The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis

Abstract

The covalent attachment of ubiquitin is an important determinant for selective protein degradation by the 26S proteasome in plants and animals. The specificity of ubiquitination is often controlled by ubiquitin-protein ligases (or E3s), which facilitate the transfer of ubiquitin to appropriate targets. One ligase type, the SCF E3s are composed of four proteins, cullin1/Cdc53, Rbx1/Roc1/Hrt1, Skp1, and an F-box protein. The F-box protein, which identifies the targets, binds to the Skp1 component of the complex through a degenerate N-terminal approximately 60-aa motif called the F-box. Using published F-boxes as queries, we have identified 694 potential F-box genes in Arabidopsis, making this gene superfamily one of the largest currently known in plants. Most of the encoded proteins contain interaction domains C-terminal to the F-box that presumably participate in substrate recognition. The F-box proteins can be classified via a phylogenetic approach into five major families, which can be further organized into multiple subfamilies. Sequence diversity within the subfamilies suggests that many F-box proteins have distinct functions and/or substrates. Representatives of all of the major families interact in yeast two-hybrid experiments with members of the Arabidopsis Skp family supporting their classification as F-box proteins. For some, a limited preference for Skps was observed, suggesting that a hierarchical organization of SCF complexes exists defined by distinct Skp/F-box protein pairs. Collectively, the data shows that Arabidopsis has exploited the SCF complex and the ubiquitin/26S proteasome pathway as a major route for cellular regulation and that a diverse array of SCF targets is likely present in plants.

Fig 1.

Phylogenetic tree of the F-box protein superfamily from Arabidopsis. The 60-aa F-box motifs from the 694 potential F-box proteins were aligned by clustalx. The alignment then was used to generate an unrooted phylogenetic tree with MEGA 2.1, using the p-distance method and a bootstrap value of 1,000. Individual members of the tree are color-coded by (A) the nature of the domain(s) C-terminal to the F-box or (B) the number of introns within the respective genes. Unknown represents F-box proteins that are truncated or have no obvious C-terminal interaction domain. The 20 groups identified from the phylogenetic analysis are marked on the right. Arrowheads denote the position of Arabidopsis F-box proteins identified previously by genetic analyses. The bar represents the branch length equivalent to 0.1 amino acid changes per residue. Expanded versions of the trees bearing the AGI numbers for each locus can be found in Figs. 7 and 8, which are published as supporting information on the PNAS web site.

Fig 2.

Sequence alignment of representative Arabidopsis F-box motifs. The 42-aa core F-box sequences from UFO, the human F-box protein Skp2, and from representatives of each of the 20 F-box protein groups from Arabidopsis were aligned by clustalx and displayed with macboxshade, using a threshold of 55%. Conserved and similar amino acids are shown in black and gray boxes, respectively. Dots denote gaps. Designations on the left identify the group and AGI number for each protein. Arrowheads mark the amino acids positions important for the Skp/F-box interactions between human Skp1 and Skp2 (27, 28). The alignment of all 694 F-box motifs can be found in Table 3, which is published as supporting information on the PNAS web site.

Fig 3.

Diagrams of representative Arabidopsis F-box proteins with information on the structure and position of the C-terminal interaction domains. Shown on the left are the types of C-terminal domains, the number of F-box proteins predicted to have those domains, and the AGI number of the representative diagramed on the right.

Fig 4.

Expanded sections of the Arabidopsis F-box protein phylogenetic tree. The sections were extracted from the complete tree created using the 60-aa F-box motif for alignment (see Fig. 1). (A) Section of the tree showing part of the C3 subfamily containing members with a C-terminal Tub domain. The phylogeny on the left is color-coded based on the number of introns in the respective genes. The diagrams on the right show the predicted organization of the corresponding proteins showing the Tub and LRR domains in cyan and green, respectively. The arrowheads in the protein diagrams mark the intron positions. Yellow, blue, and red arrowheads denote whether the intron interrupts the gene after the first, second or third nucleotide of the codon, respectively. (B) Section of the tree showing part of the C4 subfamily containing the TIR1, COI1, and ORE9/MAX2 proteins. The tree is color-coded based on the nature of the region C-terminal to the F-box. The bars represent the branch length equivalent to 0.1 amino acid changes per residue.

Fig 5.

Location of various FBX genes in chromosome III of Arabidopsis. Each gene is color-coded according to its family as shown in Fig. 1. Below is an expanded view of a ≈150-kbp segment of chromosome III showing the position and orientation of several clusters of B7 FBX genes (green arrowheads). The direction of the arrowheads indicate the 5′ to 3′ orientation of each gene. Open arrowheads identify non-FBX genes. The nucleotide location of the chromosomal segment is shown below. Similar descriptions of the other Arabidopsis chromosomes can be found in Fig. 9.

Fig 6.

Interaction of representative Arabidopsis F-box proteins with various Arabidopsis Skps (ASKs) by Y2H analysis. (A) Phylogenetic analysis of the 19 ASK proteins. Those in bold were used as bait in the Y2H analysis. (B) Y2H analyses of representative F-box protein/ASK pairs by growth selection at 28°C for 4 days on 10 mM 3-AT. See Table 4, which is published as supporting information on the PNAS web site, for complete results.