Table I.
Parameters for 20S binding and SFGFP-ssrA degradation
| Nonapeptide-cleavage stimulation |
SFGFP-ssrA degradation |
|||
|---|---|---|---|---|
| taCdc48ΔN Variant | Vmax (min−1) | Kapp (nM) | Vmax (min−1) | Kapp (μM) |
| Parent | 28 | 4.2 | 1.20 | 0.36 |
| ΔC20 | 13 | 45 | 0.84 | 1.8 |
| R575Q/ΔC20 | 12 | 60 | 0.79 | 1.8 |
| R576Q/ΔC20 | No stimulation | 0.80 | 1.2 | |
| G577A/ΔC20 | 13 | 27 | 0.19 | 2.2 |
| T579A/ΔC20 | 12 | 24 | 0.80 | 1.0 |
| S580A/ΔC20 | 13 | 43 | 0.86 | 1.6 |
| D581N/ΔC20 | No stimulation | No degradation | ||
| S582A/ΔC20 | 13 | 61 | 0.88 | 2.2 |
The interaction of the T. acidophilum 20S proteasome with T. acidophilum taCdc48ΔN and variants was assayed by titration experiments similar to those shown in Figs. 2D and 2E. For peptide cleavage, data were fit to a quadratic equation for tight binding. For protein degradation, data were fit to the Hill equation. Kapp for the peptide-cleavage experiments is the concentration of the taCdc48ΔN variant required for half-maximal stimulation. Kapp for SFGFP-ssrA proteolysis is the concentration of the ta20S proteasome required for half-maximal degradation. The ta20S proteasome alone cleaves the nonapeptide substrate at a rate of 9.5 min−1 but does not degrade SFGFP-ssrA. Values are averages (n ≥ 3) with SEM errors of 5–10%.