Fig. 6: Modeling of a patient-derived β5 mutation in yeast. | Nature Communications

Fig. 6: Modeling of a patient-derived β5 mutation in yeast.

From: Evidence supporting a catalytic pentad mechanism for the proteasome and other N-terminal nucleophile enzymes

Fig. 6: Modeling of a patient-derived β5 mutation in yeast.The alternative text for this image may have been generated using AI.

A Sequence alignment of human immunoproteasome β5i and yeast β5. The pentad serine residue is in cyan. B Phenotypic analysis of the indicated mutants, expressed from low-copy centromeric plasmids harboring the endogenous promoter/terminator elements in a strain where endogenous β5 is under the control of the pGAL1 promoter. In glucose-containing media, expression of the endogenous β5 locus is repressed and plasmid-derived β5 is the sole source of this protein. Plates were cultured at 30 °C for 2 (galactose), 1 (glucose), and 5 (canavanine 1.5 μg/mL) days. C Analysis of wild-type and mutant CP by native gel electrophoresis followed by immunoblotting with antibodies against α5, immature (propeptide-bearing) β5, and Pba1/2. D Proteasome activity assays using active site-specific fluorescent substrate probes. Background fluorescence has been subtracted and relative activity has been normalized to untreated controls. Individual points represent biologic duplicates. E Turnover of immature β5, as determined by cycloheximide chase analysis of whole extracts. Analysis was by SDS-PAGE followed by immunoblotting with the antibody that specifically recognizes immature β5. GAPDH, loading control. Asterisk, non-specific band. Similar results were obtained in 3 (B) and 2 independent experiments (C and E), respectively.

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