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Figure 2

$(A) WCE from mutants encoding proteasome lid subunits were resolved by non-denaturing-PAGE and immunoblotted with anti-Rpn12. (B) WCE from rpn8–1 and rpn11–m1 were also assayed for proteasome activity by ‘in-gel peptidase activity’. Subunits composition of indicated species is summarized in Table 3. (C) WCE from rpn8–1 was fractionated through a 10%–40% glycerol gradient and each fraction evaluated for proteasome subunits (top panels) or proteolytic activity (bottom). (D) Proteasomes from WT and rpn11–m1 were exposed to 300 mM NaCl and re-purified. Composition was estimated by immunoblotting for proteasome subunits representing each of the 19S-RP sub-complexes. (E) Migration of proteasomes from WT and rpn11–m1 in native gel before and after exposure to 300 mM NaCl.$

Participation of Rpn8 and Rpn11 C-termini in proteasome stability

(A) WCE from mutants encoding proteasome lid subunits were resolved by non-denaturing-PAGE and immunoblotted with anti-Rpn12. (B) WCE from rpn8–1 and rpn11–m1 were also assayed for proteasome activity by ‘in-gel peptidase activity’. Subunits composition of indicated species is summarized in Table 3. (C) WCE from rpn8–1 was fractionated through a 10%–40% glycerol gradient and each fraction evaluated for proteasome subunits (top panels) or proteolytic activity (bottom). (D) Proteasomes from WT and rpn11–m1 were exposed to 300 mM NaCl and re-purified. Composition was estimated by immunoblotting for proteasome subunits representing each of the 19S-RP sub-complexes. (E) Migration of proteasomes from WT and rpn11–m1 in native gel before and after exposure to 300 mM NaCl.

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