K-Rta physically associates with SUMO.

<p>(<b>A</b>) (a) Recombinant GST and GST-SUMO were purified from an <i>E. coli</i> expression system, and GST-SUMO chains were generated <i>in vitro</i>. Flag-K-Rta protein was purified from recombinant baculovirus infected Sf9 cells (right panel). (b)The GST-Pull down assay was performed by incubating GST, GST-SUMO-1, -SUMO-2, -SUMO-3, -SUMO-2 chain or –SUMO-3 chain with purified Flag-K-Rta. Association was detected by immunoblotting with anti-Flag antibody. (<b>B</b>) Mapping of SUMO-binding domain. The indicated K-Rta deletion protein was incubated with SUMO-2 chains, and the interaction was probed with an anti-SUMO monoclonal antibody (upper panel). The membrane was also stained with Ponceau to show amount of GST-K-Rta deletion protein on the membrane (bottom panel). Asterisks showed non-specific interaction between C-terminal K-Rta and IgG. (<b>C</b>) Generation of K-RtaΔSIM. (a) Multiple alignments between KSHV Rta and RRV Rta. Conserved hydrophobic clusters (putative SIM) are marked in bold letters. Mutations that showed decreased SUMO degradation are underlined. (b) Mutations introduced in this study are shown. (c) SUMO degradation by K-Rta mutants. Immunoblotting was performed after the transfection of indicated K-Rta mutant plasmids along with SUMO expression vector in 293T cells. Mutations at SIM-3, -4, or -5 impaired K-Rta SUMO degradation function. (d) K-Rta Wt but not K-RtaΔSIM degrades SUMO modified proteins. Increasing amounts of K-Rta Wt or K-RtaΔSIM3&5 (0.25, 0.5, or 1.0 ug) were cotransfected with SUMO. The SIM mutation completely abolished the SUMO degradation function of K-Rta. (<b>D</b>) (a) GST pull-down analyses with purified K-Rta Wt or K-Rta ΔSIM mutants are shown. The interaction was probed with anti-Flag antibody. (b) Ponceau staining shows the amount and purity of GST- or GST-SUMO proteins used in the assay.