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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-KraWT, HA-Kra12A or HA-Kra7A were
immunoprecipitated with IgG or anti-GFP. The presence of HA-Kra in the
precipitates was detected by western blot analysis using anti-HA.
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