Phosphatidylinositol 3,5‐Bisphosphate‐Rich Membrane Domains in Endosomes and Lysosomes

Phosphatidylinositol 3,5‐bisphosphate (PtdIns(3,5)P₂) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P₂ using freeze‐fracture electron microscopy. GST‐ATG18‐4×FLAG was used to label PtdIns(3,5)P₂ and its binding to phosphatidylinositol 3‐phosphate (PtdIns(3)P) was blocked by an excess of the p40^phox PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P₂ was concentrated in intramembrane particle (IMP)‐deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP‐deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid‐disordered domain marker. In yeast lacking either PtdIns(3,5)P₂ or its effector, Atg18p, the IMP‐deficient, PtdIns(3)P‐rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P₂ metabolism is defective. In HeLa cells, PtdIns(3,5)P₂ was significantly enriched in the vesicular domain of RAB5‐ and RAB7‐positive endosome/lysosomes of the tubulo‐vesicular morphology. This biased distribution of PtdIns(3,5)P₂ was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P₂‐rich and ‐deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality.      

Subcellular localisation of the p40phox component of NADPH oxidase involves direct interactions between the Phox homology domain and F-actin

Cytosolic components of the NADPH oxidase interact with the actin cytoskeleton. These interactions are thought to be important for the activation of this enzyme system but they are poorly characterised at the molecular level. Here we have explored the interaction between the actin cytoskeleton and p40^phox, one of the cytosolic components of NADPH oxidase. Full length p40^phox expressed in COS cells co-localised with F-actin in a peripheral lamellar compartment. The co-localisation was lost after deletion of the Phox homology (PX) domain and the PX domain in isolation (p40PX) showed the same F-actin co-localisation as the full length protein. PX domains are known lipid-binding modules however, a mutant p40PX which did not bind lipids still co-localised with F-actin suggesting that lipid-independent interactions underlie the localisation. Affinity chromatography identified actin as a binding partner for p40PX in neutrophil extracts. Pure actin interacted with both p40^phox and with p40PX suggesting it is a direct interaction. Disruption of the actin cytoskeleton with cytochalasin D resulted in actin rearrangement and concomitantly the localisation of full length p40^phox proteins and that of p40PX changed. Thus p40PX is a dual F-actin/lipid-binding module and F-actin interactions with the PX domain dictate at least in part the intracellular localisation of the cytosolic p40^phox subunit of the NADPH oxidase.     

Backbone 1H, 15N, and 13C resonance assignments for the NOXO1β PX domain

NOXO1 (Nox Organizer 1) is a homolog of the NAPDH oxidase protein p47 ^phox . NADPH oxidases transfer electrons from NADPH to molecular oxygen, generating the superoxide anion. NOXO1 contains an N-terminal PX (phox homology) domain and is one of several PX domain-containing proteins found in the cytosolic subunits of the NADPH oxidase complex. These PX domains bind to membrane lipids and target the protein to membranes, recruiting other cytosolic components to the membrane bound components and aiding formation of a active enzyme complex. This recruitment represents a level of regulation of these oxidases. Here we report the backbone assignments of NOXO1β PX.     

Crystal structure of the yeast Phox homology (PX) domain protein Grd19p complexed to phosphatidylinositol-3-phosphate

Phox homology (PX) domains have been recently identified in a number of different proteins and are involved in various cellular functions such as vacuolar targeting and membrane protein trafficking. It was shown that these modules of about 130 amino acids specifically binding to phosphoinositides and that this interaction is crucial for their cellular function. The yeast genome contains 17 PX domain proteins. One of these, Grd19p, is involved in the localization of the late Golgi membrane proteins DPAP A and Kex2p. Grd19p consists of the PX domain with 30 extra residues at the N-terminal and is homologous to the functionally characterized human sorting nexin protein SNX3. We determined the 2.0 A crystal structure of Grd19p in the free form and in complex with d-myo-phosphatidylinositol 3-phosphate (diC4PtdIns(3)P), representing the first case of both free and ligand-bound conformations of the same PX module. The ligand occupies a well defined positively charged binding pocket at the interface between the beta-sheet and alpha-helical parts of the molecule. The structure of the free and bound protein are globally similar but show some significant differences in a region containing a polyproline peptide and a putative membrane attachment site.     

The phox homology (PX) domain-dependent, 3-phosphoinositide-mediated association of sorting nexin-1 with an early sorting endosomal compartment is required for its ability to regulate epidermal growth factor receptor degradation

Recent studies have shown that phox homology (PX) domains act as phosphoinositide-binding motifs. The majority of PX domains studied show binding to phosphatidylinositol 3-monophosphate (PtdIns(3)P), an association that allows the host protein to localize to membranes of the endocytic pathway. One issue, however, is whether PX domains may have alternative phosphoinositide binding specificities that could target their host protein to distinct subcellular compartments or allow their allosteric regulation by phosphoinositides other than PtdIns(3)P. It has been reported that the PX domain of sorting nexin 1 (SNX1) specifically binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) (Zhong, Q., Lazar, C. S., Tronchere, H., Sato, T., Meerloo, T., Yeo, M., Songyang, Z., Emr, S. D., and Gill, G. N. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6767-6772). In the present study, we have shown that whereas SNX1 binds PtdIns(3,4,5)P(3) in protein:lipid overlay assays, in liposomes-based assays, binding is observed to PtdIns(3)P and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) but not to PtdIns(3,4,5)P(3). To address the significance of PtdIns(3,4,5)P(3) binding, we examined the subcellular localization of SNX1 under conditions in which plasma membrane PtdIns(3,4,5)P(3) levels were significantly elevated. Under these conditions, we failed to observe association of SNX1 with this membrane. However, consistent with the binding to PtdIns(3)P and PtdIns(3,5)P(2) being of more physiological significance was the observation that the association of SNX1 with an early endosomal compartment was dependent on a 3-phosphoinositide-binding PX domain and the presence of PtdIns(3)P on this compartment. Finally, we have shown that the PX domain-dependent/early endosomal association of SNX1 is important for its ability to regulate the targeting of internalized epidermal growth factor receptor for lysosomal degradation.     

Inhibitory regulation of EGF receptor degradation by sorting nexin 5

Endosomal trafficking of EGF receptor (EGFR) upon stimulation is a highly regulated process during receptor-mediated signaling. Recently, the sorting nexin (SNX) family has emerged as an important regulator in the membrane trafficking of EGFR. Here, we report the identification of a novel interaction between two members of the family, SNX1 and SNX5, which is mediated by the newly defined BAR domain of both SNXs. We have also shown that the PX domain of SNX5 binds specifically to PtdIns other than to PtdIns(3)P. Furthermore, the BAR domain but not the PX domain of SNX5 is sufficient for its subcellular membrane association. Functionally, overexpression of SNX5 inhibits the degradation of EGFR. This process appears to be independent of its interaction with SNX1. However, overexpression of SNX1 is able to attenuate the effect of SNX5 on EGFR degradation, suggesting the two proteins may play antagonistic roles in regulating endosomal trafficking of the receptor.     

Role of the phospholipid binding sites,PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2

In phagocytes, superoxide anion (O₂⁻), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b₅₅₈ (cyt b₅₅₈) and four cytosolic components: p40^phox, p47^phox, p67^phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b₅₅₈ to form the active enzyme.To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47^phox. We also used a fused chimera consisting of p47^phox(aa 1–286), p67^phox(aa 1–212) and Rac1Q61L, as well as mutated versions in the p47^phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b₅₅₈. They also have an impact on O₂.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.     

The Phox Domain of Sorting Nexin 5 Lacks Phosphatidylinositol 3-Phosphate (PtdIns(3)P) Specificity and Preferentially Binds to Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2)

Subcellular retrograde transport of cargo receptors from endosomes to the trans-Golgi network is critically involved in a broad range of physiological and pathological processes and highly regulated by a genetically conserved heteropentameric complex, termed retromer. Among the retromer components identified in mammals, sorting nexin 5 and 1 (SNX5; SNX1) have recently been found to interact, possibly controlling the membrane binding specificity of the complex. To elucidate how the unique sequence features of the SNX5 phox domain (SNX5-PX) influence retrograde transport, we have determined the SNX5-PX structure by NMR and x-ray crystallography at 1.5 Å resolution. Although the core fold of SNX5-PX resembles that of other known PX domains, we found novel structural features exclusive to SNX5-PX. It is most noteworthy that in SNX5-PX, a long helical hairpin is added to the core formed by a new α2′-helix and a much longer α3-helix. This results in a significantly altered overall shape of the protein. In addition, the unique double PXXP motif is tightly packed against the rest of the protein, rendering this part of the structure compact, occluding parts of the putative phosphatidylinositol (PtdIns) binding pocket. The PtdIns binding and specificity of SNX5-PX was evaluated by NMR titrations with eight different PtdIns and revealed that SNX5-PX preferentially and specifically binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). The distinct structural and PtdIns binding characteristics of SNX5-PX impart specific properties on SNX5, influencing retromer-mediated regulation of retrograde trafficking of transmembrane cargo receptors.The early work on retromer revealed its role in the trafficking of cargo proteins between endosomes and the trans-Golgi network (TGN),² although recently, retromer involvement in many other physiological and developmental processes has been uncovered (1, 2). The best studied proteins associated with retromer activity are intracellular sorting receptors such as the yeast vacuolar protein-10 (Vps10) and mammalian mannose 6-phosphate receptors (3, 4). These receptors sort acid hydrolases, enzymes essential for protein degradation, out of the TGN into the yeast vacuole or the mammalian lysosome. Upon releasing their substrates, these cargos traffic back to the TGN to mediate further rounds of cargo-hydrolase transportation. Similar retrograde trafficking of cargo proteins involving signaling molecules such as Wnt and amyloid precursor protein (APP) are thought to be critical for their secretion and function (5, 6). Retrograde transportation is highly regulated by the heteropentameric retromer complex that consists of a sorting nexin (SNX) dimer (e.g. Vps5 and Vps17 in yeast) and a Vps26/29/35 trimer (7). In mammals, the binding of the SNX dimer to specific phosphatidylinositol (PtdIns) determines its subcellular membrane association and governs the recruitment of the Vps trimer to endosomal compartments. Mammalian orthologs of the trimer have been biochemically characterized, and their interaction and function in cargo protein trafficking is well established (8). More recently, crystal structures of three Vps proteins in the trimer suggested how this trimer interacts with the SNX dimer and cargo proteins as well as with curved membranes (9–12). In the SNX dimer, SNX1 and SNX2 are thought to be interchangeable Vps5 orthologs (13, 14). The NMR structure of SNX1 revealed details of PI(3)P specific binding, thereby explaining its role in endosomal trafficking (15). The identity for SNX5 as a potential functional mammalian ortholog of Vps17, however, was not revealed until recently.Although initially identified as a Fanconi anemia complementation group A (FANCA)-binding protein (16), SNX5 was later shown to play an important role in membrane trafficking (17–19). SNX5 contains a PX domain (SNX5-PX) that is the signature feature in defining the SNX family, composed of 30 members at present (20) (Fig. 1B). In addition, SNX5 possesses a C-terminal BAR (Bin/Amphiphysin/Rvs) domain that has been reported to interact with a number of other proteins involved in endosomal trafficking (17, 21–27). It functions as a dimerization module that senses and/or induces membrane curvature (28, 29). Our previous biochemical study suggested a specific interaction between SNX5 and SNX1 through which the two SNXs mutually influence each other''s effect in endosomal trafficking of epidermal growth factor receptor upon epidermal growth factor stimulation (17). In support of this observation are several recent reports that indicate a critical role of SNX5 and the closely related SNX6, beyond that of SNX1 and SNX2, on retrograde sorting of mannose 6-phosphate receptor (24, 27). Therefore, SNX5 and SNX6 may be functionally interchangeable orthologs of Vps17 in mammalian cells (7, 24). Furthermore, in contrast to some reports (18, 30), SNX5 partially localizes to late endosomes and the TGN, exhibiting very low binding affinity for PtdIns(3)P (17), the substrate for phox domain proteins associating with early endosome association. Therefore, the subcellular localization and function of the SNX dimer in SNX5 function may depend on its unique structure that is different from other known PX domains.Open in a separate windowFIGURE 1.Amino Acid sequence alignment of phox domains and domain architecture of the mammalian sorting nexin family. A, comparative sequence alignment of PX domains for residues equivalent to Gly⁴⁹–Leu¹¹⁹ of the p40-PX domain (adapted from Worby and Dixon (21)). Prolines in the Pro-X-X-Pro motif are highlighted in yellow, and residues involved in phospholipid binding in the p40-PX domain are boxed in magenta. Arg⁵⁸ and Arg¹⁰⁵ are marked with magenta triangles, and Tyr⁵⁹ and Lys⁹² are marked with black stars at the bottom of the sequences. The two conserved Arg residues and Lys⁹² of p40-PX in other PX domains are highlighted in dark blue boxes; those corresponding to Tyr⁵⁹ are boxed in green. The secondary structure elements of p40-PX are indicated by yellow arrows (β-sheets) and red ovals (α-helices). The three sequence stretches that are unique in SNX5-PX (or SNX6-PX) are enclosed in a bright blue box. B, domain architecture of SNX family members. The four classes within the SNX family are designated as PX SNXs, PX-BAR SNXs, SH3-PX-BAR, and PX-other domain SNXs. Each individual domain is depicted in a different color and/or shape. The following domains are depicted: PX (phox), BAR (Bin-Amphiphysin-Rvs), SH3 (Src homology 3), TM (transmembrane), PXA (PX domain-associated), RGS (regulator of G-protein signaling), MIT (microtubule interacting and trafficking), B41 (band 4.1 homology), TPR (tetratricopeptide repeat), PDZ (postsynaptic protein PSD-95/SAP90, the Drosophila melanogaster septate junction protein Discs-large, and the tight junction protein ZO-1), and RA (Ras association).Most PX domains of SNX family proteins preferentially bind PtdIns(3)P (30–34), with few exceptions that interact with other PtdIns (30, 32, 35). There are about a dozen structurally characterized PX domains from the SNX family or other PX domain-containing proteins currently deposited in the Protein Data Bank (PDB) data base. Their structures all share common core features, a three-stranded β-sheet that is abutted by three α-helices and an irregular strand containing the PXXP region. Analyses of the representative p47-PX and SNX3-PX domain structures suggested that PtdIns(3)P binding involves two conserved Arg residues at positions equivalent to Arg⁵⁸ and Arg¹⁰⁵ in p40-PX (36). Because equivalent Arg residues are found in the PX domains of most SNX family members, it is generally assumed that all SNX proteins interact with the PtdIns(3)P-enriched elements of the early endocytic compartments. The amino acid sequences of the PX domains of both SNX5 and SNX6, however, lack the two conserved Arg residues that are involved in PtdIns(3)P binding as well as comprising a ∼30-residue insertion immediately after the PXXP motif (Fig. 1A). In addition, the PXXP motif is extended into a double PXXP motif with the sequence PXXPXXP. These unique sequence features set SNX5/6 apart from the other SNX family members. In the p40-PX domain and yeast SNX3, the two conserved Arg residues, the loop between the PXXP motif, and the α3-helix are involved in forming the binding pocket for the phosphate groups of PtdIns(3)P (36, 37). Therefore, changes in length and sequence in this region in SNX5/6-PX are expected to have profound impact on the specific structure and conformation required for PtdIns recognition.To elucidate how its unique sequence features influence the function of SNX5 in retromer-mediated retrograde membrane trafficking, we structurally investigated the SNX5-PX domain by NMR spectroscopy and x-ray crystallography. Using direct NMR titrations, we established the PtdIns binding specificity of SNX5-PX. The high resolution (1.5 Å) crystal structure of the domain revealed its distinct features when compared with previously known family members. Our results demonstrate that the SNX5-PX domain is indeed unique, both with respect to its structure as well as with respect to ligand binding. These findings have important implications for the function of SNX5 in the subcellular membrane trafficking and retrograde sorting.     

Solution structure of the Vam7p PX domain

PX domains have been recently found to act as phosphoinositide binding modules. In the yeast SNARE protein Vam7p, the PX domain binds to PtdIns(3)P and is required for vacuolar targeting. To gain insight into how PX domains function, the solution structure of the ligand-free Vam7p PX domain has been determined by NMR spectroscopy. The Vam7p PX domain has the same overall alpha/beta fold observed in the structures of the ligand-free p47(phox) PX domain and the PtdIns(3)P-bound p40(phox) PX domain, exhibiting several similarities and differences with these two PX domains. Most striking is the similarity between the Vam7p and p40(phox) PX domains in a subset of secondary structure elements despite the low level of sequence identity between them, suggesting that these elements form a conserved core in the PX domain fold. These similarities and the observation that a putative PtdIns(3)P binding site is already formed in the apo Vam7p PX domains suggest that ligand binding does not induce major conformational changes, contrary to what was previously thought. The proposed ligand binding site of the Vam7p PX domain includes basic side chains from the conserved structural core that also participate in PtdIns(3)P binding to the p40(phox) PX domain, and basic side chains from a variable loop that probably inserts into the membrane. These results indicate that PX domains contain a combination of conserved and variable features that allow them to have a common function and at the same time exhibit distinct specificities, mechanisms of regulation, or modes of interaction with effector molecules.     

Conformational changes in p47 upon activation highlighted by mass spectrometry coupled to hydrogen/deuterium exchange and limited proteolysis

The neutrophil NADPH oxidase is an enzymatic complex involved in innate immunity. Phosphorylation of p47^phox promotes its translocation with p67^phox and p40^phox, followed by membrane interaction and assembly with flavocytochrome b₅₅₈ into a functional complex. To characterise p47^phox conformational changes during activation, we used wild-type and the S303/304/328E triple mutant mimicking the phosphorylated state. Hydrogen/deuterium exchange and limited proteolysis coupled to mass spectrometry were used to discriminate between the various structural models. An increase in deuteration confirmed that p47^phox adopts an open and more flexible conformation after activation. Limited proteolysis correlated this change with increased auto-inhibitory region (AIR) accessibility. These results establish a structural link between the AIR release and the exposure of the Phox homology (PX) domain.     

Structural and membrane binding analysis of the Phox homology domain of Bem1p: basis of phosphatidylinositol 4-phosphate specificity

Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of the diverse PI specificity of PX domains, we determined the crystal structure of the PX domain from Bem1p that has been reported to bind phosphatidylinositol 4-phosphate (PtdIns(4)P). We also measured the membrane binding properties of the PX domain and its mutants by surface plasmon resonance and monolayer techniques and calculated the electrostatic potentials for the PX domain in the absence and presence of bound PtdIns(4)P. The Bem1p PX domain contains a signature PI-binding site optimized for PtdIns(4)P binding and also harbors basic and hydrophobic residues on the membrane-binding surface. The membrane binding of the Bem1p PX domain is initiated by nonspecific electrostatic interactions between the cationic membrane-binding surface of the domain and anionic membrane surfaces, followed by the membrane penetration of hydrophobic residues. Unlike other PX domains, the Bem1p PX domain has high intrinsic membrane penetrating activity in the absence of PtdIns(4)P, suggesting that the partial membrane penetration may occur before specific PtdIns(4)P binding and last after the removal of PtdIns(4)P under certain conditions. This structural and functional study of the PtdIns(4)P-binding Bem1p PX domain provides new insight into the diverse PI specificities and membrane-binding mechanisms of PX domains.     

Arabidopsis PCaP2 modulates the phosphatidylinositol 4,5‐bisphosphate signal on the plasma membrane and attenuates root hair elongation

Phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P₂] serves as a subcellular signal on the plasma membrane, mediating various cell‐polarized phenomena including polar cell growth. Here, we investigated the involvement of Arabidopsis thaliana PCaP2, a plant‐unique plasma membrane protein with phosphoinositide‐binding activity, in PtdIns(4,5)P₂ signaling for root hair tip growth. The long‐root‐hair phenotype of the pcap2 knockdown mutant was found to stem from its higher average root hair elongation rate compared with the wild type and to counteract the low average rate caused by a defect in the PtdIns(4,5)P₂‐producing enzyme gene PIP5K3. On the plasma membrane of elongating root hairs, the PCaP2 promoter‐driven PCaP2–green fluorescent protein (GFP), which complemented the pcap2 mutant phenotype, overlapped with the PtdIns(4,5)P₂ marker 2xCHERRY‐2xPH^PLC in the subapical region, but not at the apex, suggesting that PCaP2 attenuates root hair elongation via PtdIns(4,5)P₂ signaling on the subapical plasma membrane. Consistent with this, a GFP fusion with the PCaP2 phosphoinositide‐binding domain PCaP2^N23, root hair‐specific overexpression of which caused a low average root hair elongation rate, localized more intense to the subapical plasma membrane than to the apical plasma membrane similar to PCaP2–GFP. Inducibly overexpressed PCaP2–GFP, but not its derivative lacking the PCaP2^N23 domain, replaced 2xCHERRY‐2xPH^PLC on the plasma membrane in root meristematic epidermal cells, and suppressed FM4‐64 internalization in elongating root hairs. Moreover, inducibly overexpressed PCaP2 arrested an endocytic process of PIN2–GFP recycling. Based on these results, we conclude that PCaP2 functions as a negative modulator of PtdIns(4,5)P₂ signaling on the subapical plasma membrane probably through competitive binding to PtdIns(4,5)P₂ and attenuates root hair elongation.     

Full-length p40phox structure suggests a basis for regulation mechanism of its membrane binding

The superoxide-producing phagocyte NADPH oxidase is activated during phagocytosis to destroy ingested microbes. The adaptor protein p40phox associates via the PB1 domain with the essential oxidase activator p67phox, and is considered to function by recruiting p67phox to phagosomes; in this process, the PX domain of p40phox binds to phosphatidylinositol 3-phosphate [PtdIns(3)P], a lipid abundant in the phagosomal membrane. Here we show that the PtdIns(3)P-binding activity of p40phox is normally inhibited by the PB1 domain both in vivo and in vitro. The crystal structure of the full-length p40phox reveals that the inhibition is mediated via intramolecular interaction between the PB1 and PX domains. The interface of the p40phox PB1 domain for the PX domain localizes on the opposite side of that for the p67phox PB1 domain, and thus the PB1-mediated PX regulation occurs without preventing the PB1-PB1 association with p67phox.     

p47phox Molecular Activation for Assembly of the Neutrophil NADPH Oxidase Complex

The p47^phox cytosolic factor from neutrophilic NADPH oxidase has always been resistant to crystallogenesis trials due to its modular organization leading to relative flexibility. Hydrogen/deuterium exchange coupled to mass spectrometry was used to obtain structural information on the conformational mechanism that underlies p47^phox activation. We confirmed a relative opening of the protein with exposure of the SH3 Src loops that are known to bind p22^phox upon activation. A new surface was shown to be unmasked after activation, representing a potential autoinhibitory surface that may block the interaction of the PX domain with the membrane in the resting state. Within this surface, we identified 2 residues involved in the interaction with the PX domain. The double mutant R162A/D166A showed a higher affinity for specific phospholipids but none for the C-terminal part of p22^phox, reflecting an intermediate conformation between the autoinhibited and activated forms.     

p47phox Phox Homology Domain Regulates Plasma Membrane but Not Phagosome Neutrophil NADPH Oxidase Activation

The assembly of cytosolic subunits p47^phox, p67^phox, and p40^phox with flavocytochrome b₅₅₈ at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47^phox subunit plays a critical role in oxidase assembly. Recent studies showed that the p47^phox Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47^phox PX domain in neutrophils is unclear, we investigated its role using p47^phox knock-out (KO) mouse neutrophils to express human p47^phox and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47^phox proteins were expressed at levels similar to endogenous murine p47^phox, with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47^phox rescued p47^phox KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47^phox with Arg⁹⁰ substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47^phox Arg⁹⁰ mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47^phox PX domain for neutrophil NADPH oxidase activation.     

SNX18 tubulates recycling endosomes for autophagosome biogenesis

The role of membrane remodeling and phosphoinositide-binding proteins in autophagy remains elusive. PX domain proteins bind phosphoinositides and participate in membrane remodeling and trafficking events and we therefore hypothesized that one or several PX domain proteins are involved in autophagy. Indeed, the PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation using an image-based siRNA screen. We show that SNX18 interacts with ATG16L1 and LC3, and functions downstream of ATG14 and the class III PtdIns3K complex in autophagosome formation. SNX18 facilitates recruitment of ATG16L1 to perinuclear recycling endosomes, and its overexpression leads to tubulation of ATG16L1- and LC3-positive membranes. We propose that SNX18 promotes LC3 lipidation and tubulation of recycling endosomes to provide membrane for phagophore expansion.     

Membrane insertion of the FYVE domain is modulated by pH

The FYVE domain associates with phosphatidylinositol 3‐phosphate [PtdIns(3)P] in membranes of early endosomes and penetrates bilayers. Here, we detail principles of membrane anchoring and show that the FYVE domain insertion into PtdIns(3)P‐enriched membranes and membrane‐mimetics is substantially increased in acidic conditions. The EEA1 FYVE domain binds to POPC/POPE/PtdIns(3)P vesicles with a Kd of 49 nM at pH 6.0, however associates ～24 fold weaker at pH 8.0. The decrease in the affinity is primarily due to much faster dissociation of the protein from the bilayers in basic media. Lowering the pH enhances the interaction of the Hrs, RUFY1, Vps27p and WDFY1 FYVE domains with PtdIns(3)P‐containing membranes in vitro and in vivo, indicating that pH‐dependency is a general function of the FYVE finger family. The PtdIns(3)P binding and membrane insertion of the FYVE domain is modulated by the two adjacent His residues of the R(R/K)HHCRXCG signature motif. Mutation of either His residue abolishes the pH‐sensitivity. Both protonation of the His residues and nonspecific electrostatic contacts stabilize the FYVE domain in the lipid‐bound form, promoting its penetration and increasing the membrane residence time. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of high- curvature membranes and 3-phosphoinositides

BACKGROUND: Sorting nexins (SNXs) are phox homology (PX) domain-containing proteins thought to regulate endosomal sorting of internalized receptors. The prototypical SNX is sorting nexin-1 (SNX1), a protein that through its PX domain binds phosphatidylinositol 3-monophosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. SNX1 is associated with early endosomes, from where it has been proposed to regulate the degradation of internalized epidermal growth factor (EGF) receptors through modulating endosomal-to-lysosomal sorting. RESULTS: We show here that SNX1 contains a BAR (Bin/Amphiphysin/Rvs) domain, a membrane binding domain that endows SNX1 with the ability to form dimers and to sense membrane curvature. We present evidence that through coincidence detection, the BAR and PX domains efficiently target SNX1 to a microdomain of the early endosome defined by high curvature and the presence of 3-phosphoinositides. In addition, we show that the BAR domain endows SNX1 with an ability to tubulate membranes in-vitro and drive the tubulation of the endosomal compartment in-vivo. Using RNA interference (RNAi), we establish that SNX1 does not play a role in EGF or transferrin receptor sorting; rather it specifically perturbs endosome-to-trans Golgi network (TGN) transport of the cation-independent mannose-6-phosphate receptor (CI-MPR). Our data support an evolutionarily conserved function for SNX1 from yeast to mammals and provide functional insight into the molecular mechanisms underlying lipid-mediated protein targeting and tubular-based protein sorting. CONCLUSIONS: We conclude that through coincidence detection SNX1 associates with a microdomain of the early endosome-characterized by high membrane curvature and the presence of 3-phosphoinositides-from where it regulates tubular-based endosome-to-TGN retrieval of the CI-MPR.     

In silico phosphorylation of the autoinhibited form of p47(phox): insights into the mechanism of activation

Activation of the multicomponent enzyme NADPH oxidase requires the interaction between the tandem SH3 domain of the cytosolic subunit p47^phox and the cytoplasmic tail of membrane-bound p22^phox. In the resting state, p47^phox exists in an autoinhibited conformation stabilized by intramolecular contacts between the SH3 domains and an adjacent polybasic region. Phosphorylation of three serine residues, Ser³⁰³, Ser³⁰⁴, and Ser³²⁸ within this polybasic region has been shown to be sufficient for the disruption of the intramolecular interactions thereby inducing an active state of p47^phox. This active conformation is accessible to the cytoplasmic tail of p22^phox and initiates the formation of the membrane-bound functional enzyme complex. Molecular dynamics simulations reveal insights in the mechanism of activation of the autoinhibited form of p47^phox by in silico phosphorylation, of the three serine residues, Ser³⁰³, Ser³⁰⁴, and Ser³²⁸. The simulations highlight the major collective coordinates generating the opening and the closing of the two SH3 domains and the residues that cause the unmasking of the p22^phox binding site.     

Regulation of the NADPH-oxidase complex of phagocytic leukocytes. Recent insights from structural biology,molecular genetics,and microscopy

The NADPH-oxidase complex is a multisubunit enzyme complex that catalyzes the formation of superoxide (O₂^–) by phagocytic leukocytes. This paper reviews some of the major advances in understanding the assembly and regulation of this enzyme system that have occurred during the past decade. For example, novel domains/motifs have been identified in p47-phox (PX and super SH3 domains) and p67-phox (tetratricopeptide repeat motifs). X-ray crystallography and NMR spectroscopy have provided detailed structural data on these domains and how p47-phox and p67-phox interact with p22-phox and activated Rac, respectively. Site-directed mutagenesis and knockout experiments have identified the critical phosphorylation sites in p47-phox, revealed an activation domain in p67-phox, and demonstrated that a specific pathway exists for activating Rac to participate in oxidase assembly/activation. Cytochemistry and immunofluorescence microscopy have provided new insights into the assembly of the oxidase and reveal a level of complexity not previously appreciated.John A. Badwey has recently died