Structural,Expression and Interaction Analysis of Rice SKP1-Like Genes

The degradation of proteins by the 26S proteasome is initiated by protein polyubiquitination mediated by a three-step cascade. The specific ubiquitination of different target proteins is mediated by different classes of E3 ubiquitin ligases, among which the best known are Skp1-Cullin-F-box complexes. Whereas protists, fungi and some vertebrates have a single SKP1 gene, many animal and plant species possess multiple SKP1 homologues. In this paper, we report on the structure, phylogeny and expression of the complete set of rice SKP1 genes (OSKs, Oryza sativa SKP1-like genes). Our analyses indicated that OSK1 and OSK20 belong to a class of SKP1 genes that contain one intron at a conserved position and are highly expressed. In addition, our yeast two-hybrid results revealed that OSK proteins display a differing ability to interact with F-box proteins. However, OSK1 and OSK20 seemed to interact with most of the nine F-box proteins tested. We suggest that rice OSK1 and OSK20 are likely to have functions similar to the Arabidopsis ASK1 and ASK2 genes.     

The Arabidopsis F-Box Protein CORONATINE INSENSITIVE1 Is Stabilized by SCFCOI1 and Degraded via the 26S Proteasome Pathway

Jasmonate regulates critical aspects of plant development and defense. The F-box protein CORONATINE INSENSITIVE1 (COI1) functions as a jasmonate receptor and forms Skp1/Cullin1/F-box protein COI1 (SCF^COI1) complexes with Arabidopsis thaliana Cullin1 and Arabidopsis Skp1-like1 (ASK1) to recruit its substrate jasmonate ZIM-domain proteins for ubiquitination and degradation. Here, we reveal a mechanism regulating COI1 protein levels in Arabidopsis. Genetic and biochemical analysis and in vitro degradation assays demonstrated that the COI1 protein was initially stabilized by interacting with ASK1 and further secured by assembly into SCF^COI1 complexes, suggesting a function for SCF^COI1 in the stabilization of COI1 in Arabidopsis. Furthermore, we show that dissociated COI1 is degraded through the 26S proteasome pathway, and we identified the 297th Lys residue as an active ubiquitination site in COI1. Our data suggest that the COI1 protein is strictly regulated by a dynamic balance of SCF^COI1-mediated stabilization and 26S proteasome–mediated degradation and thus maintained at a protein level essential for proper biological functions in Arabidopsis development and defense responses.     

F-box Protein Arabidillo-1 Promotes Lateral Root Development by Depressing the Functioning of GA<Subscript>3</Subscript> in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, Arabidillo-1 and Arabidillo-2 have great sequence homology to Dictyostelium and metazoan β–catenin/Armadillo, which are important to animal and Dictyostelium development. Arabidillo-1 and Arabidillo-2 promote lateral root formation redundantly in Arabidopsis. Here, we showed that gibberellins (GA₃) has a greater inhibitory effect on lateral root growth from the null mutant arabidillo-1 than from the wild type, suggesting that the mechanism for Arabidillo-1-regulated modulation of lateral root proliferation is associated with GA₃-metabolic or signaling pathways. Our yeast two-hybrid analysis demonstrated that Arabidillo-1 interacts with ASK2 and ASK11, and that ASK2 can bind with the F-box domain of Arabidillo-1. Therefore, Arabidillo-1 is involved in the ubiquitin/26S proteasome-mediated proteolytic pathway. Based on these results, we conclude that Arabidillo-1 can degrade some positive regulator of the GA₃ signaling pathway through selective protein degradation of ubiquitin/26S. Moreover, that process is believed to be the mechanism for Arabidillo-1 promotion of lateral root development in Arabidopsis.     

ASK1 physically interacts with COI1 and is required for male fertility inArabidopsis

Jasmonates are a new class of plant hormones that play important roles in plant development and plant defense. TheCOI1 gene was previously shown to be required for jasmonate-regulated plant fertility and defense. We demonstrated for the first time that COI1 interacts with theArabidopsis SKP1-LIKE1 (ASK1) to form a complex that is required for jasmonate action inplanta. Functional analysis by antisense strategy showed thatASK1 is involved in male fertility.     

The P25 pathogenicity factor of Beet necrotic yellow vein virus targets the sugar beet 26S proteasome involved in the induction of a hypersensitive resistance response via interaction with an F-box protein

P25, a Beet necrotic yellow vein virus (BNYVV) pathogenicity factor, interacts with a sugar beet protein with high homology to Arabidopsis thaliana kelch repeat containing F-box family proteins (FBK) of unknown function in yeast. FBK are members of the Skp1-Cullin-F-box (SCF) complex that mediate protein degradation. Here, we confirm this sugar beet FBK-P25 interaction in vivo and in vitro and provide evidence for in planta interaction and similar subcellular distribution in Nicotiana tabacum leaf cells. P25 even interacts with an FBK from A. thaliana, a BNYVV nonhost. FBK functional classification was possible by demonstrating the interaction with A. thaliana orthologs of Skp1-like (ASK) genes, a member of the SCF E3 ligase. By means of a yeast two-hybrid bridging assay, a direct effect of P25 on SCF-complex formation involving ASK1 protein was demonstrated. FBK transient Agrobacterium tumefaciens-mediated expression in N. benthamiana leaves induced a hypersensitive response. The full-length F-box protein consists of one F-box domain followed by two kelch repeats, which alone were unable to interact with P25 in yeast and did not lead to cell-death induction. The results support the idea that P25 is involved in virus pathogenicity in sugar beet and suggest suppression of resistance response.     

Conservation and divergence of ASK1 and ASK2 gene functions during male meiosis in Arabidopsis thaliana

Selective proteolysis of regulatory proteins mediated by the ubiquitin pathway is an important mechanism for controlling many biological events. The SCF (Skpl-Cullin-F-box protein) class of E3 ubiquitin ligases controls the ubiquitination of a wide variety of substrates, thereby mediating their degradation by the 26S proteasome. The Arabidopsis genome contains 21 genes encoding Skp1-like proteins that are named as ASKs (Arabidopsis Skp1-like). So far, only the ASK1 gene has been characterized genetically, and is known to be required for male meiosis, flower development, and auxin response. The ASK2 gene is most similar to ASK1 in terms of both the amino acid sequence and expression pattern. To compare ASK2 with ASK1 functionally in male meiosis, different transgenic lines over-expressing ASK1 and ASK2 were tested for their ability to complement the male meiosis defect of the ask1-1 mutant. The genomic ASK1 rescued the ask1-1 mutant defects. The 35S::ASK1 transgene restored male fertility to the ask1-1 mutant, although the percentages of normal pollen grains and tetrads were reduced. 35S::ASK2 lines in the ask1-1 background exhibited partial fertility with even fewer normal pollen grains and tetrads than those of the 35S::ASK1 lines. Detailed analysis of chromosome behavior during male meiosis demonstrated that 35S::ASK1 and 35S::ASK2 lines had different fractions of pollen mother cells undergoing normal meiosis. Our results suggest that ASK2 partially substitutes for ASK1 if expressed at higher than normal levels.     

Expression and interaction analysis of Arabidopsis Skp1-related genes

Specific protein degradation has been observed in several aspects of development and differentiation in many organisms. One example of such proteolysis is regulated by protein polyubiquitination that is promoted by the SCF complex consisting of Skp1, cullin, and an F-box protein. We examined the activities of the Arabidopsis Skp1-related proteins (ASKs). Among 19 annotated ASK genes, we isolated 16 of the corresponding cDNAs (ASK1, 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19), and examined their gene products for interactions with 24 representatives of F-box proteins carrying various classes of the C-terminal domains using the yeast two-hybrid system. As a result, we found diverse binding specificities: ASK1, ASK2, ASK11 and ASK12 interacted well with COI1, FKF1, UFO-like protein, LRR-containing F-box proteins, and other F-box proteins with unknown C-terminal motifs. We also observed specific interaction between F-box proteins and ASK3, ASK9, ASK13, ASK14, ASK16 and ASK18. In contrast, we detected no interaction between any of the 12 ASK proteins and F-box proteins containing CRFA, CRFB or CRFC domains. Both histochemical and RT-PCR analysis of eight ASK genes expression revealed unique expression patterns for the respective genes.     

SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase

Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD-protein. Here we show that SKP1/ASK1, a conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1-binding proteasomal protein, alpha4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs. SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts. SKP1/ASK1, cullin and proteasomal alpha-subunits show nuclear co-localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division. Detection of SnRK in purified 26S proteasomes and co-purification of epitope- tagged SKP1/ASK1 with SnRK, cullin and proteasomal alpha-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and alpha4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis.     

The ASK1 and ASK2 genes are essential for Arabidopsis early development

The requirement of CUL1 for Arabidopsis embryogenesis suggests that Skp1-CUL1-F-box protein (SCF) complexes play important roles during embryo development. Among the 21 Arabidopsis Skp1-like genes (ASKs), it is unknown which ASK gene(s) is essential for embryo development. In this study, we demonstrate a vital role for ASK1 and ASK2 in Arabidopsis embryogenesis and postembryonic development through analysis of the ask1 ask2 double mutant. Our detailed analysis indicates that the double mutations in both ASK1 and ASK2 affect cell division and cell expansion/elongation and cause a developmental delay during embryogenesis and lethality in seedling growth. The expression patterns of ASK1 and ASK2 were examined further and found to be consistent with their roles in embryogenesis and seedling development. We propose that mutations in ASK1 and ASK2 abolish all of the ASK1- and ASK2-based SCF and non-SCF complexes, resulting in alteration of gene expression and leading to defects in growth and development.     

A Comprehensive Analysis of Interaction and Localization of Arabidopsis SKP1-LIKE (ASK) and F-Box (FBX) Proteins

F-Box (FBX) proteins are encoded by a multigene family present in major lineages of eukaryotes. A number of FBX proteins are shown to be subunits of SCF complex, a type of E3 ligases composed of SKP1, CULLIN, FBX and RBX1 proteins. The Arabidopsis SKP-LIKE (ASK) proteins are also members of a family and some of them interact with FBX proteins directly. To clarify how FBX and ASK proteins combine, we carried out a large-scale interaction analysis between FBX and ASK proteins using yeast two-hybrid assay (Y2H) in Arabidopsis thaliana. FBX proteins randomly chosen from those proteins that interacted with more than one ASK protein were further analyzed for their subcellular localization and in vivo interaction with ASK proteins. Furthermore, the expression profiles of FBX and ASK genes were compared. This work reveals that FBX proteins had a preference for interacting with ASK proteins depending on the domains they contain such as the FBX-associated (FBA) domain, the Kelch domain and leucine rich repeat (LRR). In addition, it was found that a single FBX protein could form multiple SCF complexes by interacting with several ASK proteins in many cases. Furthermore, it was suggested that the variation of SCF complexes were especially abundant in tissues related to male gametophyte and seed development. More than half of the FBX proteins studied did not interact with any of the ASK proteins, implying the necessity for certain regulations for their interaction in vivo and/or distinct roles from subunits of the SCF complex.     

ASK1 physically interacts with COI1 and is required for male fertility in<Emphasis Type="Italic">Arabidopsis</Emphasis>

Jasmonates are a new class of plant hormones that play important roles in plant development and plant defense. TheCOI1 gene was previously shown to be required for jasmonate-regulated plant fertility and defense. We demonstrated for the first time that COI1 interacts with theArabidopsis SKP1-LIKE1 (ASK1) to form a complex that is required for jasmonate action inplanta. Functional analysis by antisense strategy showed thatASK1 is involved in male fertility.     

Members of two gene families encoding ubiquitin-conjugating enzymes, AtUBC1-3 and AtUBC4-6, fromArabidopsis thaliana are differentially expressed

Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plantArabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designatedAtUBC1-3 andAtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of theAtUBC1-3 andAtUBC4-6 genes by the histochemical analysis of transgenicArabidopsis containing the corresponding promoters fused to the -glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between theAtUBC1-3 andAtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s inArabidopsis.     

<Emphasis Type="Italic">Drosophila</Emphasis> Bys is nuclear and shows dynamic tissue-specific expression during development

Although the bys-like family of genes has been conserved from yeast to humans, it is not apparent to what extent the function of Bys-like proteins has been conserved across phylogenetic groups. Human Bystin is thought to function in a novel cell adhesion complex involved in embryo implantation. The product of the yeast bys-like gene, Enp1, is nuclear and has a role in pre-ribosomal RNA (pre-rRNA) splicing and ribosome biogenesis. To gain insight into the function of the Drosophila melanogaster bys-like family member, termed bys, we examined bys mRNA expression and the localization of Bys protein. In embryos, bys mRNA is expressed in a tissue-specific pattern during gastrulation. In the larval wing imaginal disc, bys mRNA is expressed in the ventral and dorsal regions of the wing pouch, regions that give rise to epithelia that adhere to one another after the wing disc everts. The bys mRNA expression patterns could be interpreted as being consistent with a role for Bys in events requiring cell-cell interactions. However, embryonic bys mRNA expression patterns mirror those of genes that are potential targets of the growth regulator Myc and encode nucleolar proteins implicated in cell growth. Additionally, in Schneider line 2 (S2) cells, an epitope-tagged Bys protein is localized to the nucleus, suggesting that Drosophila Bys function may be conserved with that of yeast Enp1.Edited by D.A. Weisblat