Zhang, Wenjing , Ito, Hironori , Gray, William M. .
Investigating the role of ETA2/CAND1 in regulating SCF complex activity.
The eta2-1 mutant was identified in a genetic screen for enhancers of the tir1-1 auxin (eta) response defect. eta2-1 plants exhibit several phenotypes related to impaired auxin response. Molecular studies found that these phenotypes are the result of reduced SCFTIR1 activity in eta2-1 mutants. Isolation of the ETA2 gene revealed that it encodes an Arabidopsis ortholog of human CAND1 (Cullin-Associated and Neddylation-Dissociated). Biochemical studies with mammalian cell lines suggest that CAND1 acts as a negative regulator of SCF function by sequestering unmodified CUL1 away from SKP1 and the F-box protein, thus preventing assembly of the SCF complex. In contrast, we find that the eta2-1 mutation diminishes the ability of CAND1 to interact with CUL1, demonstrating that the interaction between these two proteins is required for SCF activity and that CAND1 positively regulates SCF function. These paradoxical findings have been explained by a model invoking CAND1 regulation of a dynamic cycle of assembly and disassembly of the SCF complex in vivo, through association and dissociation with CUL1. Double mutant analysis with the axr6-2 and axr6-3 alleles of CUL1 reveals additional insight into the interactions between CAND1 and CUL1. Whereas eta2-1 and axr6-3 interact synergistically, eta2-1 axr6-2 double mutants show mutual suppression of eta2-1 and axr6-2. Although the eta2-1 mutation itself is recessive, its suppression of axr6-2 is dominant, indicating a heightened sensitivity to CAND1 dosage level. This genetic study, together with co-immunoprecipitation experiments suggests that the axr6-2 mutation may inhibit dissociation of CUL1 from the CAND1-CUL1 complex. The result that the eta2-1 mutation suppresses the rub-modification defect in axr6-2 implies that in order to be modified by rub CUL1 needs to be first dissociated from CAND1. Gel-filtration experiments show some surprising differences in the fractionation patterns of SCF subunits between eta2-1 mutants and wild-type, shedding new light on CAND1ís function. Molecular studies examining the effects of these mutations on SCF homeostasis are underway.
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1 - University of Minnesota, Plant Biology, 250 Biological Sciences Center, 1445 Gortner Ave., Saint Paul, MN, 55108, USA
2 - University of Minnesota, Plant Biology
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM