Fig. 2. Recombinant forms of Xp54 and identification of potential NES and phosphorylation sites. (A) A diagram of recombinant proteins expressed in this study. The black boxes represent the conserved motifs seen in all DEAD-box helicases. Locations of a potential NES (cross-hatched) and four potential CK2 phosphorylation sites (PKS, white boxes) are indicated. Various fragments of Xp54 (grey areas) were expressed either with a T7 epitope-tag or as GFP fusions containing a 6-myc tag. The white areas to the right in T7-
CT and T7-V/CT represent replacement of the C-terminus of Xp54 with an unrelated sequence encoded by the plasmid. (B) An immunoblot of T7-p54 and T7-V, overexpressed in stage VI oocytes, showing cytoplasmic (C) but no nuclear (N) reaction. (C) Comparison of the leucine-rich sequence of Xp54 with the NES of HIV Rev (Fornerod et al., 1997), PKI (Hauer et al., 1999) and An3 (Askjaer et al., 2000). Positions of critical leucines (black circles) and other similarities (open circles) are indicated. The numbers refer to the residue positions starting from the initiating methionine. (D) The four potential CK2 phosphorylation sites (S/TxxD/E, where x is any non-basic residue) are located near the C-terminus of Xp54.
