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First published online 20 March 2007
doi: 10.1242/jcs.006924

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The AAA-ATPase p97-Ufd1-Npl4 is required for ERAD but not for spindle disassembly in Xenopus egg extracts

Department of Molecular Cell Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

View larger version (33K): [in this window] [in a new window]   Fig. 1. p97 is not essential for spindle disassembly. (A,B) CSF-extracts were immunodepleted with the antibodies anti-Ufd1, anti-Npl4, anti-Ufd1 and anti-Npl4, anti-p47 or unspecific IgG and immunoblotted after the first and second depletion. Equivalent amounts and one-tenth of untreated extracts served as controls. Asterisks denote unspecific bands. (C) CSF-extracts were supplemented with Xenopus, murine or human p97 isoforms (wild type or dominant-negative mutants at various final concentrations) or stable human cyclinB1. Alternatively, extracts were depleted () with the antibodies anti-Ufd1, anti-Npl4, anti-Ufd1 and anti-Npl4, or anti-p47 and, where indicated, supplemented with soluble anti-Ufd1 or anti-Npl4 antibodies. Sperm-induced spindles were assembled for 30 minutes at 20°C. After triggering release into interphase, Rhodamine-labelled MTs and DAPI-stained sperm chromatin were examined. The two micrographs are typical examples of spindles immediately before (– Ca2+) and 60 minutes after release (+ Ca2+). Bars, 10 µm. (D) Translation of CFTRF508 mRNA in rabbit reticulocyte lysate or Xenopus low-speed extracts followed by CFTR western blotting. Arrowheads indicate CFTR-specific bands, all unlabeled bands are unspecific. (E) p97-QQ-supplemented versus buffer-supplemented extracts, incubated with CFTRF508 mRNA for 3 hours before quantifying CFTR relative to -tubulin by western blotting. Shown is the mean (± s.d.) of three independent experiments. (F) Egg extract was incubated with human CFTRF508 mRNA for 90 minutes at 20°C prior to addition of Xenopus p97-QQ (final 1 mg/ml) or reference buffer. Fifteen minutes later, translation was stopped with cycloheximide and degradation of CFTRF508 was analyzed by western blotting. Lanes 7 and 14 show steady-state levels for one out of three experiments quantified in E. (G) ATPase assays of recombinant p97 proteins. The OD615 values at 0 minutes were substracted from OD615 values at 15 and 30 minutes.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Spindle disassembly is not delayed in the absence of functional p97. (A) CSF-extracts pre-incubated with murine wild-type p97, the E305Q-E578Q mutant p97-QQ (both at 1 mg/ml final concentration) or reference buffer for 30 minutes at 4°C were supplemented with Rhodamine-tubulin and 500 sperm/µl. Following release (t=0 minutes) kinetics of spindle disassembly were monitored. (B) Ufd1-Npl4-depleted CSF extract from 1A was supplemented with soluble anti-Npl4 antibody. Kinetics of spindle disassembly relative to mock-depleted extract plus unspecific IgG or to untreated extract plus reference buffer were monitored as in A.

View larger version (48K): [in this window] [in a new window]   Fig. 3. Normal disassembly of centrosome-induced asters in the absence of functional p97. (A) CSF-extract was pre-treated with murine wild-type p97, the E305Q-E578Q mutant p97-QQ (both at 1 mg/ml final concentration) or reference buffer. Thirty minutes after addition of human centrosomes, meiotic exit was triggered and Rhodamine-labelled microtubules were visualized. Micrographs show representative centrosomes 0 and 60 minutes after release into interphase. Relative to the top row of images, the bottom row of images are overexposed. Bars, 10 µm. (B) CSF-extracts were depleted of Ufd1 and Npl4 or mock-depleted, and combined with centrosomes to induce aster formation (released and analyzed as in A). (C) Analysis of depleted extracts from B, carried out as described in Fig. 1A.