Arabidopsis AtCUL3a and AtCUL3b Form Complexes with Members of the BTB/POZ-MATH Protein Family

Abstract

The ubiquitin proteasome pathway in plants has been shown to be important for many developmental processes. The E3 ubiquitin-protein ligases facilitate transfer of the ubiquitin moiety to substrate proteins. Many E3 ligases contain cullin proteins as core subunits. Here, we show that Arabidopsis (Arabidopsis thaliana) AtCUL3 proteins interact in yeast two-hybrid and in vitro pull-down assays with proteins containing a BTB/POZ (broad complex, tramtrack, bric-a-brac/pox virus and zinc finger) motif. By changing specific amino acid residues within the proteins, critical parts of the cullin and BTB/POZ proteins are defined that are required for these kinds of interactions. In addition, we show that AtCUL3 proteins assemble with the RINGfinger protein AtRBX1 and are targets for the RUB-conjugation pathway. The analysis of AtCUL3a and AtCUL3b expression as well as several BTB/POZ-MATH genes indicates that these genes are expressed in all parts of the plant. The results presented here provide strong evidence that AtCUL3a and AtCUL3b can assemble in Arabidopsis with BTB/POZ-MATH and AtRBX1 proteins to form functional E3 ligases.The ubiquitin proteasome pathway participates in a broad variety of physiologically and developmentally controlled processes in plants (for an overview, see Smalle and Vierstra, 2004). A critical step involves E3 ubiquitin ligases that facilitate the transfer of ubiquitin moieties to a substrate protein, leading to degradation via the 26S proteasome.In Arabidopsis (Arabidopsis thaliana), the bestcharacterized E3s are the SCF (Skp1-cullin-F-box) complexes that consist of at least four subunits: a cullin protein (AtCUL1), an ASK (Arabidopsis Skp1 ortholog) protein, a RING finger protein (RBX1), and an F-box protein (Gray et al., 1999Lechner et al., 2002;Shen et al., 2002). The cullin is a scaffolding subunit for the SCF and binds the ASK-F-box and RBX1 subunits within NH 2 -and COOH-terminal domains, respectively (Zheng et al., 2002). F-box proteins are substrate adaptors that confer specificity to the assembled SCF complexes (Gagne et al., 2002). AtCUL1-dependent E3s are crucial regulators for phytohormone responses, flowering, embryo development, and other processes (Smalle and Vierstra, 2004).At least 11 cullins are encoded in the Arabidopsis genome . However, only six members contain the conserved RUB (related to ubiquitin)-modification site characteristic of cullins that assemble into an SCF or related E3 complex (del Querido et al., 2001;del Pozo et al., 2002). Based on homology to cullins in other organisms, these six Arabidopsis cullins can be classified into three groups. The first group is the CUL1 family, which includes AtCUL1 (At4g02570), AtCUL2 (At1g02980), and AtCUL5 (At1g43140). It is likely that all three members participate in an SCF complex Risseeuw et al., 2003). The second group comprises the CUL3 family with AtCUL3a (At1g26830) and AtCUL3b (At1g69670). These two cullins are approximately 88% identical to each other and represent potential Arabidopsis orthologs of Caenorhabditis...

Cullins 3a and 3b Assemble with Members of the Broad Complex/Tramtrack/Bric-a-Brac (BTB) Protein Family to Form Essential Ubiquitin-Protein Ligases (E3s) in Arabidopsis

“…In a recent report, Weber et al (50) provided further support by showing that another Arabidopsis BTB protein containing a MATH domain interacts with CUL3a/b. Residues important for BTB docking by CUL3 and for CUL3 docking by the BTB domain in the yeast and animal versions (15)(16)(17)(18) are strongly conserved in the Arabidopsis and rice counterparts, strongly suggesting that a similar interaction lattice is used to bind the plant BTB proteins to the CUL3a/b-RBX1 subcomplex.…”

“…49) were found in four separate clades (A1, C, D2, and E), implying that other proteins within these same clades also interact with CUL3a/b. Weber et al (50) recently supported this notion by showing that another member of the MATH domain-containing A1 family also binds CUL3a/b. For the remaining clades, representative BTB proteins were tested for binding with CUL3a/b by Y2H assay, but interactions were not apparent when compared with negative controls (data not shown).…”

Arabidopsis BTB/POZ protein-dependent PENETRATION3 trafficking and disease susceptibility

“…Our study unravels a specific requirement of a BTB/POZdomain protein for PEN3 membrane trafficking. The BTB/POZdomain is an evolutionarily conserved motif and protein-protein interaction domain found in proteins involved in a wide range of cellular functions [13][14][15][16][17] . The Arabidopsis genome encodes for around 80 BTB/POZ-domain proteins, of which a small subset act as substrate adaptors for E3 ubiquitin ligase complexes by interacting with CULLIN3 (CUL3) [13][14][15][16] .…”

“…The BTB/POZdomain is an evolutionarily conserved motif and protein-protein interaction domain found in proteins involved in a wide range of cellular functions [13][14][15][16][17] . The Arabidopsis genome encodes for around 80 BTB/POZ-domain proteins, of which a small subset act as substrate adaptors for E3 ubiquitin ligase complexes by interacting with CULLIN3 (CUL3) [13][14][15][16] . Whereas no BTB/POZ-domain protein has previously been associated with ER membrane trafficking in plants, a precedent exists in mammals: Transport of procollagen I from the ER involves monoubiquitylation-dependent regulation of the COPII component SEC31, which requires the KLHL12 BTB/ POZ-domain protein as a CUL3 adaptor 13,17,18 .…”

“…However, KLHL12 and EAP3 are molecularly unrelated except for harbouring a BTB/ POZ domain. Moreover, the BTB/POZ domain of EAP3 displays poor conservation of the residues required for CUL3 binding and is not likely to function as an E3 ligase adaptor 15,16 . Thus, we hypothesize that EAP3 mediates protein-protein interactions at the cytosolic face of the ER, where the ATP-binding cassette domains of PEN3 are located 1 .…”