Mutation ofE1-CONJUGATING ENZYME-RELATED1Decreases RELATED TO UBIQUITIN Conjugation and Alters Auxin Response and Development

Abstract

The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes. RUB conjugation to specific target proteins is necessary for the development of many organisms, including Arabidopsis (Arabidopsis thaliana). Here, we report the isolation and characterization of e1-conjugating enzyme-related1-1 (ecr1-1), an Arabidopsis mutant compromised in RUB conjugation. The ecr1-1 mutation causes a missense change located two amino acid residues from the catalytic site cysteine, which normally functions to form a thioester bond with activated RUB. A higher ratio of unmodified CUL1 relative to CUL1-RUB is present in ecr1-1 compared to wild type, suggesting that the mutation reduces ECR1 function. The ecr1-1 mutant is resistant to the auxin-like compound indole-3-propionic acid, produces fewer lateral roots than wild type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. In addition, ecr1-1 hypocotyls fail to elongate normally when seedlings are grown in darkness, a phenotype shared with certain other RUB conjugation mutants that is not general to auxin-response mutants. The suite of ecr1-1 molecular and morphological phenotypes reflects roles for RUB conjugation in many aspects of plant growth and development. Certain ecr1-1 elongation defects are restored by treatment with the ethylene-response inhibitor silver nitrate, suggesting that the short ecr1-1 root and hypocotyl result from aberrant ethylene accumulation. Further, silver nitrate supplementation in combination with various auxins and auxin-like compounds reveals that members of this growth regulator family may differentially rely on ethylene signaling to inhibit root growth.

MLN4924 Is an Efficient Inhibitor of NEDD8 Conjugation in Plants

“…The even stronger decrease in hypocotyl elongation together with the visible increase in anthocyanin accumulation suggested that axr1 as well ecr1 mutants are already sensitized in this pathway (Fig. 4, A and B; Leyser et al, 1993;Woodward et al, 2007). We then also found that HY5 protein but not HY5 transcript level increases in dark-grown MLN4924-treated seedlings and that this correlates with an increase in transcript abundance of light-induced genes in these seedlings (Fig.…”

“…In Arabidopsis, NEDD8 is known as RUB and is encoded by three different genes (Bostick et al, 2004); NAE is composed of the NAE1 subunit AXR1 or its homolog AXR1-LIKE (AXL) and the UBA3 subunit E1 C-TERMINAL RELATED (ECR1; del Pozo et al, 2002;Dharmasiri et al, 2007). axr1 and ecr1 mutants are insensitive to the root growth-inhibiting effect of the phytohormone auxin and have reduced root gravitropism (Leyser et al, 1993;Woodward et al, 2007). axr1 mutants are partially impaired in NEDD8 conjugation, and the effect of the axr1 mutation on NEDD8 conjugation and development is enhanced when the AXR1 homolog AXL is dysfunctional, resulting in embryo lethality (Dharmasiri et al, 2007;Hotton et al, 2011).…”

DENEDDYLASE1 Deconjugates NEDD8 from Non-Cullin Protein Substrates in Arabidopsis thaliana

“…Like ubiquitin, NEDD8 is conjugated to substrate proteins through an enzymatic cascade that includes the E1 NEDD8 activating enzyme (NAE); in Arabidopsis, NAE is a heterodimer of AXR1 (AUXIN RESISTANT1) or AXL (AXR1-LIKE) and ECR1 (E1 C-TERMINAL RELATED1). The NEDD8-conjugating cascade also includes an E2 conjugating enzyme; in Arabidopsis, this is RUB1 CONJUGATING ENZYME1 (RCE1; Pozo et al, 1998;del Pozo and Estelle, 1999;del Pozo et al, 2002;Dharmasiri et al, 2007;Woodward et al, 2007). NEDD8 is ultimately conjugated to its protein substrate with the help of E3 NEDD8 ligases like RBX1 (RING BOX1), a constitutive subunit of cullin-RING E3 ubiquitin ligases (CRLs), and DEFECTIVE IN CULLIN NEDDYLATION (DCN; Gray et al, 2002;Duda et al, 2008;Kurz et al, 2008).…”

Isolation and Characterization ofcul1-7, a Recessive Allele ofCULLIN1That Disrupts SCF Function at the C Terminus of CUL1 inArabidopsis thaliana

“…In many cases, slowed degradation of the mutant protein was experimentally verified (Timpte et al 1994;Leyser et al 1996;Rouse et al 1998;Nagpal et al 2000;Tian et al 2002;Tatematsu et al 2004;Yang et al 2004). Other auxin response mutants include plants with defective subunits of the SCF ubiquitin E3 ligase or mutations that cause misregulation of SCF activity/ assembly (Ruegger et al 1998;Gray et al 2003;Hellmann et al 2003;Cheng et al 2004;Chuang et al 2004;Feng et al 2004;Dharmasiri et al 2005b;Quint et al 2005;Alonso-Peral et al 2006;Walsh et al 2006;Moon et al 2007;Woodward et al 2007).…”

“…This attachment follows an enzymatic cascade similar to that of ubiquitin, beginning with RUB1/2 activation by a heterodimeric E1, then transfer to the RUB1/2 E2, and finally transfer to CUL1 using RBX1 as part of the RUB1/2 E3 (Del Pozo et al 1998Kamura et al 1999;Gray et al 2002;Bostick et al 2004;Larsen and Cancel 2004;Woodward et al 2007). One of the original auxin resistant mutants, axr1, is an allele of the N-terminal half of the RUB1/2 E1 heterodimer (Leyser et al 1993;Del Pozo et al 1998).…”