Figure 3. Rpt6 tail binding to the CP is required for Rpt3 tail-mediated release of Nas6 from the proteasome holoenzyme.

(a) Increased retention of Nas6 in the proteasomes purified from rpt3-Δ1rpt6-Δ1 cells. Proteasomes were isolated from indicated strains via a Protein A tag that is appended to CP subunit Pre1⁴⁴. Purified proteasomes (1 μg) were subjected to 12% SDS-PAGE and immunoblotting (IB) for the indicated proteins: Nas6, Rpn14, chaperones; Rpn8, Rpn12, lid subunits; Rpt5, base subunit; α7, CP subunit. Pre1-TEV-ProA tag was chosen to exclude the purification of free RP. Note that Nas6 abundance remains unchanged in these cells (Fig. 1b). (b) Increased retention of Nas6 in the proteasomes in whole cell extracts from rpt3-Δ1rpt6-Δ1 cells. Whole cell lysates (40 μg) from indicated strains were resolved by 3.5% native gel (top panel). Following proteasome activity assay with LLVY-AMC, the native gel region containing the proteasome holoenzymes (RP₂-CP and RP₁-CP) was excised into a horizontal strip and directly subjected to 12% SDS-PAGE and immunoblotting for indicated proteins (bottom panel). Rpn8 (a lid subunit) and Rpt5 (a base subunit) serve as loading controls for proteasome levels in the native gel strip. Note that decreased proteasome activities in rpt3-Δ1rpt6-Δ1 cells are due to gate opening defects (Fig. 2a,b). (c) Nas6 level in the proteasomes from rpt3-Δ1rpt4-Δ1 cells is comparable to that in the proteasomes from rpt3-Δ1 cells. Experiments were carried out as in (b) to assess the level of Nas6 in the proteasome holoenzymes from indicated cells. Rpn8 (a lid subunit) and Rpt5 (a base subunit) serve as loading controls for proteasome levels in the native gel strip. (d) Phenotypic analysis of indicated yeast strains showing that the Rpt3 tail exhibits stronger genetic interaction with the Rpt6 tail than the Rpt4 tail. Four-fold serial dilutions of the indicated cells were spotted onto synthetic complete medium (SC) and grown for 2–3 days at 30 °C and 33 °C. For testing sensitivity to canavanine (an arginine analog), arginine was omitted from the SC medium. Canavanine was included at 1 μg/ml final concentration.