Reference "Cloning of a 16-kDa ubiquitin carrier protein from wheat and Arabidopsis thaliana. Identification of functional domains by in vitro mutagenesis"

Reference ID

9211

Title

Cloning of a 16-kDa ubiquitin carrier protein from wheat and Arabidopsis thaliana. Identification of functional domains by in vitro mutagenesis

Source

The Journal of biological chemistry, 1991, vol. 266, pp. 23878-23885

Authors (2)

Sullivan-M-L

Vierstra-R-D

Abstract

Ubiquitin carrier proteins (E2s) are involved in the covalent attachment of
ubiquitin to a variety of cellular target proteins in eukaryotes. Here, we
report the cloning of genes from wheat and Arabidopsis thaliana that encode 16-
kDa E2s and a domain analysis of E2s by in vitro mutagenesis. The genes for
E216kDa, which we have designated wheat and At UBC1, encode proteins that are
only 33% identical (58% similar) with a 23-kDa E2 from wheat (encoded by the
gene now designated wheat UBC4), but are 63% identical (82% similar) with the E2
encoded by the Saccharomyces cerevisiae DNA repair gene, RAD6. Unlike the
proteins encoded by RAD6 and wheat UBC4, the UBC1 gene products lack acidic C-
terminal domains extending beyond the conserved core of the proteins and are
incapable of efficient in vitro ligation of ubiquitin to histones. From
enzymatic analysis of the UBC1 and UBC4 gene products mutagenized in vitro, we
have identified several domains important for E2 function, including the active
site cysteine and N-terminal and C-terminal domains. Cysteine residues 88 and 85
in the UBC1 and UBC4 gene products, respectively, are necessary for formation of
the ubiquitin-E2 thiol ester intermediate. Whereas the UBC1 gene product does
not require its additional cysteine residue at position 116 for thiol ester
formation, alteration of cysteine 143 in the UBC4 gene product greatly
diminishes this ability. The N terminus of UBC1 contains two domains that affect
activity: a proximal region containing hydroxylated and uncharged residues whose
removal increases the rate of thiol ester formation and a distal tract rich in
basic residues. Deletion or substitution of these basic residues with neutral
residues diminishes the rate of thiol ester formation. We have demonstrated also
that C-terminal extensions can function to confer substrate specificity to E2s.
When the acidic extension was deleted from UBC4, the protein was unable to
efficiently conjugate ubiquitin to histones in vitro. Furthermore, fusion of the
UBC4 acidic extension to the C terminus of UBC1 resulted in a chimeric protein
capable of efficient histone conjugation, as did fusion of short tracts of
alternating aspartate and glutamate residues. This result suggests that the
target protein specificity of E2s can be altered by the addition of appropriate
C-terminal extensions, thus providing a way to modify the selectivity of the
ubiquitin system.