Figure 4.
Rfp2 is polyubiquitinated in cells in a RING-dependent manner. (A) Polyubiquitination of Rfp2. Left panel, cotransfection of GST-Rfp2 and HA-ubiquitin into HEK293 cells was followed by cell lysis under denaturing conditions to disrupt noncovalent interactions. Rfp2 was precipitated using glutathione beads, and Western blot analysis with HA antibodies was used to detect high-molecular-weight smears indicative of ubiquitinated Rfp2 conjugates. In cells treated with MG-132 (30 μM) ubiquitinated Rfp2 conjugates accumulate. Right panel, Western blot of whole cell lysate using 5% of the input used for glutathione precipitation. Rfp2 antibodies detect both unubiquitinated and polyubiquitinated forms of Rfp2. Nonubiquitinated Rfp2 is marked with a solid arrowhead, and asterisks indicate Rfp2 conjugated with single ubiquitin molecules. Numbers indicate protein size in kDa. (B) RING-dependent polyubiquitination of Rfp2. Top panel, HEK293 cells were transfected with indicated constructs and immunoprecipitated with Rfp2 antibodies. Where indicated MG-132 (30 μM) was added for 4 h before harvesting. Ubiquitinated proteins were visualized with HA antibodies. Polyubiqutinated smears (marked with brackets) are seen in samples transfected with full-length Rfp2 but not with the RING deletion mutant Rfp2-ΔRING. Solid arrowhead indicates immuno-globulin heavy chain from the immunoprecipitation. Bottom panel, Western blot of whole cell lysate using 10% of the input used for immunoprecipitation showing stabilization of the Rfp2-ΔRING mutant. Rfp2 and Rfp2-ΔRING are marked with solid arrowheads. Numbers indicate protein size in kDa. (C) Stabilization of the Rfp2[13] mutant. Left panel, Western blot analysis of HEK293 cells ectopically expressing wild-type Rfp2 or Rfp2[13] with Rfp2 antibodies showing stabilization of the mutant protein. Actin was used as a loading control. Right panel, steady state levels of transfected GST-tagged Rfp2 and Rfp2[13] in HEK293 cells at various indicated time points after cycloheximide treatment in the presence or absence of MG-132. Western blot with GST antibodies shows stabilization of the Rfp2 point mutant irrespective of proteasomal inhibition. Tubulin was used as a loading control.