (GGAATATAAATCAAAGCTTTAAATTTCTTCTGCCTTGCGTG). The GFP-UFO1-UIMs was subcloned into the same plasmid by using the primers UfoUIMBamF (GCACTCTTAGGATCCCAGGAGGCGCAAGC) and UfoUIMBamR (GCGGATCCTCAATTGATTTCACTCAATGAC). The genomic UIM domain was replaced with a TAP tag (36) by using the primers UfoUIMTAPF (CGGAATCAAGCTGAAGCGGGGGAGCCTGTCGGAGATGATGTCCATGGAAA AGAGAAG) and UfoUIMTAPR (CTATAAATAAAATATTTAACATATGCTCTTCCAAATGTACATACTTTCATACGACTCACTATAGGG) to amplify the tag from plasmid pBS1479 (Euroscarf). The clone for overexpressing GFP-F-box residues 1 to 233 was made by deleting all residues between the HindIII site at bp 700 of the UFO1 open reading frame and a similar site in the vector. Ufo1 deleted for its F-box was cloned into pOBD using primers WDF (GGATTCAATATTAATGCTGCAGTG) and UIMR (GATCCCCGGGAATTGCCATGTCAATTGATTTCACTCAATGACAACGCAAT). The fusion of the Ufo1 WD40 domain in pOAD is described in reference 21.
CLN2 and SIC1 were cloned as GFP fusion proteins in YCPGAL by using the primers YCpGALCLN2BamF (CCATTCATTCATTAAATTTAAGGATCCACAATGGCTAGTGC) and YCpGALCLN2BamR (GGTACGTTTGGCAAATTGGGATCCATTTATCATGAAAAGAACAGG) and the primers YCpGAL-SIC1BamF (CGGAATTTTGACCCTGGATCCAGGGACTATTACACGAAAATG) and YCpGAL-SIC1BamR (GTAAATAAAATATAATGGATCCAGAAACTTTTTTTTTTCATTTC), respectively.
Subclones of Ddi1 with the UbL or UbA domain deleted were LexA fusions (3). 2µm plasmids with SKP1 from a genomic library, or CDC4 cloned as a PCR product amplified from genomic DNA, were a gift from D. Kornitzer. GST-Ddi1 was a gift of D. Skowyra and GST-Rad23 and GST-Dsk2 were obtained from S. Elsasser (8).
Yeast two-hybrid system. UFO1 was fused to the Gal4 activation domain of pOAD (4). Rad23, Dsk2, and Ddi1 fusions to the Gal4 DNA-binding domain were from Ito et al. (17). The plasmids were cotransformed into yeast strain PJ694a, where three reporter genes are under the control of the GAL promoter (19) and cells in which the two proteins interact can grow on selective plates lacking histidine and adenine. Transformations of yeast cells were performed by using lithium acetate (1).
Protocols for metabolic labeling, immunoprecipitation, and pulse-chase are described in reference 21, as are the methods for coimmunoprecipitation and immunoblotting. In these experiments, GFP-UFO1 was induced from the GAL promoter by overnight growth in minimal medium with 2% galactose. In the experiment with G1-arrested cells, overnight cultures were diluted into medium with 2 µg of 
-factor/ml and 2% galactose, followed by incubation for 3 h before promoter shutoff by the addition of 3% glucose. An overnight 50-ml culture of 108 cells/ml served as the source of a 300-µl extract that had 80 µg of protein/µl. A total of 190 µl (15 mg of protein) was taken for immunoprecipitation (IP) with anti-Ddi1 or anti-GFP, and the immunoprecipitate was run in a single lane for Western blotting (WB) with the appropriate antisera. Anti-Ddi1 was a gift from J. Gerst and was used at dilutions of 1:1,000 for IP and 1:5,000 for WB; anti-GFP antiserum from Roche Molecular Biochemicals and anti-myc (mouse monoclonal antibody 9E10) from Santa Cruz Biotechnology were used at 1:200 for IP and at 1:1,000 for WB. Goat anti-mouse and goat anti-rabbit antibodies were used at 1:1,000 and were from Santa Cruz Biotechnology. Protein A-Sepharose was purchased from Amersham and was used at 50%; 30-µl portions were added to each sample.
Microscopy. Cells expressing green fluorescent protein (GFP)-tagged proteins were observed with a Nikon fluorescence microscope fitted with GFP-specific filters (dichromic 505 nm, excitation 450 to 490 nm, emission 515 nm). Images were captured with a Micromax 512 BFT camera (Roper Scientific) by using WinView32 imaging software.
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RESULTS |
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UIM). pGFP-UFO1 transformants grew normally on galactose plates, whereas those that expressed pGFP-UFO1UIM were growth inhibited. Growth could be restored by ectopic expression of SKP1 (Fig. 1A). pGFP-UFO1UIM cells in liquid culture arrested with long buds, similar to the G1/S arrest phenotype of skp1-3 mutants at 37°C, implying a lack of SCF function (Fig. 1B). Quantification showed about half the GFP-UFOUIM-expressing cells arrested with long buds. These experiments relied on high-level expression of UFO1 and its truncated form. We therefore investigated the effect of deleting the UIMs in genomic UFO1. This was done by integrating a PCR product encoding a TAP tag and the TRP1 marker gene (36) either just prior to the stop codon of UFO1 (UFO11-668::TAP) or in place of its UIMs (UFO11-538uim::TAP). Cells expressing UFO11-668::TAP grew normally, whereas those expressing UFO11-538uim::TAP formed microcolonies with cells arrested with long buds. These cells did not continue to grow, and the transformants died shortly after reaching the stage shown in Fig. 1C. This is in contrast to ufo1 mutants that have no obvious growth phenotype.
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UIM is active in Ho degradation.
The G1/S arrest could be due to lack of degradation of a substrate of Ufo1 that has a role in cell cycle progression. However, we found that the Ufo1 substrate, Ho endonuclease, was degraded with a similar half-life in the presence of full-length Ufo1 or Ufo1UIM, indicating that Ufo1UIM is capable of forming active SCF complexes. This was done by cotransforming ufo1 cells with pGFP-UFO1 or pGFP-UFO1UIM together with pTET-HO-LACZ and measuring the half-life of Ho-LacZ by pulse-chase and IP (Fig. 2A).
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UIM.
The lethality caused by deletion of the genomic UFO1 UIMs implies that Ufo1 is exerting a dominant-negative effect on the SCF pathway. If the deletion of the Ufo1 UIMs is interfering directly with SCFUfo1 turnover, we would expect cells producing Ufo1UIM to accumulate SCF degradation substrates of other F-box proteins such as Cln2 (ubiquitylated by SCFGrr1), and the Cdk inhibitor Sic1 (ubiquitylated by SCFCdc4) (38). Indeed, Western blotting demonstrated significant accumulation of Cln2 in cells expressing pGFP-UFO1UIM but not in cells expressing full-length UFO1 (Fig. 2B). Furthermore, in an anti-GFP immunoprecipitation and Western blot experiment, we could show directly that the turnover of Cln2 is retarded when GFP-UFO1UIM, but not GFP-UFO1, is expressed (Fig. 2C). These data are further supported genetically by the observation that the cell cycle arrest upon GFP-UFO1UIM expression was ameliorated in a sic1 mutant and by the accumulation of Sic1 in these cells (Fig. 2D).
Suppression of the arrest caused by deletion of the UIMs of Ufo1.
The data presented above show that Ufo1UIM has a dominant-negative effect on SCF activity, suggesting that it may sequester core subunits of the SCF. We therefore tested whether the arrest can be suppressed by coexpression of subunits of the SCF. F-box proteins are recruited to the SCF by binding the Skp1 adaptor; however, we showed previously that the Ufo1 F-box domain binds both Skp1 and Cdc53 and that a WD40 domain subclone binds Cdc53 (21). Thus, Ufo1 may be sequestering both core components of the SCF and preventing them from recycling. In accordance with this we found that overexpression of the F-box protein, CDC4, suppressed the phenotype of cells with a deletion of the genomic UFO1 UIMs, UFO11-538uim::TAP. Presumably, the large amounts of Cdc4 could compete for binding the SCF core subunits, implying that they are indeed limiting for substrate degradation (Fig. 3A and B). Similarly, when we coexpressed SKP1 with UFO1UIM we found marked restoration of growth with only 16% arrested cells with long buds. Likewise, coexpression of CDC53 suppressed the cell cycle arrest induced by UFOUIM by half. The Ufo1 WD40 domain alone fused to the Gal4 DNA activating domain did not cause cell cycle arrest, and high-level expression of an Ufo1 F-box clone and putative linker region (residues 1 to 233) led to only 13% arrested cells compared to ca. 50% when Ufo1UIM was produced from the same plasmid (Fig. 3A and B). A mutant version of Ufo1UIM in which we introduced two point mutations into a putative nuclear localization sequence of basic residues between the F-box and the WD40 domains, Ufo1UIMK134A,K141A, was not affected in nuclear import of Ufo1 but did lead to suppression of the cell cycle arrest induced by Ufo1UIM (Fig. 3C). Taken together, the results confirm that it is the truncated Ufo1UIM protein itself that is directly inhibiting cell cycle progression and that contacts between the Ufo1 F-box domain and both Skp1 and Cdc53 are involved in the dominant-negative effect on function of other SCF complexes. Moreover, this experiment emphasizes the difference between the inhibitory effect on the cell cycle of Ufo1UIM, which consists of the F-box and WD40 domains, compared to the rescue by Cdc4 comprising the same two domains.
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-factor-arrested G1 cells, excluding the possibility that Ufo1 degradation requires progression into S phase. Furthermore, Ufo1 was stabilized in cdc53 mutants in which SIC1 was deleted to enable cell cycle progression (Fig. 5C). These results indicate that the main pathway for ubiquitylation of Ufo1 involves SCFUfo1, followed by degradation in the proteasome.
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The Ufo1 UIMs are required for degradation of Ufo1.
The experiments described above suggest that the cell cycle arrest may be due to the Ufo1UIM protein sequestering the endogenous Skp1-Cdc53 subunits of the SCF and acting as a dominant negative, preventing assembly of other SCF complexes. In this model, the UIMs of Ufo1 are critical for its degradation. To explore directly whether Ufo1 degradation is dependent on its UIMs, we collected aliquots of cells overexpressing pGAL-GFP-UFO1 and pGAL-GFP-UFO1UIM over a 3-h period after transfer from galactose to glucose medium to obtain promoter shutoff. Ufo1 was present at the 1-h time point but had disappeared by 2 h. In contrast, Ufo1UIM was markedly stabilized and could still be found at the 3 h time point. Thus, in the absence of its UIMs, the half-life of Ufo1 is extended, and this could explain inactivation of SCF function when it is overexpressed; it could also explain the lethality induced by deletion of the genomic UFO1 UIMs. Ufo1UIMK134A,K141A in which the cell cycle arrest is suppressed, has a half-life comparable to that of Ufo1, further strengthening the correlation between extended half-life and cell cycle arrest (Fig. 6A).
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UIM to form active SCF complexes as evidenced by normal degradation of Ho endonuclease (Fig. 2). However, Ufo1UIM had a considerably extended half-life (Fig. 6A). Therefore, deletion of the Ufo1 UIMs may be interfering with a function downstream of ubiquitylation. We recently found that the ubiquitin domain protein Ddi1 is required for the final stages of degradation of Ho and that, in the absence of Ddi1, Ho accumulates in a stable ubiquitylated form (23). Ddi1 belongs to a family of UbL-UbA proteins proposed to act as proteasome receptors for ubiquitylated substrates (11, 35). We therefore tested whether any of the UbL-UbA proteins—Rad23, Dsk2, or Ddi1—is required for Ufo1 degradation. We induced pGFP-UFO1 in galactose medium overnight in rad23, dsk2, and ddi1 deletion mutants and collected aliquots of cells at 30-min intervals after transfer to glucose for promoter shutoff. Ufo1 is highly stabilized in ddi1 mutants, whereas it was degraded with a half-life similar to that observed in wild-type cells in rad23 and dsk2 mutants (Fig. 6B). These results imply that Ddi1 has a role in Ufo1 degradation and thus in the turnover of SCFUfo1 complexes.
Ufo1 binds Ddi1 in two-hybrid and co-IP experiments.
In accordance with this, when we tested for complex formation between Ufo1 and the UbL-UbA proteins Ddi1, Rad23, and Dsk2, we found that Ufo1 interacts only with Ddi1 and not with either Rad23 or Dsk2 in the two-hybrid protein interaction trap (Fig. 7A). Deletion of the UIMs of Ufo1 abrogated its interaction with Ddi1 in a co-IP experiment (Fig. 7B). An analysis of the domains of Ddi1 involved in interaction with Ufo1 showed that deletion of the UbL of Ddi1 abrogated binding to Ufo1 and that deletion of the UbA of Ddi1 reduced the interaction considerably (Fig. 7C). These results suggest that Ddi1 may have a role in the turnover of Ufo1 and, by implication, of SCFUfo1 complexes. This leads to the prediction that expressing full-length GFP-UFO1 in ddi1 cells would phenocopy expression of GFP-UFO1UIM in DDI1 cells. Indeed, we found that, whereas full-length UFO1 overexpression in wild-type cells did not affect cell cycle progression and it was only truncation of the Ufo1 UIM that led to arrest, in ddi1 cells a very similar level of cell cycle arrest was obtained irrespective of the presence or absence of the Ufo1 UIMs (Fig. 7D). Ddi1 itself is very stable, with little sign of degradation over 3 h; its turnover was not affected by deletion of Ufo1 (Fig. 7E).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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UIM is overexpressed and even moreso when the UIMs of the genomic UFO1 gene are deleted is an indication that the levels of the core SCF components are stringently regulated and that there is not a large pool of free subunits. Skp1 participates in different complexes, the centromeric complex (6), and in a complex with the Golgi complex-related F-box protein, Rcy1 (10), that do not involve Cdc53. Our data imply that its partitioning must be very tightly regulated since it is not rapidly mobilized from either of these complexes. Transformants in which the UIMs of genomic UFO1 are deleted reach the stage of microcolonies and then die, whereas after about 20 h in liquid culture, cells expressing ectopic UFO1UIM resume growth, and this could be due to upregulation of biosynthesis of the core SCF elements. To date, research on substrate degradation by the proteasome has focused on how substrates are marked for recruitment by F-box proteins. An extreme case is the CKI, Sic1, that requires a minimum of six phosphorylations to form a stable complex with Cdc4. The actual residues phosphorylated are less important than their total number, and this is interpreted to be a mechanism that links nutritional availability with entry into the cell cycle (32). The underlying assumption is that once a stable complex between phosphorylated Sic1 and Cdc4 is formed, substrate degradation followed by SCF turnover are assured. In contrast, we find that extraction of Ufo1 from SCFUfo1 complexes is a highly regulated process and depends specifically on its unique UIMs.
The SCF alternates between an active and inactive state regulated by neddylation of Cdc53 (43). In Schizosaccharomyces pombe and higher eukaryotes the signalosome (CSN) is responsible for inactivating SCF function by deneddylation. In conditions when SCF activity is limiting, the deneddylation by the CSN and the deubiquitylating activity of its associated DUB are critical for survival. This is due to their stabilization of the F-box protein, which would otherwise be rapidly eliminated in an autocatalytic reaction on binding active SCF core elements (42). Our experiments show that Ufo1 degradation requires the SCF functions Cdc53 and its associated E2, Cdc34. Stabilization of Ufo1 deleted for its F-box domain indicates the importance of incorporation of Ufo1 into SCF complexes for its degradation. Deletion of the Ufo1 UIMs leads to extension of the half-life of Ufo1. Ufo1UIM resembles canonical F-box proteins that do not have UIMs, and its extended half-life may perhaps be attributed to the UIMs superseding internal degron sequences similar to those identified in Cdc4 and Met30 (9, 31, 46). The S. pombe ortholog of Ufo1, Pof10, is also very stable, and in this species overexpression of the full-length protein leads to loss of viability (16). The finding that Ufo1UIMK134A,K141A does not lead to cell cycle arrest despite the absence of UIMs suggests that interactions of Ufo1UIM with other protein(s) are important. These mutations are in the vicinity of the F-box domain and may affect the strength of binding between the Ufo1 F-box do- main and Skp1 and/or result in a different positioning of Ufo1UIMK134A,K141A within the SCFUfo1 complex. For example, in S. pombe a defined point mutation in the F-box binding region of Skp1 differentially affects its binding affinity for different F-box proteins (28). Alternatively, F-box proteins may have a unique pathway of degradation: the proteasome receptor, Cic1, that binds an subunit of the 20S CP mediates degradation of the F-box proteins Cdc4 and Grr1. Cic1 is not required for degradation of Ufo1 (not shown); however, the existence of a different 20S CP-interacting proteasome receptor cannot be excluded (18). Mutant Ufo1UIMK134A,K141A may be treated as an SCF ubiquitylated substrate rather than as a component of the SCF complex and may be degraded via a different postubiquitylation pathway.
There is growing evidence for regulation of additional stages in the assembly of active SCF complexes. Entry of Skp1 into SCF complexes is inhibited by Cadmium: in the presence of Cadmium the F-box protein, Met30, binds its substrate Met4 but no longer binds Skp1 (2, 44). A further downstream regulated interaction is demonstrated between the Skp1-like BTB-adaptor, Keap1, that binds its substrate, the transcription factor Nrf2, but no longer binds the Cul3 scaffold under stress conditions. This abrogates ubiquitylation of Nrf2 and leads to its stabilization (45). These experiments show that in addition to F-box protein recruitment of a substrate that is marked for degradation by signal transduction pathways, assembly of the substrate-F-box protein complex into active SCF complexes is a highly regulated process.
The interaction of Ufo1 with Ddi1 is important for the degradation of Ufo1 and for the turnover of SCFUfo1 complexes. This may be by recycling the SCF core components Skp1 and Cdc53 into alternative SCF complexes. Ddi1 is a very stable protein and is not a degradation substrate of Ufo1. The Ufo1 UIMs bind all yeast UbL-UbA proteins indiscriminately; however, full-length Ufo1 only binds Ddi1. The UIMs are required for its interaction with Ddi1. The importance of the interaction between Ufo1 and Ddi1 is shown most clearly by the extended half-life of Ufo1 in ddi1 mutants and by the cell cycle arrest of ddi1 cells induced by overexpression of full-length UFO1. These genetic data are supported by experimental data obtained with the two-hybrid system and in co-IP experiments that demonstrate that both the UbL and UbA domains of Ddi1 are required for interaction with Ufo1. Regulation of the SCF pathway is a novel function for an UbL-UbA protein. The UbL domain of Ddi1 is required for interaction with the 19S RP of the proteasome, and one model for activation of an UbL-UbA protein could be that binding of a K48-linked ubiquitin chain to the UbA domain frees the UbL domain to bind the proteasome. This model is suggested by our finding that Ddi1 can bind ubiquitylated Ho in the absence of its UbL domain (23). Furthermore, the UbL of Rad23 interacts with its UbA domain, and this may represent an inactive form of the protein (37). The UbA domain of Ddi1 is a cis-stabilization element (13), and we have shown that it interacts with ubiquitin-conjugated Ho (23). Here, too, we find that it is important for the interaction of Ddi1 with Ufo1. Furthermore, it has been shown that the UbL-UbA KPC2 subunit is required to stabilize the KPC1 RING subunit of the heterodimeric E3 (12). This mechanism regulates function of the E3 for p27, a cyclin-dependent kinase inhibitor in mammalian cells. It will be interesting to decipher how these competing interactions for the Ddi1 UbL are regulated. Our experiments identify further regulatory steps that determine whether the ubiquitylated substrate will be processed further by functions of the pathway that leads to its degradation. In addition, Ddi1, alias Vsm1, has a role in intracellular membrane transport (29, 30), and it is not clear whether this role involves its association with Ufo1.
We propose a model in which Ufo1 and Ufo1UIM are incorporated into SCF complexes and can support substrate ubiquitylation, but after that their fates differ. Interaction between the Ufo1 UIMs and the UbL of Ddi1 could be required for activation of Ddi1 for destabilization of the SCFUfo1 complex. In this model the UbL and UbA domains would play a regulatory role and gate access to the Ddi1 core. (In this scenario one possible explanation for the marked reduction [ca. 90%] in the binding of Ufo1 to Ddi1 lacking its UbA domain may reflect a similar lack of accessibility to the Ddi1 core.) The interaction between the Ufo1 UIMs and Ddi1 is not required for substrate extraction but is specifically required for turnover of SCFUfo1 complexes. This may involve repositioning of Ufo1 by Ddi1 within the SCF complex so that it now becomes accessible to the E2 and/or on the proteasome so that it can be deubiquitylated, unfolded, and degraded. In the absence of the Ufo1 UIMs or of Ddi1 the half-life of Ufo1 is extended, and this leads to inhibition of the SCF pathway of substrate degradation (Fig. 9).
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ACKNOWLEDGMENTS |
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These experiments were supported by the Israel Cancer Research Fund, by the Association for International Cancer Research, by the German-Israel Foundation for Scientific Research and Development, by the Israel Cancer Association, and by FP5 EU contract HPRN-CT-2002-00238.
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FOOTNOTES |
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