Figure 3. CIDEA targets the LD monolayer through a cationic amphipathic helix.
(A) Secondary structure of CIDEA predicted by SWISS-MODEL server. (B) Helical wheel representation of the putative amphipathic α-helix (163–180) generated at http://heliquest.ipmc.cnrs.fr/. (C) Circular dichroism (CD) spectra of a 28-aa peptide corresponding to the 158–185 sequence of CIDEA (41 μM) solubilized in 50 mM potassium phosphate, pH 6.2 plus 0.1% n-dodecyl-β-D-maltopyranoside. (D) A Hela cell expressing HA-CIDEA-(1–120)-v5 (red) or HA-CIDEA-(1–117)-(163–180) (red), showing the inclusion of aas 163–180 enhances LD localization and the ability to promote LD docking. The phenotypic distribution was performed in a minimum of three independent experiments for every construct (n>50 cells). HA signal in LDs was only detected in a proportion of the cells where HA-CIDEA constructs had induced LD enlargement or clustering, possibly due to the formation of CIDEA complexes reducing antibody accessibility to the HA epitope at the N-term. (E) A Hela cell expressing CIDEA-(F166R/V169R/L170R)-v5 (red) showing aa substitutions to compromise amphipathicity of the helix disrupt LD targeting, and a Hela cell expressing CIDEA-(K167E/R171E/R175/E)-v5 (red) showing amino acid (aa) substitutions to invert the charge of the helix but maintaining amphipathicity retains predominantly LD localization.
Figure 3—figure supplement 1. Conservation of amino acids (aas) for CIDEA amphipathic helix across vertebrate species.
