Figure 1.

Uba5, a novel E1-like enzyme. (A) Schematic representation of Uba1 and Uba5 in H. sapiens. Uba1 is divided into several domains, including I, Ib, II, III, and IV boxes, which are conserved in other E1-like enzymes, and other regions without obvious similarity, described previously (Komatsu et al, 2001). Note that Uba5 is of a relatively small size and includes the box I and two other parts. The box I region of Uba1 (amino acids 459–611) has 48.4% similarity and 22.3% identity to amino acids 72–229 of Uba5, which includes the conserved ATP-binding motif (GXGXXG). The sequence of Uba5 is available from GenBanK™ under the accession number AK026904. hs, H. sapiens; ce, C. elegans; dm, D. melanogaster; at, A. thaliana. (B) Sequence alignment of hsUba5 and its homologs of other species (dm, NM_132494; ce, NM_058847; at, NM_100414). The amino-acid sequence of hsUba5 is compared by the ClustalW program. Asterisks, identical amino acids; single and double dots, weakly and strongly similar amino acids, respectively, determined by the criteria of ClustalW program. Open box indicates an ATP-binding motif. The putative active site Cys residue is boxed in black. The metal-binding motif is underlined. (C) Identification of the intermediate linked to Uba5 in HEK293 cells. Both Uba5 and Uba5^C250S, in which the predicted active site Cys positioned at 250 was changed to Ser by site-directed mutagenesis, were tagged with Flag peptide at N-terminus, resulting in Flag-Uba5 and Flag-Uba5^C250S, respectively. Each Flag-Uba5 and Flag-Uba5^C250S was expressed in HEK293 cells. The cell lysates were subjected to SDS–PAGE and analyzed by immunoblotting with anti-Flag antibody.