Efficient ASK-assisted system for expression and purification of plant F-box proteins.

Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins.

Expression, purification and crystallization of the FP domain of the human F-box protein Fbxo7.

Fbxo7 is a conserved protein in higher eukaryotes that belongs to the F-box protein family. Fbxo7 is the substrate-recognition component of the SCFFbxo7 (Skp1-Cul1-Fbox protein) E3 ubiquitin ligase. Besides the F-box motif, Fbxo7 also contains a C-terminal proline-rich region, an N-terminal ubiquitin-like domain and a novel FP (Fbxo7/PI31) domain preceding the F-box motif. The FP domains of Fbxo7 and the PI31 proteasome inhibitor mediate interaction between the two proteins. For structure determination of the FP domain of Fxbo7, a protein construct (amino acids 181-335) corresponding to the FP domain was expressed, purified and crystallized. The native and selenomethionine-labeled proteins crystallized in different crystal forms. Native and single-wavelength anomalous dispersion data sets with diffraction to 2.1 and 2.0 Šresolution, respectively, have been collected and structure determination is in progress.

Comprehensive identification of substrates for F-box proteins by differential proteomics analysis.

Although elucidation of enzyme-substrate relations is fundamental to the advancement of biology, universal approaches to the identification of substrates for a given enzyme have not been established. It is especially difficult to identify substrates for ubiquitin ligases, given that most such substrates are immediately ubiquitylated and degraded as a result of their association with the enzyme. We here describe the development of a new approach, DiPIUS (differential proteomics-based identification of ubiquitylation substrates), to the discovery of substrates for ubiquitin ligases. We applied DiPIUS to Fbxw7α, Skp2, and Fbxl5, three of the most well-characterized F-box proteins, and identified candidate substrates including previously known targets. DiPIUS is thus a powerful tool for unbiased and comprehensive screening for substrates of ubiquitin ligases.

Characterization of novel F-box proteins in plants induced by biotic and abiotic stress.

Plants protect against pathogen infections by a combination of constitutive and induced strategies. The induction of plant defense involves the recognition of compounds derived from the pathogen or the plant itself, called elicitors. Looking for new genes involved in plant defense responses, we isolated a cDNA clone corresponding to an elicitor-induced mRNA from Phaseolus vulgaris cell suspension cultures. This clone, PvFBS1, encodes a protein with an F-box, therefore a putative component of an SCF ubiquitin ligase complex. PvFBS1 mRNA accumulates in leaves of whole plants in response to wounding or osmotic stress, as well as, following the application of methyl jasmonate (MeJA). salicylic acid (SA) or abscisic acid (ABA). Several sequences related to PvFBS1 were found in the GenBank. In Arabidopsis thaliana there are 4 genomic sequences coding for proteins with similarity to PvFBS1. One of them, AtFBS1, displays a pattern of induction analogous to the one observed for PvFBS1. A yeast two-hybrid assay proved that AtFBS1 was able to interact with ASK1, the component of the SCF complex that binds the F-box. A deletion of the F-box in AtFBS1 abolishes the ability of this protein to interact with ASK1. This demonstrates the functionality of the F-box contained in AtFBS1. Gene fusions to the GUS reporter gene revealed a complex regulation for AtFBS1 expression.

Molecular analysis and expression of a floral organ-relative F-box gene isolated from 'Zigui shatian' pummelo (Citrus grandis Osbeck).

F-box proteins are a large family of eukaryotic proteins that contained a conserved motif of approximately 40 amino acids. They play an important role in the processing of degradation of cellular regulatory proteins. In this study we isolated a full-length of cDNA encoding a putative F-box protein from Citrus grandis Osbeck CV 'Zigui shatian' pummelo and designated as CgF-box. The cDNA sequence of CgF-box was 920 bp containing a 585 bp open reading frame encoding a precursor protein of 194 amino acid residues. The deduced protein comprised a conserved F-box domain at the position from the 40th to 84th amino acids. Cluster analysis suggested that CgF-box was more closely related to the grape F-Box protein. Southern hybridization verified CgF-box existed in the genome as multiple copies. The expression analysis revealed that the expression level of CgF-box gene remarkably increases during the flower developmental process of 'Zigui shatian' pummelo, such as high level of expression was noted in style, petal and anther, on the other hand low level of expression was found in ovary and leaf. For further verifying the different expression in different tissue of this gene, in situ hybridization was conducted, strong expression signal could be observed in the style, stigma and anther, low even no signal was observed in ovary. According to their findings we can conclude that CgF-box was not only involved in flower maturation, but also showed different roles in different tissue.

Affinity purification of Candida albicans CaCdc4-associated proteins reveals the presence of novel proteins involved in morphogenesis.

Candida albicans CDC4 is nonessential and plays a role in suppressing filamentous growth, in contrast to its evolutionary counterparts involved in the G1-S transition of the cell cycle. Genetic epistasis analysis has indicated that proteins besides Sol1 are targets of C. albicans Cdc4. Moreover, no formal evidence suggests that C. albicans Cdc4 functions through the ubiquitin E3 ligase of the Skp1-Cul1/Cdc53-F-box complex. To elucidate the role of C. albicans CDC4, C. albicans Cdc4-associated proteins were sought by affinity purification. A 6xHis epitope-tagged C. albicans Cdc4 expressed from Escherichia coli was used in affinity purifications with the cell lysate of C. albicans cdc4 homozygous null mutant. Candida albicans Cdc4 and its associated proteins were resolved by SDS-PAGE and visualized by silver staining. The candidate proteins were recovered and trypsin-digested to generate MALDI-TOF spectra profiles, which were used to search against those of known proteins in the database to reveal their identities. Two out of four proteins encoded by GPH1 and THR1 genes were further verified to interact with C. albicans Cdc4 using a yeast two-hybrid assay. We conclude that in vitro affinity purification using C. albicans Cdc4 generated from E. coli as the bait and proteins from cell lysate of C. albicans cdc4 homozygous null mutant as a source of prey permit the identification of novel proteins that physically interact and functionally associate with C. albicans Cdc4.

Molecular cloning and expression analysis of an F-box protein gene responsive to plant hormones in Brassica napus.

F-box protein family is characterized by an F-box motif that has been shown to be critical for the controlled degradation of regulatory proteins. In plant, F-box protein plays an important role in signal pathways and involved in various signal transduction systems. A full-length cDNA encoding a putative F-box protein, designated as BnSLY1, was isolated from Brassica napus. The full-length cDNA of BnSLY1 was 809 bp containing a 438 bp open reading frame encoding a precursor protein of 138 amino acid residues. Comparative and bioinformatic analyses revealed that BnSLY1 showed high degree of homology with F-box proteins from other plant species and contained F-box, GGF and LSL conserved motifs. The expression of BnSLY1 under exogenous gibberellins acid-3 (GA3), abscisic acid (ABA) and GA biosynthetic inhibitor paclobutrazol (PAC) was analyzed using real-time PCR. The results showed that the expression of BnSLY1 was down-regulated after GA3 treatment and prominently induced by ABA in the low concentrations. Moreover, BnSLY1 was also induction in the high concentrations of PAC. These results suggest that the expression of BnSLY1 was regulated by the exogenous GA3, ABA and PAC and may be related to endogenous level of GA in B. napus.

DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth.

BACKGROUND: The growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. Genetic studies have demonstrated that the ubiquitin ligase Highwire restrains synaptic terminal growth by down-regulating the MAP kinase kinase kinase Wallenda/dual leucine zipper kinase (DLK). To investigate the mechanism of Highwire action, we have identified DFsn as a binding partner of Highwire and characterized the roles of DFsn in synapse development, synaptic transmission, and the regulation of Wallenda/DLK kinase abundance. RESULTS: We identified DFsn as an F-box protein that binds to the RING-domain ubiquitin ligase Highwire and that can localize to the Drosophila neuromuscular junction. Loss-of-function mutants for DFsn have a phenotype that is very similar to highwire mutants - there is a dramatic overgrowth of synaptic termini, with a large increase in the number of synaptic boutons and branches. In addition, synaptic transmission is impaired in DFsn mutants. Genetic interactions between DFsn and highwire mutants indicate that DFsn and Highwire collaborate to restrain synaptic terminal growth. Finally, DFsn regulates the levels of the Wallenda/DLK kinase, and wallenda is necessary for DFsn-dependent synaptic terminal overgrowth. CONCLUSION: The F-box protein DFsn binds the ubiquitin ligase Highwire and is required to down-regulate the levels of the Wallenda/DLK kinase and restrain synaptic terminal growth. We propose that DFsn and Highwire participate in an evolutionarily conserved ubiquitin ligase complex whose substrates regulate the structure and function of synapses.

Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases.

The ubiquitin-mediated proteolysis of cyclin E plays a central role in cell-cycle progression, and cyclin E accumulation is a common event in cancer. Cyclin E degradation is triggered by multisite phosphorylation, which induces binding to the SCF(Fbw7) ubiquitin ligase complex. Structures of the Skp1-Fbw7 complex bound to cyclin E peptides identify a doubly phosphorylated pThr380/pSer384 cyclin E motif as an optimal, high-affinity degron and a singly phosphorylated pThr62 motif as a low-affinity one. Biochemical data indicate that the closely related yeast SCF(Cdc4) complex recognizes the multisite phosphorylated Sic1 substrate similarly and identify three doubly phosphorylated Sic1 degrons, each capable of high-affinity interactions with two Cdc4 phosphate binding sites. A model that explains the role of multiple cyclin E/Sic1 degrons is provided by the findings that Fbw7 and Cdc4 dimerize, that Fbw7 dimerization enhances the turnover of a weakly associated cyclin E in vivo, and that Cdc4 dimerization increases the rate and processivity of Sic1 ubiquitination in vitro.

DRE-1: an evolutionarily conserved F box protein that regulates C. elegans developmental age.

During metazoan development, cells acquire both positional and temporal identities. The Caenorhabditis elegans heterochronic loci are global regulators of larval temporal fates. Most encode conserved transcriptional and translational factors, which affect stage-appropriate programs in various tissues. Here, we describe dre-1, a heterochronic gene, whose mutant phenotypes include precocious terminal differentiation of epidermal stem cells and altered temporal patterning of gonadal outgrowth. Genetic interactions with other heterochronic loci place dre-1 in the larval-to-adult switch. dre-1 encodes a highly conserved F box protein, suggesting a role in an SCF ubiquitin ligase complex. Accordingly, RNAi knockdown of the C. elegans SKP1-like homolog SKR-1, the cullin CUL-1, and ring finger RBX homologs yielded similar heterochronic phenotypes. DRE-1 and SKR-1 form a complex, as do the human orthologs, hFBXO11 and SKP1, revealing a phyletically ancient interaction. The identification of core components involved in SCF-mediated modification and/or proteolysis suggests an important level of regulation in the heterochronic hierarchy.

In vitro reconstitution of SCF substrate ubiquitination with purified proteins.

The development of in vitro systems to monitor ubiquitin ligase activity with highly purified proteins has allowed for new insights into the mechanisms of protein ubiquitination to be uncovered. This chapter describes the methodologies employed to reconstitute ubiquitination of the budding yeast cyclin-dependent kinase inhibitor Sic1 by the evolutionarily conserved ubiquitin ligase SCF(Cdc4) and its ubiquitin-conjugating enzyme Cdc34. Based on our experience in reconstituting Sic1 ubiquitination, we suggest some parameters to consider that should be generally applicable to the study of different SCF complexes and other ubiquitin ligases.

The F-box protein TIR1 is an auxin receptor.

The plant hormone auxin regulates diverse aspects of plant growth and development. Recent studies indicate that auxin acts by promoting the degradation of the Aux/IAA transcriptional repressors through the action of the ubiquitin protein ligase SCF(TIR1). The nature of the signalling cascade that leads to this effect is not known. However, recent studies indicate that the auxin receptor and other signalling components involved in this response are soluble factors. Using an in vitro pull-down assay, we demonstrate that the interaction between transport inhibitor response 1 (TIR1) and Aux/IAA proteins does not require stable modification of either protein. Instead auxin promotes the Aux/IAA-SCF(TIR1) interaction by binding directly to SCF(TIR1). We further show that the loss of TIR1 and three related F-box proteins eliminates saturable auxin binding in plant extracts. Finally, TIR1 synthesized in insect cells binds Aux/IAA proteins in an auxin-dependent manner. Together, these results indicate that TIR1 is an auxin receptor that mediates Aux/IAA degradation and auxin-regulated transcription.

Plant responses to ethylene gas are mediated by SCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor.

Plants use ethylene gas as a signal to regulate myriad developmental processes and stress responses. The Arabidopsis EIN3 protein is a key transcription factor mediating ethylene-regulated gene expression and morphological responses. Here, we report that EIN3 protein levels rapidly increase in response to ethylene and this response requires several ethylene-signaling pathway components including the ethylene receptors (ETR1 and EIN4), CTR1, EIN2, EIN5, and EIN6. In the absence of ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F box proteins, EBF1 and EBF2. Plants containing mutations in either gene show enhanced ethylene response by stabilizing EIN3, whereas efb1 efb2 double mutants show constitutive ethylene phenotypes. Plants overexpressing either F box gene display ethylene insensitivity and destabilization of EIN3 protein. These results reveal that a ubiquitin/proteasome pathway negatively regulates ethylene responses by targeting EIN3 for degradation, and pinpoint EIN3 regulation as the key step in the response to ethylene.

EIN3-dependent regulation of plant ethylene hormone signaling by two arabidopsis F box proteins: EBF1 and EBF2.

The plant hormone ethylene regulates a wide range of developmental processes and the response of plants to stress and pathogens. Genetic studies in Arabidopsis led to a partial elucidation of the mechanisms of ethylene action. Ethylene signal transduction initiates with ethylene binding at a family of ethylene receptors and terminates in a transcription cascade involving the EIN3/EIL and ERF families of plant-specific transcription factors. Here, we identify two Arabidopsis F box proteins called EBF1 and EBF2 that interact physically with EIN3/EIL transcription factors. EBF1 overexpression results in plants insensitive to ethylene. In contrast, plants carrying the ebf1 and ebf2 mutations display a constitutive ethylene response and accumulate the EIN3 protein in the absence of the hormone. Our work places EBF1 and EBF2 within the genetic framework of the ethylene-response pathway and supports a model in which ethylene action depends on EIN3 protein stabilization.