Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Rpn5-1p is expressed at the restrictive temperature.

  Wild-type (W303-1A) and rpn5-1 (YEK100) cells were grown 7 hours at either 25°C or 37°C to late-log phase and total proteins were extracted by the alkaline method (Kushnirov, 2000), and subjected to Western blotting using the antibodies indicated on the left. Cdc28p was detected by anti-Cdc2 (Santa Cruz) as a loading control. n5: rpn5-1, n2: ·N rpn2

• Supplemental Figure 2 - A complete lid is formed in ·N rpn2 cells grown at the restrictive temperature. Rpn11-3xFLAG tagged wild-type (YYS40) and ·N rpn2 (YAT3507) cells were cultured 6 hours at either 25°C or 37°C. Proteasomes were affinity-purified using anti-FLAG immobilized beads and resolved on a 12.5% polyacrylamide gel and stained with CBB. A wild-type lid was purified as described before (Isono et al. , 2004) (left panel). Bands were cut out and subjected to mass spectrometric analysis using 4800 MALDI TOF/TOF (Applied Bioshystems). Identified proteins are indicated on the left of the gel (right three panels). The smallest lid component, Sem1(Rpn15)p, could not be identified by LC/MS and its incorporation was verified by Western blotting using anti-Sem1p antibody (left, bottom panel).
• Supplemental Figure 3 - Rpn7p-3xFLAG is expressed in rpn5-1 mutant cells. Rpn7p-3xFLAG tagged wild-type and rpn5-1 cells (YEK221 and YEK225, respectively) were cultured as in Fig. 2D. Total proteins were extracted by the alkaline method (Kushnirov, 2000) and subjected to Western blotting using anti-FLAG M2 antibody (Sigma). CP was detected as a loading control.
• Supplemental Figure 4 - GFP-fused components are incorporated into the proteasome.

  A, production of GFP-fused components were verified by Western blotting.

  B, total lysates of GFP tagged wild-type, rpn5-1 and rpn7-3 cells cultured for 7 hours at 37°C were resolved by non-denaturing PAGE and analyzed by subsequent immuno-blotting. The results of Rpn1p-GFP tagged strains are shown. Positions of various proteasome species are indicated on the left of the panel.

• Supplemental Figure 5 - Proteasomes are delocalizeed in the srp1-49 mutant under restrictive condition.

  A, srp1-49 mutants are temperature-sensitive. The srp1-49 mutation was introduced into a W303 strain and growth was tested at 25°C and 37°C on YPDAU plates. The temperature-sensitivity was rescued by introducing a single copy SRP1 plasmid (pEK228).

  B, localization of Pre6p-GFP, Rpn1p-GFP and Rpn11p-GFP in srp1-49 (YEK206, YEK207 and YEK208, respectively) were observed. Log-phase cells cultured for 8 hours at either 25°C or 37°C were observed under a confocal microscope.

• Supplemental Material