Figure 2

The expression and assembly of HsN3 in transfected COS1 cells. A. A cartoon that illustrates the multiple steps known to involve in the assembly of the 26S proteasome. See text for details. B. A cartoon that illustrate the importance of β subunit prosequence in proteasome assembly. See text for details. C. The different assembly properties of wild type HsN3 and mutant HsN3 lacking its prosequence in transfected COS1 cells. COS1 was transfected with N3-F, a construct that contains the full-length wild type HsN3 and a Flag-epitope at the C-terminus (lane 2), or with F-ΔN3 (lane 3), a construct that lacks its prosequence at the N-terminus, instead contains an N-terminal Flag-epitope, or with T7-ΔN3, a construct that similarly lacks the prosequence but contains an N-terminal T7-epitope (lane 4). Cells were metabolically labeled with ³⁵S-methionine for 4 hrs before cells were harvested and analyzed by immunoprecipitation, as indicated. The two major bands in lane 2 were detected by Western blot using anti-Flag and marked out as prosequence-containing HsN3 (Pro-N3-F) and mature HsN3 (mature N3-F). The endogenous proteasome components at the molecular weight range of 19S proteasome (marked by small dots) or 20S proteasome in lane 2 are marked with brackets. A distinct set of eight bands that commonly detected to be associated with F-ΔN3 or T7-ΔN3 is marked with short lines at the right side of the panel. The transfection efficiency was monitored by co-transfection with a GFP construct. Equal transfection efficiency (70–80%) among different samples in a single experiment was the pre-requisite for metabolic labeling and the subsequent immunoprecipitation assays. Equal total amount of proteins were used for immunoprecipitation.