Plant Physiology 139:1597-1611 (2005)
© 2005 American Society of Plant Biologists
GENOME ANALYSIS
Genome Analysis and Functional Characterization of the E2 and RING-Type E3 Ligase Ubiquitination Enzymes of Arabidopsis1,[W]
Edward Kraft2,
Sophia L. Stone2,
Lingeng Ma,
Ning Su,
Ying Gao,
On-Sun Lau,
Xing-Wang Deng and
Judy Callis*
Section of Molecular and Cellular Biology, Division of Biological Sciences (E.K., S.L.S., J.C.) and Plant Biology Graduate Group Program (E.K., J.C.), University of California, Davis, California 95616; and Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520–8104 (L.M., N.S., Y.G., O.-S.L., X.-W.D.)
Attachment of ubiquitin to substrate proteins is catalyzed by the three enzymes E1, E2 (ubiquitin conjugating [UBC]), and E3 (ubiquitin ligase). Forty-one functional proteins with a UBC domain and active-site cysteine are predicted in the Arabidopsis (Arabidopsis thaliana) genome, which includes four that are predicted or shown to function with ubiquitin-like proteins. Only nine were previously characterized biochemically as ubiquitin E2s. We obtained soluble protein for 22 of the 28 uncharacterized UBCs after expression in Escherichia coli and demonstrated that 16 function as ubiquitin E2s. Twelve, plus three previously characterized ubiquitin E2s, were also tested for the ability to catalyze ubiquitination in vitro in the presence of one of 65 really interesting new gene (RING) E3 ligases. UBC22, UBC19-20, and UBC1-6 had variable levels of E3-independent activity. Six UBCs were inactive with all RINGs tested. Closely related UBC8, 10, 11, and 28 were active with the largest number of RING E3s and with all RING types. Expression analysis was performed to determine whether E2s or E3s were expressed in specific organs or under specific environmental conditions. Closely related E2s show unique patterns of expression and most express ubiquitously. Some RING E3s are also ubiquitously expressed; however, others show organ-specific expression. Of all the organs tested, RING mRNAs are most abundant in floral organs. This study demonstrates that E2 diversity includes examples with broad and narrow specificity toward RINGs, and that most ubiquitin E2s are broadly expressed with each having a unique spatial and developmental pattern of expression.
1 This work was supported by the National Science Foundation (2010 grant nos. MCB–00115870 and MCB–0519970 to J.C. and X.-W.D.); by the National Institutes of Health (grant no. GM–47850 to X.-W.D. and grant no. GM0007377–27 to E.K.); and by the Natural Sciences and Engineering Research Council of Canada and the International Human Frontier Science Program Organization fellowships (to S.L.S.). Y.J. was the recipient of a Yale University Joseph F. Cullman Jr. fellowship and L.M. was a long-term postdoctoral fellow of the Human Frontier Science Program.
2 These authors contributed equally to the paper.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Judy Callis (jcallis{at}ucdavis.edu).
[W] The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.105.067983.
* Corresponding author; e-mail jcallis{at}ucdavis.edu; fax 530–752–3085.
Received July 1, 2005;
returned for revision September 30, 2005;
accepted October 17, 2005.
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