Figure 5. A coiled coil motif in Lem8 is important for its interactions with 14-3-3ζ.

(A) Lem8 harbors a putative coil motif. A predicted coiled coil motif located in the amino end of Lem8 (top panel). The sequence ranges from the 45th residue to the 73rd residue with a coiled coil probability of 100% according to MARCOIL (lower panel, left). Replacement of Leu₅₈ and Glu₅₉ with glycine (highlighted in red) is predicted to reduce the coiled coil probability to about 10% (lower panel, right). (B) The predicted coiled coil motif is critical for Lem8-mediated yeast toxicity. Yeast cells inducibly expressing Lem8 or mutant Lem8_GG were serially diluted and spotted onto the indicated media for 48 hr (top panel). The expression of Lem8 and Lem8_GG was examined and PGK1 was probed as a loading control (lower panel). (C) Lem8_GG loses the capacity to cleave Phldb2 in mammalian cells. Lysates of HEK293T cells expressing Lem8 or Lem8_GG were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and detected by immunoblotting with antibodies specific for Phldb2 and HA, respectively. Tubulin was used as a loading control. Results shown were one representative from three independent experiments with similar results. (D) The predicted coiled coil motif is required for self-processing of Lem8. The indicated alleles of Lem8-GFP were individually expressed in HEK293T cells by transfection. Samples resolved by SDS–PAGE were detected by immunoblotting with GFP-specific antibodies. Results shown were one representative from three independent experiments with similar results. (E) Interactions between 14-3-3ζ and the Lem8_GG mutant. Lysates of 293T cells expressing Flag-14-3-3ζ with HA-Lem8 or HA-Lem8_GG were subjected to immunoprecipitation with the anti-HA antibody and the presence of 14-3-3ζ in the precipitates was probed with the Flag-specific antibody. Results shown were one representative from three independent experiments with similar results.