FIGURE 2.

Identification of the SUMO acceptor sites on arrestin-3. A, SUMOylation analysis of arrestin-3 by co-expressing SUMO-1 and Ubc9 in COS-1 cells. COS-1 cells grown in six-well plates were transfected either with empty vector (pcDNA) or HA-arrestin-3, plus either empty vector (pcDNA), FLAG-Ubc9 alone, His-SUMO-1 alone, or together with FLAG-Ubc9. Twenty-fours hours later, the cells were lysed directly in 2× sample buffer, and equal amounts were analyzed by IB. Unmodified and SUMOylated arrestins are indicated. Shown are representative IBs from one of three independent experiments. B, SUMOylation analysis of arrestin-3 lysine mutants. Lysine residues 295 and 400, both of which reside within SUMOylation consensus sites, were changed individually to arginine residues. Lysine residues 398 and 400 were changed simultaneously to create the 2K/R mutant, and lysine residues 293 and 295 were changed simultaneously to arginine residues in the 2K/R background to create the 4K/R mutant. The SUMOylation status of these mutants was assessed as described in A. Shown are representative IBs from one of three independent experiments.