The C terminus of Blm10 stimulates the CP peptidase activity and thus mediates gate opening. A, left panel, sequence alignments of the C-terminal segments of S. cerevisiae (yeast), human, and archaeal (M. jannaschii, PAN) proteasomal ATPases. HbYX motifs are indicated in bold letters. Right panel, sequence alignments of the C-terminal segments of Blm10 orthologs. S.c., Saccharomyces cerevisiae; C.a., Candida albicans; C.e., Caenorhabditis elegans; H.s., Homo sapiens; A.t., Arapidopsis thaliana. B, heptapeptides derived from the wild-type Blm10 C terminus (ct-Blm10), as well as a peptide where the penultimate tyrosine has been replaced by alanine (ct-Blm10 Y-A) were tested for their ability to stimulate CP peptidase activity against three fluorogenic peptides assaying the chymotryptic (Suc-GGL-AMC), the tryptic (Suc-LRR-AMC), and the caspase-like activity (Ac-nLPnLD-AMC) of the proteasome. The assays were performed with 0.2 μg of purified wild-type CP and an open gate mutant, where the N termini of α3 and α7 were deleted. The open gate mutant was purified from either WT cells or cells deleted for BLM10. The data are normalized for the activity of wild-type CP with the respective substrate. C, to compare ct-Blm10-mediated stimulation with ct-PAN and ct-PA26, the same assay as in A was performed with Suc-LRR-AMC, the proteasome substrate that showed the highest stimulation for the C-terminal peptide of Blm10. DMSO, dimethyl sulfoxide.

Impact of Blm10 on the different proteasomal peptidase activities. Substrate kinetics were determined using purified CP and Blm10-CP with varying concentrations of Suc-LLVY-AMC to determine the chymotrypsin-like activity (A), Ac-RLR-AMC for trypsin-like activity (B), and Ac-nLPnLD-AMC for caspase-like activity (C). The kinetic parameters of each reaction are shown in the tables to the right.

Proteasome peptidase activity in singly and doubly capped Blm10-CP. A, equal molar quantities of CP or Blm10 CP were separated by native gel electrophoresis. Peptidase activity was visualized by an in-gel activity assay (top panel), and protein concentration was assessed with Sypro Ruby (second panel). The picture of the Sypro Ruby stain is presented in a black and white inverted version. Parallel gels were subjected to immunoblotting using anti-Blm10 (third panel) or anti-CP antibodies. B, signals obtained from A were quantified using ImageJ. The means of three independent experiments are presented. The different values obtained for free CP present in lanes 1 and 2 represent differences in the latency of the CP.

The penultimate tyrosine within the Blm10 YYX motif is required for proteasome binding. A, WT, BLM10 deleted cells (blm10Δ), and genomically integrated point mutants within the last five residues of Blm10 as indicated by bold letters to the right were spotted on YPD plates in the absence (left panel) or in the presence of 0.3 μg cycloheximide (right panel) and were grown for 3–5 days at 30 °C. The C-terminal sequences are indicated to the right. B, the same strains as in A were tested for proteasome association in unfractionated lysates. The cell lysates were separated by native gel electrophoresis. Active proteasomes are visualized by an in-gel activity assay using Suc-LLVY-AMC.

Phenotypic characterization of cells overexpressing Blm10 or a gating incompetent Blm10PA26 chimera. A, the strains indicated were grown in YPD and spotted on complete media with glucose (YPD) as control, galactose (YPGal) to induce Blm10 overexpression, and glycerol as carbon source for analysis of nonfermentative growth. The plates were incubated either at 30 °C (upper panel) or at 37 °C (lower panel). B, the strains indicated were subjected to the same analysis as described for Fig. 2B to visualize the different proteasome holocomplexes.

Accelerated in vitro degradation of tau-441 by Blm10 proteasomes. A, equal molar amounts of Blm10-CP or CP were separated by SDS-PAGE and stained with Sypro Ruby to demonstrate that equal amounts of CP were present in the assay. B, proteasomal complex composition was assessed by native gel electrophoresis followed by an in-gel activity assay. C, tau-441 was incubated with equal molar amounts of CP or Blm10-CP in the absence or presence of MG132. Aliquots were taken at the times indicated, separated by SDS-PAGE. Tau-441 was detected by immunoblotting with a tau-specific antibody. D, the mean values of the densitometric analysis of three independent degradation assays are presented.