The PX-BAR membrane-remodeling unit of sorting nexin 9

Abstract

Sorting nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting nexin 9 (SNX9) is involved in the late stages of clathrin-mediated endocytosis in non-neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane-remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro. Elucidation of the protein superdomain structure, together with mutational analysis and biochemical and cell biological experiments, demonstrated how the SNX9 PX and BAR domains work in concert in targeting and tubulation of phosphoinositide-containing membranes. The study provides insights into the SNX9-induced membrane modulation mechanism.

Figure 1

Membrane binding and tubulation by SNX9 PX-BAR. (A) Epifluorescence micrographs of HeLa cells transfected with myc-tagged wild-type (PXBAR) and mutated (Y287A and K522E/K528E) PX-BAR, stained with anti-myc antibodies. While wild-type protein created distinct elongated tubular structures, the PX and BAR domain mutants gave diffuse cytoplasmic staining. Scale bars are 10 μm. (B) Electron micrographs of liposomes and tubules. Liposomes were made from brain lipid extract and passed through 800-nm filters by extrusion. Liposomes were mixed with 10 μM purified PX-BAR (PXBAR+lip) or buffer (lip), and processed for electron microscopy. Scale bar is 100 nm. (C) Liposomes were made from brain lipid extract (brain lip), a 10:10:40:40 (w/w) mixture of phosphatidylserine, cholesterol, phosphatidylethanolamine, and phosphatidylcholine (PS), or with 5% of PtdIns-3-phosphate (PI3P) or PtdIns-4,5-bisphosphate (PI4,5P₂) in the PS mixture at the expense of phosphatidylserine. Liposomes were extruded through 800-nm filters and mixed with 0.7 μM protein as indicated, or protein was incubated without liposomes (no lip). Samples were centrifuged and supernatants (S) and pellets (P) were analyzed by SDS–PAGE and quantitated by densitometry. The bars show the means (±s.e.m.) from four experiments. (D) Electron micrographs of PI4,5P₂- and PI3P-containing liposomes generated as in panel C and incubated with 10 μM purified PX-BAR. Scale bar is 100 nm. (E) Epifluorescence micrographs of HeLa cells transfected with myc-tagged PX-BAR construct and treated with wortmannin or ionomycin as indicated. Cells were stained with anti-myc antibodies and costained with antibodies for EEA1. Scale bar is 10 μm.

Figure 2

(A) Side view of the SNX9 PX-BAR dimer structure ribbon diagram. BAR domains are shown in red, PX in blue, and the novel Yoke (Y) subdomain in yellow. N- and C-termini are labeled. (B) Top view of the SNX9 PX-BAR dimer structure. The domains are labeled. (C) Ribbon representation of SNX9 Yoke (Y) subdomain structure. The Yoke domain consists of two parts: Y_N derived from amino-acid residues 214–250 and Y_C from 375–390. Secondary structure elements are labeled. (D) Sequence alignment of human SNX9 (Q9Y5X1), SNX18 (AAH67860), and SNX30 (ABN09670) PX-BAR domains. The proteins represent a subfamily of PX-BAR SNXs. Secondary structure elements corresponding to the SNX9 structure are monitored. Coloring corresponds to that from panels A–C. Helices are labeled with H, β-strands with S, and P denotes a proline-rich motif. The amino-acid residues involved in the dimerization are labeled (+, hydrophobic bar–bar contacts; ^*, H-bond bar–bar contacts), as well as residues involved in interdomain interaction (§, hydrophobic bar-px:y contacts; &, H-bonds bar-px:y contacts). Symbols of consensus sequence are: 2, E/Q; 3, T/S; 4, K/R; 5, Y/F; and 6, hydrophobic.

Figure 3

(A) SNX9 BAR domain monomer in rainbow colors from the N-terminus in blue to C-terminus in red. α-helices are labeled. (B) SNX9 BAR domain. The core and arm regions are monitored, and the proline amino-acid residues at the α-helices kinks are labeled.

Figure 4

(A) Ribbon diagram of the SNX9 PX domain in rainbow colors, from PX N-terminus (251) in blue to PX C-terminus (372) in red. Secondary structure elements are labeled. (B) SO₄ ion bound to the SNX9 PX domain PIP-binding pocket. Amino-acid residues contacting the ion are shown and labeled. (C) PI(3)P molecule bound to SNX9 PX domain PIP-binding pocket. Amino-acid residues contacting the PI(3)P are shown and labeled. H-bonds are depicted as dashed lines. (D) PI(3)P molecule in the SNX9 PX domain PIP-binding pocket shown as a molecular surface. The pocket is large enough to accommodate a PIP molecule phosphorylated at the fourth and fifth positions. (E) The PI(3)P molecule fills the SNX3 PI(3)P-binding pocket (PDB ID 1OCU).

Figure 5

Membrane tubulation is dependent on an amphipatic helix upstream of the Y_N part of yoke sub-domain. (A) Epifluorescence micrographs of HeLa cells transfected with myc-tagged ¹⁸⁵DA, ²⁰¹MK, and ²⁰⁴PL protein constructs stained with anti-myc antibodies. Scale bar is 10 μm. (B) Sequence comparison of the potential amphipatic helix region (helix 0) in SNX9 from selected species as indicated. Numbers refer to amino-acid position in human SNX9. Arrows depict the N-terminus of protein constructs ¹⁸⁵DA, ²⁰¹MK, ²⁰⁴PL, and ²¹⁴PG. Amino acids M201-G215 are presented as a helical wheel with positively charged amino acids in blue and hydrophobic amino acids in yellow. Conserved amino acids are encircled. (C) Liposome binding assay where indicated proteins were incubated with liposomes generated from total brain lipids (lip), or incubated without liposomes (no lip). Where indicated (lip+NaCl), 0.5 M NaCl was added to the protein/liposome mixture. Samples were centrifuged and supernatants (S) and pellets (P) were analyzed by SDS–PAGE. The F208A/F211A protein was made by mutation of the ¹⁸⁵DA construct. (D) Epifluorescence micrograph of myc-tagged amphipatic helix mutant F208A/F211A expressed in HeLa cells (left panel, scale bar is 10 μm). Electron microscopic analysis of liposomes incubated with purified proteins as indicated (right panel, scale bar is 100 nm). Note the inability of the F208A/F211A mutant to tubulate membranes both in vivo and in vitro. (E) A 3D model of the N-terminal SNX9 helix 0. Structurally resolved amino acids are shown in yellow and the modeled part is in magenta.

Figure 6

(A, B) Electrostatic potential surface of SNX9 PX-BAR. (A) The concave face of the dimer is mostly positively charged. PI(3)P moieties are shown in yellow and red. (B) The convex face is negatively charged. (C) Subfamily conserved basic amino-acid residues (black) forming clusters on the concave face of SNX9 PX-BAR dimer. The residues from one monomer are labeled. (D) Side view of panel C. (E) Membrane binding of PX-BAR mutants. The indicated proteins (0.7 μM) were incubated together with liposomes from total brain lipids (+) or without liposomes (−), and samples were centrifuged and the supernatant (S) and pellet (P) fractions were analyzed by SDS–PAGE. (F) Indicated proteins were incubated with PI(3)P- or PI(4,5)P₂-containing liposomes made as in Figure 1C, and the samples were processed as in panel E. (G) The PX-BAR unit of SNX9 is curvature sensitive. Liposomes were made from a 30:70 (w/w) mixture of phosphatidylserine and phosphatidylcholine and extruded through filters of pore size 800, 400, 100, and 50 nm. PX-BAR (3 μM) was added and the samples were centrifuged, and the supernatants (S) and pellets (P) were analyzed by SDS–PAGE and quantitated by densitometry. The bars show the means from two experiments, with the maximum value indicated for each set.