The RUB1/NEDD-8 family of ubiquitin-related genes is widely represented among eukaryotes. Here we report that Cdc53p in Saccharomyces cerevisiae, a member of the Cullin family of proteins, is stably modified by the covalent attachment of a single Rub1p molecule. Two genes have been identified that are required for Rub1p conjugation to Cdc53p. The first gene, designated ENR2, encodes a protein with sequence similarity to the amino-terminal half of the ubiquitin-activating enzyme. By analogy with Aos1p, we infer that Enr2p functions in a bipartite Rub1p-activating enzyme. The second gene is SKP1, shown previously to be required for some ubiquitin-conjugation events. A deletion allele of ENR2 is lethal with temperature-sensitive alleles of cdc34 and enhances the phenotypes of cdc4, cdc53, and skp1, strongly implying that Rub1p conjugation to Cdc53p is required for optimal assembly or function of the E3 complex SCF Cdc4. Consistent with this model, both enr2⌬ and an allele of Cdc53p that is not Rub1p modified, render cells sensitive to alterations in the levels of Cdc4p, Cdc34p, and Cdc53p.

The plant hormone auxin has a central role in many aspects of plant growth and development. By screening for mutants of Arabidopsis that are resistant to exogenous auxin, we have identified several genes that are required for normal auxin response. One of these genes, AXR1, is defined by recessive mutations that confer auxin resistance to the roots, rosettes and inflorescences of mutant plants. In addition, axr1 mutants display a variety of morphological defects that are consistent with a reduction in auxin sensitivity. Here we isolate the AXR1 gene using a map-based approach and report that AXR1 encodes a new protein with significant sequence similarity to the ubiquitin-activating enzyme E1. The AXR1 protein is highly diverged from previously characterized E1 enzymes, however, and lacks a key cysteine residue that is essential for E1 activity. AXR1 may therefore define a new class of enzymes in the ubiquitin pathway or it may have a novel function in cellular regulation which is unrelated to ubiquitin conjugation.

A screen for suppressors of the auxin resistant mutant axr1 in Arabidopsis thaliana has identified at least three second site suppressor loci called Suppressor of Auxin Resistance (SAR). In this study we focus on the SAR1 gene. Previous studies have documented the effects of the axr1 mutations on auxin-inhibition of root growth, auxin-induced gene expression, seedling morphology and aerial morphology. In this study, we show that the axr1 mutations also affect root hair development and epidermal cell length. The sar1-1 mutation suppresses at least partially, every aspect of the axr1 phenotype. Genetic experiments indicate that this suppression is gene specific. When crossed with the auxin-resistant mutant aux1-7, the suppressor has little affect on auxin response. However, the morphology of sar1-1 aux1-7 inflorescences is different from either of the single mutants indicating that both genes play a role in auxin mediated development of the inflorescence. The sar1-1 mutation also affects morphology in an AXR1 background. sar1-1 plants are shorter than wild-type, have altered leaf morphology, flower earlier than wild-type plants and appear to have reduced cell division in the primary root. In most respects sar1-1 axr1 and sar1 AXR1 plants are indistinguishable, indicating that sar1 both suppresses and is epistatic to axr1. Based on these results, we propose that SAR1 acts after AXR1 and that a major function of AXR1 is to relieve SAR1 mediated repression of auxin response.