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. 2004 Apr 8;23(9):1977–1986. doi: 10.1038/sj.emboj.7600205

Figure 4.

Figure 4

Ufc1, a novel E2-like enzyme. (A) Sequence alignment of hsUfc1 and its homologs. The sequence of Ufc1 is available from GenBanK™ under the accession number BC005187 (dm, NM_137230; ce, NM_066654; at, BT001180). The homology analysis was performed as described in Figure 1B. The putative active site Cys residue is boxed in black. (B) Immunoblotting analysis. Each Myc-tagged Ufm1 (Myc-Ufm1) and Myc-Ufm1ΔC3 was expressed alone (lanes 2 and 3, respectively), and coexpressed with Flag-Ufc1 (lanes 5 and 6, respectively) or Flag-Ufc1C116S (lanes 8 and 9, respectively). Each Flag-Ufc1 and Flag-Ufc1C116S was also expressed alone (lanes 4 and 7, respectively). The cell lysates were subjected to SDS–PAGE and analyzed by immunoblotting with anti-Flag antibody. The bands corresponding to Flag-Ufc1, Flag-Ufc1C116S, and Flag-Ufc1C116S intermediates are indicated on the right. (C) Immunoblotting analysis after immunoprecipitation. Each Myc-Ufm1 and Myc-Ufm1ΔC3 was expressed alone (lanes 2 and 3, respectively), and coexpressed with Flag-Ufc1C116S (lanes 5 and 6, respectively). Flag-Ufc1C116S was also expressed alone (lane 4). The cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS–PAGE and analyzed by immunoblots with anti-Flag and anti-Myc antibodies. The bands corresponding to Flag-Ufc1C116S, Flag-Ufc1C116S–endogenous Ufm1, and Flag-Ufc1C116S–Myc-Ufm1 intermediates are indicated. (D) In vitro thioester bond formation assay of Ufm1 by Ufc1. Purified recombinant GST-Ufm1ΔC2 (2 μg) (lanes 1–8) was incubated for 30 min at 25°C with the following: purified recombinant GST-Uba5 (0.2 μg) (lanes 2–9), GST-Ufc1 (2 μg) (lanes 3–6, 8, and 9), GST-Ufc1C116S (2 μg) (lane 7), and 5 mM ATP (lanes 1, 2, and 4–9). Lane 9 was conducted similar to lane 8, except that GST-Ufm1ΔC3 was used instead of GST-Ufm1ΔC2. Reactions were then incubated with SDS loading buffer lacking reducing agent (lanes 1–4 and 6–9) or containing 100 mM DTT (lane 5). The presence or absence of various components is indicated above the lanes. The bands corresponding to free GST-Ufm1ΔC2 (mature Ufm1), GST-Ufm1ΔC3, GST-Uba5, GST-Ufc1, GST-Ufc1C116S, and GST-Ufc1–GST-Ufm1ΔC2 thioester product are indicated on the right.