Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis.
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.
Kraft
Edward
E
Section of Molecular and Cellular Biology, Division of Biological Sciences , University of California, Davis, California 95616, USA.
Stone
Sophia L
SL
Ma
Lingeng
L
Su
Ning
N
Gao
Ying
Y
Lau
On-Sun
OS
Deng
Xing-Wang
XW
Callis
Judy
J
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D020869:Gene Expression Profiling; D018745:Genome, Plant; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D010802:Phylogeny; D044767:Ubiquitin-Protein Ligases / Q000145:classification / Q000235:genetics* / Q000378:metabolism
D044767:Ubiquitin-Protein Ligases