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Fig. 2. FIGL-1 and MEI-1 belong to subgroup seven of the AAA-ATPases. (A) Members of subgroup seven of the AAA-ATPases are depicted in a phylogenetic tree. The closer two proteins cluster, the more recently they diverged during evolution. Whereas human and mouse contain fidgetin and fidgetin-like 1, the C. elegans genome only contains one gene F32D1.1, which was named figl-1 and encodes Fidgetin-like 1 protein (Yakushiji et al., 2006). Species abbreviations are as follows: Hs, Homo sapiens; Sc, S. cerevisiae; Ce, C. elegans. (B) Alignment of MEI-1 and FIGL-1. Identical residues are shown in black and conserved substitutions on a gray background. The catalytic domain of AAA-ATPases (predicted by SMART) is underlined. Note that MEI-1 and FIGL-1 show some sequence homology outside the ATPase domain. (C) The indicated concentration (µM) of purified tubulin was polymerized in vitro, incubated with GST–FIGL-1 or yeast coronin (Crn1) as a positive control, and separated by high-speed centrifugation over a glycerol cushion. The entire pellet (P) fraction was analyzed by SDS-PAGE, whereas only 10% of the top supernatant (S1) and the bottom supernatant (S2) were loaded. The co-sedimentation of FIGL-1 with polymerized tubulin was quantified at 0 µM, 2 µM and 4 µM tubulin (n=1 for 2 µM and 4 µM tubulin, n=2 for 0 µM), and shown as a percentage (%) of the total input recovered in the pellet (bound) fraction (lower panel). In the human Cul3 negative control, only 7% (for 2 µM, n=2) and 6% (for 4 µM, n=2) of in vitro-translated Cul3 was recovered under these conditions (data not shown).
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