Figure 3: YME1L degrades substrates in a degron-dependent manner.
From: Engineered AAA+ proteases reveal principles of proteolysis at the mitochondrial inner membrane
(a) SDS–PAGE showing degradation of I27 variants (20 μM) by hexYME1L (1 μM). Rapid degradation is observed only for variants bearing the β20 degron. (b) Loss of I27 variants over time from degradation reactions in a. (c) Initial degradation rates of I27-β20 and CMI27-β20 by hexYME1L calculated from linear fits to early time points (0–20 min) from b. (d) SDS–PAGE showing the processive degradation of mDHFR-I27-β20 (10 μM) by hexYME1L (2 μM) in the presence and absence of methotrexate (100 μM). Degradation in the presence of methotrexate results in the accumulation of a protected intermediate corresponding to the molecular weight of the mDHFR protein. (e) Accumulation of the intermediate in the presence and absence of methotrexate from experiments in d. (f) Relative stimulation of the ATPase rate of hexYME1L (0.25 μM) by the addition of I27 variants (20 μM). Significant stimulation is seen in the presence of folded I27-β20 (26%) and unfolded CMI27-β20 (48%) but not in the presence of folded I27 (6%) or unfolded CMI27 (14%). *P≤0.05, **P≤0.01 calculated from an unpaired Student’s t-test (two-tailed). All data shown are from independent experiments and error bars indicate±s.e.m. (n=3).
