Fig. 1. Shot is physically associated with the translation initiation factor Kra/eIF5C. (A) Domain structure of the evolutionarily conserved Shot isoforms, Shot L(A) and Shot L(B). ABD, actin-binding domain; CH, calponin homology motif; EF, EF-hand motif; GAS2, GAS2 homology motif. (B) Sequence comparison of Kra with human Kra/BZAP45 (GenBank accession number BAA02795). Sequence identities (51%) are indicated by white letters on a black background, and sequence similarities (+1 or more in a PAM 250 matrix) are indicated by black letters on a gray background. N-terminal leucine zipper and C-terminal W2 domains are indicated above the sequences. Multiple alanine substitutions 12A and 7A, which were introduced into the AA-boxes 1 and 2, respectively, are also shown within the W2 domain. The boundaries of the kra cDNAs isolated from the yeast two-hybrid screen are marked by bent arrows. (C) Shot physically interacts with Kra. Soluble extracts of S2 cells transiently expressing HA-tagged Kra with Shot L(A)-GFP or C-Shot L-GFP were immunoprecipitated with IgG or anti-GFP antibody. The precipitates were subjected to SDS-PAGE and western blot analysis using anti-HA. (D) A region covering the EF-hand motifs of Shot is largely responsible for its interaction with Kra. In pull-down assays, GST-Kra was incubated with [³⁵S]methionine-labeled C-terminal fragments of Shot containing AA residues 4688-5201 (C-Shot L), 4688-4915 (EF-GAS2), 4688-4858 (EF), 4859-4984 (GAS2) and 4916-5201 (EF-GAS2, an arrowhead). The relative input of the labeled proteins (20% of the amount used in each reaction) is shown in the bottom panel. (E) The AA-box 2 in Kra is essential for its interaction with Shot. Soluble extracts from S2 cells expressing C-Shot L-GFP with HA-Kra^WT, HA-Kra^12A or HA-Kra^7A were immunoprecipitated with IgG or anti-GFP. The presence of HA-Kra in the precipitates was detected by western blot analysis using anti-HA.