The Arabidopsis thaliana UBC7/13/14 genes encode a family of multiubiquitin chain-forming E2 enzymes.
Covalent modification of proteins by attachment of multiubiquitin chains serves as an essential signal for selective protein degradation in eukaryotes. The specificity of ubiquitin-protein conjugation is controlled in part by a diverse group of ubiquitin-conjugating enzymes (E2s or UBCs). We have previously reported that the product of the wheat TaUBC7 gene recognizes ubiquitin as a substrate for ubiquitination in vitro, catalyzing the condensation of free ubiquitin into multiubiquitin chains linked via lysine 48 (van Nocker, S., and vierstra, R. D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10297-10301). Based on this activity, this E2 may play a central role in the ubiquitin proteolytic pathway by assembling chains in vivo. Here, we describe the cloning and characterization of a three-member gene family from Arabidopsis thaliana (designated AtUBC7/13/14) encoding structural homologs of TaUBC7. Like TaUBC7, recombinant AtUBC7/13/14 proteins formed multiubiquitin chains in vitro. AtUBC7/13/14 mRNAs were found in all tissues examined, and unlike related UBCs from yeast, the levels of mRNA were not elevated by heat stress or cadmium exposure. Transgenic Arabidopsis were engineered to express increased levels of active AtUBC7, for the first time altering the level of an E2 in a higher eukaryote. Plants expressing high levels of AtUBC7 exhibited no phenotypic abnormalities and were not noticeably enriched in multiubiquitinated conjugates. These findings indicate that the in vivo synthesis of multiubiquitin chains is not rate-limited by the abundance of AtUC7 and/or involves other, yet undefined components.
van Nocker
S
S
Department of Horticulture, University of Wisconsin-Madison, 53706, USA.
Walker
J M
JM
Vierstra
R D
RD
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D017343:Genes, Plant; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D044763:Ubiquitin-Conjugating Enzymes
D018076:DNA, Complementary; D011994:Recombinant Proteins; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases