Figure 6.

One APC11 protein molecule binds three Zn²⁺ ions. (A) Sequence alignment of APC11 and Rbx1 from various organisms (Hs, Homo sapiens; Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae; and Sp, S. pombe). The residues that coordinate the two Zn²⁺ ions of the canonical RING finger motif are labeled as open and closed circles, respectively. The residues for coordinating the third Zn²⁺ ion are labeled as open triangles. (B) Autoubiquitination assay of the APC11 mutants. ³⁵S-labeled APC11 mutants were translated in vitro in reticulocyte lysate and incubated with a mixture of ubiquitin, ATP, and E1 in the presence and absence of Ubc4. The reaction mixture was separated on SDS-PAGE followed by autoradiography. The Zn²⁺-coordinating residues are labeled as in A. The mutations that affect Zn²⁺-binding are indicated by +, whereas the mutations that do not affect zinc binding are marked by −.