Fig. 7: Modeling of a patient-derived β2 mutation in yeast.

A Sequence alignment of human immunoproteasome β2i and yeast β2. The pentad aspartate residue is in cyan. B Position of the corresponding G191 residue in yeast CP. An amino acid side chain at this position would be predicted to disrupt the Ser/Asp pair of the catalytic pentad. C Phenotypic analysis of the indicated mutants, expressed from low-copy centromeric plasmids harboring the endogenous promoter/terminator elements in a strain where endogenous β2 is under the control of the pGAL1 promoter. In glucose-containing media, expression of the endogenous β2 locus is repressed and plasmid-derived β2 is the sole source of this protein. Plates were cultured at 30 °C for 2 (galactose and glucose) and 7 (canavanine, 2 μg/mL) days. D Analysis of wild-type and mutant CP by native gel electrophoresis followed by immunoblotting with α5 antibody. E 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. Similar results were obtained in 3 (C) and 2 independent experiments (D), respectively.