Caldendrin and Importin-a Bind to Jacob in a Competitive Manner

(A) Mapping the Caldendrin–Jacob interaction using the yeast two-hybrid assay. Upper panel: deletion and point mutation constructs of Jacob tested for interaction with full-length Caldendrin. Lower panel: deletion constructs of Caldendrin tested for interaction with full-length Jacob. Yeast two-hybrid interactions were quantified based on the time course of induction of the reporter gene β-galactosidase. Triple plus signs (+++) indicate blue colonies after 1 h; double plus signs (++) indicate blue colonies after 3 h; a plus sign (+) indicates blue colonies after 6 h; a negative sign (–) indicates no signal after 6 h.

(B) Interaction of Jacob with GST-tagged Caldendrin (Cald-GST) in a pull-down assay at different Ca²⁺ concentrations indicated. A Triton X-100–extracted P2 fraction was incubated with glutathione sepharose loaded with a GST fusion protein containing the C-terminal half (aa 137–298) of Caldendrin or with GST alone. pull-down, pellet fraction; unbound, supernatant.

(C) Interaction of Jacob with GST-tagged Caldendrin in a competitive pull-down assay with recombinant CaM at 100 μM Ca²⁺. A Triton X-100–extracted P2 fraction was incubated with glutathione sepharose loaded with a GST-fusion protein containing the C-terminal half (aa 137–298) of Caldendrin or with GST alone in the presence or absence of 40-μg recombinant CaM. PD, pull-down fraction; UB, unbound material. After washing, bound proteins were detected by western blotting using the antibody anti-JB150.

(D) WT-Jacob-GFP does not bind to CaM in a pull-down assay. Jacob binding to CaM or Caldendrin-GST sepharose was tested in the presence of 100 μM Ca²⁺ or 2 mM EGTA. GST sepharose was used as the control. WT-Jacob-GFP was detected by western blotting with a GFP antibody.

(E) Co-immunoaffinity purification of Jacob and Caldendrin. Left panel: 15 mg of protein from the 100,000 × g supernatant of a rat brain membrane extract were loaded either to an anti-Jacob affinity column (anti-Jac) or to an anti-Caldendrin affinity column (anti-Cald). Jacob is co-eluted with Caldendrin and vice versa from the corresponding affinity columns. A number sign (#) indicates Jacob-immunoreactive bands and an asterisk (*) Caldendrin-immunoreactive bands. Right panel: no specific binding was seen to normal serum control beads. 1: Deoxycholic acid (DOC) extract from rat brain cortex; 2: nonbinding proteins (flow-through); 3: specifically bound proteins (eluate).

(F) Caldendrin coimmunoprecipitates with Jacob from a rat brain extract in a Ca²⁺-dependent manner. Rabbit anti-Jacob bound to protein A sepharose specifically precipitates Jacob independent from the conditions used (5 mM of EGTA or 100 μM Ca²⁺ in the precipitation buffer). Rabbit IgG is used as a negative control (upper panel). Caldendrin was only detected in the precipitate under Ca²⁺ conditions (lower panel).

(G) Molecular modelling of the Caldendrin–Jacob interaction. Caldendrin binds Ca²⁺-dependently to the first incomplete IQ motif, and blocks the NLS. As indicated, Jacob contains two incomplete IQ motifs. The matching residues are marked in bold. The helical structure has been modelled using the coordinates of Myosin I (pdb: 1wdc), showing that Jacob has a similar CaM site distribution as the template structure. In contrast to myosin I/CaM, Caldendrin binds to site 1 in a complex more closely related to the compact CaMKII/CaM complex. This is in agreement with Caldendrin's binding at high Ca²⁺ concentration, whereas IQ motif proteins bind independently of Ca²⁺ concentrations. Crucial residues for the interaction are indicated.

(H) Immunoprecipitation of Importin-α1 from a soluble rat brain protein fraction with a Protein-A sepharose-coupled Jacob antibody (JB-150). Importin-α1 was only found in the immunoprecipitate (IP) of the Protein-A sepharose coupled Jacob-antibody, whereas it remained in the prewashing supernatant (S1) in the IgG and Protein-A sepharose control.

(I) GST-Importin-α1 pull-down of myc/his-tagged WT and DNLS-Jacob extracted from transfected HEK-293 cells. Only WT Jacob, but not DNLS-Jacob, is found in the pull-down, indicating that the presence of the NLS is essential for the Importin-α1/Jacob interaction. PD, pull-down fraction; UB, unbound material.

(J) GST-Importin-α1 pull-down of myc/his-tagged WT Jacob in the presence of equimolar amounts of Caldendrin. Pull-down of Jacob is attenuated in the presence of 2 mM Ca²⁺, but not in the presence of 2 mM EGTA. PD, pull-down fraction; UB, unbound material.