Fig. 8

The αC-helix of the Popeye domain mediates heteromeric complex formation between POPDC1 and POPDC2. a A model of heteromeric complex formation of the Popeye domains of POPDC1 (cyan) and POPDC2 (blue) using the structure of a CAP dimer (translucent; PDB: 1G6N) as a template. b Sequence alignment of the αC-helix of POPDC1, POPDC2 and POPDC3 from multiple vertebrate species and CAP protein from E. coli. A set of highly conserved hydrophobic amino acids are highlighted in red. c and d Overlay of the predicted αC-helical structures of POPDC1 (cyan), POPDC2 (blue) and POPDC3 (purple) with the side chains of the highly conserved hydrophobic residues. c Amino terminal and d side view. e and f The ratio of plasma membrane to cytoplasm expression levels of POPDC1-ECFP and POPDC2-EYFP in HEK293 cells, where either e POPDC1-ECFP or f POPDC2-EYFP was subjected to site-directed mutagenesis to introduce aspartic acid in place of a conserved hydrophobic residue within the αC-helix sequence. Total number of cells analyzed: POPDC1: L245D n = 71, F249D n = 56, I253D n = 27, I257D n = 40, L261D n = 47, L264D n = 42; POPDC2: I229D n = 48, F233D n = 20, L237D n = 45, I241D n = 42, L245D n = 63, L248D n = 56, N ≥ 2. Min. 2 transfections per group. Bars show median ± 95% CI. Groups were compared using Kruskal–Wallis followed by Dunn’s test using the wild-type pair as a comparison; **p < 0.01, ****p < 0.0001. g and h Relationship between plasma membrane versus cytoplasm expression and each mutation in g POPDC1 and h POPDC2. i The predicted αC-helical Popeye domain interface between POPDC1 and POPDC2. Hydrophobic residues whose mutation to aspartic acid led to severely impaired plasma membrane localization of both POPDC proteins are labelled in red