Interaction of Ynl155w with the proteasome.
A, native gel electrophoresis of purified wild-type and spg5Δ proteasomes (3 μg), followed by in-gel activity assay with the fluorogenic proteasome substrate, suc-LLVY-AMC. B, proteasomes from panel A were excised from the gel and analyzed by mass spectrometry, yielding three peptides from Ynl155w. C, interaction of purified Ynl155w (2.3 μg) with purified 26 S proteasome (5 μg). Upper panel, Coomassie-stained SDS-PAGE gel. Lower panel, anti-Ynl155w SDS-PAGE immunoblot. D, interaction of purified Ynl155w (70 μg) with RP and base subcomplexes of the proteasome (3 nmol for each subcomplex). Upper panel, anti-Ynl155w immunoblot. Lower panel, Coomassie-stained gel. Input, 0.14%; eluates, 2.2% of total. E, interaction of purified Ynl155w (40 μg) with proteasome subunit Rpn2 (5.3 μg), as visualized by anti-Ynl155w immunoblot. Coomassie staining of eluates confirmed comparable amounts of GST, GST-Rpn1, and GST-Rpn2 (data not shown). Input, 1%; eluates, 2.2% of total.