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Figure 1.
Figure 1: Characterization of the Ulp1 enzyme. a, The
His[6]–ubiquitin–Smt3–HA substrate. Ub, ubiquitin. b,
Cleavage of His[6]–Ub–Smt3–HA by Ulp1 and Ubp1, analysed
by SDS–PAGE. Lane 1, no added enzyme; lane 10, mixture of
separately purified proteins. Preincubations with inhibitors
(NEM, PMSF and Ald, for Ub aldehyde) were done for 15 min at
room temperature. Ubp1 was used as a control for cleavage after
Ub in the substrate and to confirm the inhibitory activity of Ub
aldehyde. c, Cleavage of SUMO1–ranGAP1 synthesized in vitro.
Lane 1, ranGAP1-K526R mutant; lanes 2–6, human ranGAP1
incubated at 30 °C with purified GST–Ulp1 for indicated
times (min); lane 8, GST–Ulp1 pretreated with NEM. Relative
molecular mass standards are indicated (in thousands). d, In
vitro cleavage by Ulp1 of yeast Smt3–protein conjugates. A
longer exposure was needed to visualize free Smt3. Yeast
extracts (25  g)
were from ulp1-ts cells grown at 23 °C (lanes 1, 2) and at
37 °C (lanes 3–5); lane 3, GST added. Immunoblot used a
rabbit anti-Smt3 antibody raised against His[6]–Smt3. PGK,
phosphoglycerate kinase; Smt3-aty, Smt3 precursor; Smt3, mature
Smt3.
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