Phosphoinositides differentially regulate protrudin localization through the FYVE domain

Abstract

Protrudin is a FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia. Our image-based screening of the lipid binding domain library revealed novel plasma membrane localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance analysis showed that this FYVE domain preferentially binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) unlike canonical FYVE domains that specifically bind phosphatidylinositol 3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and plasma membrane via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of the protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth.

FIGURE 1.

Identification of PM-localized LBDs. EYFP-tagged (A) 94 different LBDs were imaged and grouped into five localization patterns (B). C, six distinct LBDs showed PM localization. The outline of the cell is marked as an orange line. Bars, 10 μm. D, quantitative analysis of the proportion of PM-localized LBDs in C. E, summary of image-based screening results.

FIGURE 2.

Determination of K_d for binding of the protrudin-FYVE domain to POPC/POPS/PtdIns(3,4,5)P₃ (77:20:3) vesicles. A, the protrudin-FYVE domain was injected at 5 ml/min at varying concentrations (10, 30, 50, 100, 200, 300, 500, 700, 800, 900, and 1000 nm from bottom to top) over the POPC/POPS/PtdIns(3,4,5)P₃ (77:20:3) surface and R_eq values were measured. B, a binding isotherm was generated from the R_eq (average of triplicate measurements) versus the concentration of FYVE plot. A solid line represents a theoretical curve constructed from R_max (=875 ± 35) and K_d (=190 ± 30 nm) values determined by nonlinear least-squares analysis of the isotherm using the equation: R_eq = R_max/(1 + K_d/P_o), where R_max indicates the maximal R_eq value. All measurements were performed at 23 °C in 20 mm Tris-HCl buffer, pH 7.4, with 0.16 m KCl.

FIGURE 3.

Amino acids responsible for lipid-binding specificity of the FYVE domain. A, amino acid sequence of the protrudin-FYVE domain was compared with that of the EEA1-FYVE domain. Lipid-binding pocket (purple) of the EEA1-FYVE domain is composed of a WXXD motif, R + HHC + XCG, and RVC regions. Corresponding sites in the protrudin-FYVE domain are colored pink. The CXXC motif or the predicted amino acids in the protrudin-FYVE domain were distinctly marked as green and red. B, a model of PtdIns(4,5)P₂ binding to the protrudin-FYVE domain. The PtdIns(4,5)P₂ molecule is shown in stick, and the FYVE domain in an electrostatic surface model. The positively charged residues (blue) presumed to interact with PtdIns(4,5)P₂ are labeled. The structure is shown using PyMOL software (DeLano Scientific LLC). C, images of cells expressing the wild type FYVE domain, K367A/R369A, R386A, and 4A. Bars, 10 μm. D, quantitative analysis of the expression level of the FYVE domain on the PM (WT, n = 10; K367A, n = 28; K367A/R369A, n = 11; R381A, n = 20; R386A, n = 19; 2A, n = 12; 4A, n = 10). Error bars, mean ± S.E. E, images of cells expressing the full-length of protrudin (WT), protrudin containing K367A/R369A and 4A (K367A/R369A/R381A/K386A) mutations, and the FYVE domain-deleted protrudin (ΔFYVE), respectively. Bars, 10 μm.

FIGURE 4.

The role of PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ in protrudin localization through the FYVE domain. A, NIH3T3 cell expressing the protrudin-FYVE domain was treated with 5 nm PDGF. Bars, 10 μm. B, quantitative analysis for translocation of the Btk-PH domain (red, n = 5), Akt-PH domain (blue, n = 7), and protrudin-FYVE domain (orange, n = 12) to the PM under PDGF treatment. Fluorescence fold-change in cytosol (F^*_cytosol/F_cytosol) indicates the degree of translocation of each domain from PM to cytosol upon PDGF treatment: i.e. a lower value means more PM translocation. Error bars, mean ± S.E. PDGF-triggered translocation of protrudin (WT) (C) and ΔFYVE (D). E, quantitative analysis of the level of protrudin (WT, orange, n = 6) and ΔFYVE (black, n = 4) at the PM after PDGF treatment. The fold-change of fluorescence intensity of a protein at the PM caused by each treatment was quantified as (F^*_PM/F^*_cytosol)/(F_PM/F_cytosol) in which F_PM/F_cytosol and F^*_PM/F^*_cytosol, indicate the fluorescence ratio before and after each treatment, respectively. Error bars represent S.E.

FIGURE 5.

The roles of PtdIns(4,5)P₂ in protrudin localization through the FYVE domain. A, Akt-PH domain interacting PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ stimulated by PDGF was completely removed from the PM under LY29. Bars, 10 μm. B, quantitative analysis of the level of controls, PLCδ-PH domain and Akt-PH domain, and protrudin-FYVE domain at the PM after LY29 or LY30 treatments. The fold-change of fluorescence intensity of each domain at the PM caused by chemical treatment was quantified as (F^*_PM/F^*_cytosol)/(F_PM/F_cytosol), in which F_PM/F_cytosol and F^*_PM/F^*_cytosol indicate the fluorescence ratio before and after each treatment, respectively. Error bars represent S.E. Cells expressing protrudin (WT) were sequentially treated with PDGF and LY29 (C) or vice versa (D). Yellow arrowhead indicates the PM. Bars, 10 μm. ARF6(Q67L)-EYFP (green) (E) or EYFP-Rab5B (Q79L) (green) (F) were cotransfected with the 2× FYVE domain (PtdIns(3)P biosensor, red), PLCδ-PH domain (PtdIns(4,5)P₂ biosensor, red), and the protrudin-FYVE domain (red), respectively. ARF6(Q67L)-EYFP (green) (G) and ARF6(T27N)-EYFP (green) (H) were cotransfected with protrudin (WT, red).

FIGURE 6.

The effect of the FYVE domain in neurite outgrowth promoted by protrudin. A, images of primary cultured hippocampal neurons expressing Lyn-mCherry together with control (citrine only), citrine-protrudin (WT), or citrine-protrudin (without FYVE). Lyn-mCherry, a PM marker, was used to clearly show neurites. Bars, 10 μm. B, total and maximum neurite length was measured by NeuronJ (control, n = 16, WT, n = 13; K367A/R369A, n = 16; 4A, n = 28; without FYVE, n = 13). Error bars indicate S.E. C, a hypothetical model of the PM to endosome translocation regulated by different PtdInsPs.