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 −.