Identification of a motif enriched in ferritins that do not have a classical secretion signal. (A) A taxonomy-based phylogenetic tree of ferritins containing or lacking the classical ER SP targeting sequence. The organisms marked red are SP positive, and the organisms marked black are SP negative. The pink area is a dominantly SP-positive cluster, and the green area is a dominantly SP-negative cluster. Mus musculus and Homo sapiens locations are marked in yellow and shown in the enlargement. (B) A 13-amino-acid motif detected by the MEME⁶⁰  and FIMO⁶⁰  tools. The motif includes residues 74 to 86 of the H subunit and residues 70 to 82 of the L subunit. (C) The motif is situated along an unstructured loop of the ferritin subunits and sits at the interface between 2 subunits (colored in cyan and gold), creating a continuous long zipper-like structure (ie, a motif dimer) with its neighbor motif. (D) R79, F81, and Q83 of the motif received high ODA scores for protein–protein interaction (high score is labeled in red and low score in blue). (E) Visualization of protein–protein interaction patch, calculated by ODA score. (F) Visualization of protein–protein interaction patches calculated by ODA score shown in the context of the whole ferritin multimer. Our motif, R22, and an additional unidentified group of residues are marked in red. (G) Motif distribution and location throughout the entire ferritin 24-mer. The motif forms a long dimer (colored in green) at the interface of 2 subunits. R79 is marked in red, and R22 is marked in orange. (H) Human ferritin H- and L-subunit–knockout HeLa cells were transfected with plasmids coding for either WT murine H-ferritin (FTH^WT) or FTH^R79A, FTH^F81A, or FTH^Q83A. Nontransfected HeLa cells (NT) were analyzed as a negative control. WT RAW264.7 (nontransfected) cells were analyzed as a positive control for both L- and H-ferritin subunits (lane 12 in “cell lysates” gel and lane 17 in “media” gel). 24 hours after transfection, cells were metabolically labeled with ³⁵S (2-hour pulse) and chased for 0, 4, and 18 hours. Cell lysates were collected after 4 and 18 hours (lanes 2-6 and 7-11 in cell lysates gel, respectively), whereas media samples were collected after 0, 4, and 18 hours (lanes 2-6, 7-11, and 12-16 in media gel, respectively). Ferritin was immunoprecipitated with an anti-murine H-ferritin antibody. All samples were precleared (PC) by incubation with protein A sepharose beads alone to clear samples from nonspecific binding to the beads. Two of these samples, 4 hours of WT cell lysates and 18 hours of WT medium, where most nonspecific binding was expected, were analyzed on the gels (lanes 1). All samples were separated by SDS-PAGE and visualized by phosphorimaging.