FIG. 3.

Conjugation of Smt3 to Dorsal is reversible. (A) Alignment of yeast and human Ulp1 with Drosophila Ulp1. Only the C-terminal 181 amino acids are shown. The conserved catalytic triad residues (arrowheads) and oxyanion hole Gln residue (asterisk) are indicated. (B) Deconjugation by Ulp1. 529SU cells were untransfected (lane 1) or transiently transfected with 10 μg of Dorsal and 0 (lane 2), 0.5 (lane 3), 1 (lane 4), or 2 (lane 5) μg of a Ulp1 expression vector. Whole-cell lysates were analyzed as described for Fig. 1A. (C) Processing of Smt3-GFP fusion by Ulp1. The contents of each lane were incubated in the presence of 150 mM NaCl, 1 mM dithiothreitol, 10 mM Tris (pH 8.0), and 0.2% Triton X-100. The reactions were stopped at the times indicated above the lanes (in minutes). Lane 1, HA-Smt3; lane 2, HA-Smt3-GFP; lanes 3 to 8, HA-Smt3-GFP and crude GST-Ulp1 (or GST). In lane 8, the GST-Ulp1 was preincubated with 20 mM NEM at room temperature for 15 min prior to its addition to the reaction mixture. Lane 9 contained the same ingredients as lane 7 except that GST-Ulp1 was replaced with GST. The reactions were stopped by boiling in SDS loading buffer, subjected to SDS-12% PAGE, and analyzed by anti-HA immunoblotting. A similar blot was also probed with an anti-GFP antibody, demonstrating that the appearance of HA-Smt3 was accompanied by the parallel appearance of GFP (data not shown). (D) Biphasic response of Dorsal to Ulp1. The DE5 reporter was cotransfected without Dorsal and Twist expression vectors or with Dorsal and Twist expression vectors plus 0, 1, 2, 5, or 10 μg of a Ulp1 expression vector. Data were analyzed as described for Fig. 2A.