FIGURE 6.

The N-terminal region of HBZ directly interacts with the 26 S proteasome. A, identification of Rpn5 as a target for the N-terminal region of HBZ using a yeast two-hybrid screen. The yeast strain Y190 was transformed as indicated. Transformants were grown in -Leu/-Trp/-His medium and selected for histidine prototrophy. B, HBZ associates with Rpn5 in vitro. GST, GST-HBZ-N, or GST-HBZ-N-3.8.10 fusion proteins were incubated with ³⁵S-labeled Rpn5. Bound proteins were detected by autoradiography. The abundance of GST proteins is shown by CBB staining. C, HBZ associates with Rpn5 in cells. HEK-293T cells were transfected as indicated. Cell lysates were immunoprecipitated with an anti-FLAG antibody and then immunoblotted with an anti-HA antibody. D, HBZ associates with the 26 S proteasome. HEK-293T cells were transfected with pcDNA3-FLAG-HBZ or pcDNA3-FLAG-HBZ-3.8.10 and then treated with MG132 for 12 h. Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with the antibodies indicated. E, cells transiently expressing HA-tagged HBZ were fractionated by 10-40% glycerol density gradient centrifugation. Each fraction was analyzed by immunoblotting with anti-HA, anti-Rpn10, or anti-α3 antibodies. The peaks corresponding to the 26 S and 20 S proteasomes are indicated.