Skip to main content
. Author manuscript; available in PMC: 2010 May 29.
Published in final edited form as: Cell. 2009 May 14;137(5):887–899. doi: 10.1016/j.cell.2009.04.061

Fig. 3.

Fig. 3

The rpt4-G106D and nas2Δ mutants have proteasome assembly defects.

A. Immunoblot analysis of nondenaturing PAGE-separated proteins from yeast whole-cell extracts. The faint central band in the anti-Rpt5 blot is of unknown composition.

B. Nondenaturing gel separation of proteins from yeast extracts followed by gel overlay with the fluorogenic substrate Suc-LLVY-AMC to assay proteasomal activity. Left panel, effect of low-copy (YCp) and high-copy (YEp) expression of NAS2 on functional proteasome formation. Right panel, effect of nas2Δ on assembly. Addition of SDS to the reaction buffer activates the otherwise latent CP and Blm10-CP (unlabeled band above free CP).

C. A C-terminal Flag tag on Rpn5 does not disrupt the Rpn5-containing RP subcomplex in rpt4-G106D cells (arrowhead).

D. Nondenaturing gel separation of Flag-affinity purified proteins from rpt4-G106D RPN5-FLAG cells. MS/MS analysis of the indicated band from the GelCode Blue-stained gel demonstrated that it was the 9-subunit RP lid. Asterisk, a band that is likely the free RP but was not analyzed. Unlike panel C, gel separation included a stacking gel and was run longer.

E. A triplicated Flag tag on Rpt4 does not disrupt the Rpt4-containing RP subcomplex in rpt4-G106D cells (arrowhead).

F. Nondenaturing gel separation of Flag-affinity purified proteins from rpt4-G106D RPT4-3xFLAG cells. MS/MS analysis of the indicated band from the GelCode Blue-stained gel yielded Rpt4 and Rpt5 from the RP base. Asterisk, as in panel D. PAGE was done with a 5.5% PA separating gel instead of the 4% used in other panels, and a stacking gel was used.