Identification of a cytoplasmic motif of Surf4 that is critical for the trafficking of progranulin and prosaposin. (A) A stretch of 15 amino acids within the cytoplasmic C-terminus of Surf4 proteins is conserved across species. The region shown in brackets was deleted to generate the GFP-Surf4Δmotif mutant. Amino acids with high consensus (MultAlin) shown in red, the low consensus in blue, and neutral in black. (B and D) Immunofluorescence images of progranulin (B) and prosaposin and LAMP1 (D) in Surf4 KO cells expressing GFP-Surf4 WT, GFP-Surf4Δmotif, and GFP-Surf4 FF→AA (mutation of the diphenylalanine within the conserved motif). Scale bar, 10 µm; inset, 3.38 µm wide. (C) Manders colocalization coefficients of progranulin with LAMP1. Data were collected from 20 cells. Error bars show mean ± SEM. Kruskal–Wallis test with Dunn’s multiple comparisons test; ****, P < 0.0001. (E) Manders colocalization coefficients of prosaposin with LAMP1. Data were collected from three independent experiments with 20 cells. Error bars show mean ± SEM. Kruskal–Wallis test with Dunn’s multiple comparisons test; ****, P < 0.0001.