Ubiquitin is conjugated to its target proteins by the following sequential reactions. Firstly, ubiquitin is processed by a specific protease to expose a glycine residue at its carboxy-terminus. The exposed glycine is activated by an E1 enzyme and is then transferred to an E2 enzyme. Finally, usually supported by an E3 enzyme, ubiquitin is conjugated to its target proteins (Varshavsky, 1997
). Many ubiquitin-like modifiers have been reported and they all seem to be conjugated to their targets via
a mechanism similar to ubiquitination (Welchman et al.
). Autophagy, the bulk degradation process of cytosolic components by the vacuolar/lysosomal system (Ohsumi, 2001
), has also been shown to have similar modification systems, such as ubiquitination in the yeast Saccharomyces cerevisiae
(Mizushima et al.
; Ichimura et al.
). A 21 kDa protein, Atg12, is one such modifier essential for autophagy (Mizushima et al.
). The calboxy-terminal glycine of Atg12 is activated by Atg7, an E1-like enzyme, through ATP hydrolysis (Tanida et al.
) and Atg12 is then transferred to Atg10, an E2-like enzyme (Shintani et al.
). Finally, an isopeptide linkage is formed between the carboxy-terminal glycine of Atg12 and the Lys149 side chain of Atg5. The Atg12 conjugation system is conserved in eukaryotes, including mammals, and has been shown to be essential for autophagy in both yeast and mammals.
In contrast to ubiquitin, which has many target proteins, the target for Atg12 is restricted to Atg5 alone. Furthermore, no E3-like enzyme has been reported for the Atg12 conjugation system. Therefore, Atg10 seems to recognize both Atg12 and Atg5 directly and to catalyze their conjugation reaction. Atg12 has a ubiquitin fold despite having low sequence homology with ubiquitin (Suzuki et al.
). Atg5 has also been shown to possess ubiquitin folds (Matsushita et al.
). Structural information on Atg10 would be helpful in order to understand how this enzyme recognizes two ubiquitin-fold proteins simultaneously and catalyzes their conjugation reaction. In this report, we describe the purification, crystallization and preliminary crystallographic analysis of S. cerevisiae