Phox Homology Research Articles

Mechanism of cargo recognition by retromer-linked SNX-BAR proteins.

Endocytic recycling of internalized transmembrane proteins is essential for many important physiological processes. Recent studies have revealed that retromer-related Sorting Nexin family (SNX)–Bin/Amphiphysin/Rvs (BAR) proteins can directly recognize cargoes like cation-independent mannose 6-phosphate receptor (CI-MPR) and Insulin-like growth factor 1 receptor (IGF1R); however, it remains poorly understood how SNX-BARs select specific cargo proteins and whether they recognize additional ligands. Here, we discovered that the binding between SNX-BARs and CI-MPR or IGF1R is mediated by the phox-homology (PX) domain of SNX5 or SNX6 and a bipartite motif, termed SNX-BAR-binding motif (SBM), in the cargoes. Using this motif, we identified over 70 putative SNX-BAR ligands, many of which play critical roles in apoptosis, cell adhesion, signal transduction, or metabolite homeostasis. Remarkably, SNX-BARs could cooperate with both SNX27 and retromer in the recycling of ligands encompassing the SBM, PDZ-binding motif, or both motifs. Overall, our studies establish that SNX-BARs function as a direct cargo-selecting module for a large set of transmembrane proteins transiting the endosome, in addition to their roles in phospholipid recognition and biogenesis of tubular structures.

PX Domain-Containing Kinesin KIF16B and Microtubule-Dependent Intracellular Movements.

As a member of the kinesin-3 family, kinesin family member 16B (KIF16B) has a characteristic PhoX homology (PX) domain that binds to membranes containing phosphatidylinositol-3-phosphate (PI(3)P) and moves along microtubule filaments to the plus end via a process regulated by coiled coils in the stalk region in various cell types. The physiological function of KIF16B supports the transport of intracellular cargo and the formation of endosomal tubules. Ras-related protein (Rab) coordinates many steps of membrane transport and are involved in the regulation of KIF16B-mediated vesicle trafficking. Data obtained from clinical research suggest that KIF16B has a potential effect on the disease processes in intellectual disability, abnormal lipid metabolism, and tumor brain metastasis. In this review, we summarize recent advances in the structural and physiological characteristics of KIF16B as well as diseases associated with KIF16B disorders, and speculating its role as a potential adaptor for intracellular cholesterol trafficking.

The novel zebrafish model pretzel demonstrates a central role for SH3PXD2B in defective collagen remodelling and fibrosis in Frank-Ter Haar syndrome.

ABSTRACTFrank-Ter Haar syndrome (FTHS, MIM #249420) is a rare skeletal dysplasia within the defective collagen remodelling spectrum (DECORS), which is characterised by craniofacial abnormalities, skeletal malformations and fibrotic soft tissues changes including dermal fibrosis and joint contractures. FTHS is caused by homozygous or compound heterozygous loss-of-function mutation or deletion of SH3PXD2B (Src homology 3 and Phox homology domain-containing protein 2B; MIM #613293). SH3PXD2B encodes an adaptor protein with the same name, which is required for full functionality of podosomes, specialised membrane structures involved in extracellular matrix (ECM) remodelling. The pathogenesis of DECORS is still incompletely understood and, as a result, therapeutic options are limited. We previously generated an mmp14a/b knockout zebrafish and demonstrated that it primarily mimics the DECORS-related bone abnormalities. Here, we present a novel sh3pxd2b mutant zebrafish, pretzel, which primarily reflects the DECORS-related dermal fibrosis and contractures. In addition to relatively mild skeletal abnormalities, pretzel mutants develop dermal and musculoskeletal fibrosis, contraction of which seems to underlie grotesque deformations that include kyphoscoliosis, abdominal constriction and lateral folding. The discrepancy in phenotypes between mmp14a/b and sh3pxd2b mutants suggests that in fish, as opposed to humans, there are differences in spatiotemporal dependence of ECM remodelling on either sh3pxd2b or mmp14a/b. The pretzel model presented here can be used to further delineate the underlying mechanism of the fibrosis observed in DECORS, as well as screening and subsequent development of novel drugs targeting DECORS-related fibrosis.This paper has an associated First Person interview with the first author of the article.

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers.

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Two isotypes of phosphatidylinositol 3-phosphate-binding sorting nexins play distinct roles in trogocytosis in Entamoeba histolytica.

Phosphatidylinositol phosphates (PIPs) function as important second messengers in many cellular events. In the human intestinal protist Entamoeba histolytica, where phagocytosis/trogocytosis plays an indispensable role in proliferation and pathophysiology during infection, various PIPs are involved in multiple steps of phago/trogocytosis. PI3‐phosphate (PI3P) plays a pivotal role in the biogenesis of phagosome/trogosomes via recruitment of PI3P effectors. Because no known PI3P downstream effectors are conserved in E. histolytica, we exploited a unique method to identify the proteins PI3P dependently recruited to phagosomes. We rationalised that overexpression of PI3P‐binding GFP‐HrsFYVE competes for PI3P on phagosomal membranes and results in dissociation of PI3P effectors from phagosomes. EhVps26 and EhVps35, but not sorting nexins (SNXs), of the retromer complex were detected from phagosomes only without GFP‐HrsFYVE overexpression. Two potential SNXs, EhSNX1 and EhSNX2, identified in the genome, possess only phox homology domain and specifically bound to PI3P, but retromer components, EhVps26 and EhVps35, did not bind to PI3P. Live and immunofluorescence imaging showed that EhSNX1 was recruited to the trogocytic cup and tunnel‐like structures, and subsequently, EhSNX2 was recruited to trogosomes. Furthermore, EhSNX1, but not EhSNX2, specifically bound to Arp2/3 and EhVps26, which were localised to the tunnel‐like structures and the trogosomes, respectively. EhSNX2 gene silencing increased trogocytosis, suggesting that EhSNX2 plays an inhibitory role in trogocytosis.

Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44.

The serum- and glucocorticoid-regulated kinase (SGK) isoforms contribute resistance to cancer therapies targeting the PI3K pathway. SGKs are homologous to Akt and these kinases display overlapping specificity and phosphorylate several substrates at the same residues, such as TSC2 to promote tumor growth by switching on the mTORC1 pathway. The SGK3 isoform is up-regulated in breast cancer cells treated with PI3K or Akt inhibitors and recruited and activated at endosomes, through its phox homology domain binding to PtdIns(3)P. We undertook genetic and pharmacological phosphoproteomic screens to uncover novel SGK3 substrates. We identified 40 potential novel SGK3 substrates, including four endosomal proteins STX7 (Ser126) and STX12 (Ser139), RFIP4 (Ser527) and WDR44 (Ser346) that were efficiently phosphorylated in vitro by SGK3 at the sites identified in vivo, but poorly by Akt. We demonstrate that these substrates are inefficiently phosphorylated by Akt as they possess an n + 1 residue from the phosphorylation site that is unfavorable for Akt phosphorylation. Phos-tag analysis revealed that stimulation of HEK293 cells with IGF1 to activate SGK3, promoted phosphorylation of a significant fraction of endogenous STX7 and STX12, in a manner that was blocked by knock-out of SGK3 or treatment with a pan SGK inhibitor (14H). SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing the SNARE complex and promoted plasma membrane localization. Our data reveal novel substrates for SGK3 and suggest a mechanism by which STX7 and STX12 SNARE complexes are regulated by SGK3. They reveal new biomarkers for monitoring SGK3 pathway activity.

Molecular identification of a BAR domain-containing coat complex for endosomal recycling of transmembrane proteins.

Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargos with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargos including the cation-independent mannose-6-phosphate receptor and semaphorin 4C by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a β-hairpin that binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargos share this motif and require SNX5/SNX6 for their recycling. These include cargos involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the ‘Endosomal SNX-BAR sorting complex for promoting exit 1’ (ESCPE-1), which couples sorting motif recognition to BAR domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.

Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy.

Temporal regulation of cell polarity via the interaction of the Ras GTPase Rsr1 and the scaffold protein Bem1.

The Cdc42 guanosine triphosphatase (GTPase) plays a central role in polarity development in species ranging from yeast to humans. In budding yeast, a specific growth site is selected in the G1 phase. Rsr1, a Ras GTPase, interacts with Cdc42 and its associated proteins to promote polarized growth at the proper bud site. Yet how Rsr1 regulates cell polarization is not fully understood. Here, we show that Rsr1-GDP interacts with the scaffold protein Bem1 in early G1, likely hindering the role of Bem1 in Cdc42 polarization and polarized secretion. Consistent with these in vivo observations, mathematical modeling predicts that Bem1 is unable to promote Cdc42 polarization in early G1 in the presence of Rsr1-GDP. We find that a part of the Bem1 Phox homology domain, which overlaps with a region interacting with the exocyst component Exo70, is necessary for the association of Bem1 with Rsr1-GDP. Overexpression of the GDP-locked Rsr1 interferes with Bem1-dependent Exo70 polarization. We thus propose that Rsr1 functions in spatial and temporal regulation of polarity establishment by associating with distinct polarity factors in its GTP- and GDP-bound states.

Classification of the human phox homology (PX) domains based on their phosphoinositide binding specificities

Phox homology (PX) domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides, markers of organelle identity in the endocytic system. Although many PX domains bind the canonical endosome-enriched lipid PtdIns3P, others interact with alternative phosphoinositides, and a precise understanding of how these specificities arise has remained elusive. Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays, biolayer interferometry and isothermal titration calorimetry. These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns3P, non-specifically to various di- and tri-phosphorylated phosphoinositides, bind both PtdIns3P and other phosphoinositides, or associate with none of the lipids tested. A comprehensive evaluation of PX domain structures reveals two distinct binding sites that explain these specificities, providing a basis for defining and predicting the functional membrane interactions of the entire PX domain protein family.

The SNARE protein FolVam7 mediates intracellular trafficking to regulate conidiogenesis and pathogenicity in Fusarium oxysporum f. sp. lycopersici.

SummarySoluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) are conserved in fungi, plants and animals. The Vam7 gene encodes a v‐SNARE protein that involved in vesicle trafficking in fungi. Here, we identified and characterized the function of FolVam7, a homologue of the yeast SNARE protein Vam7p in Fusarium oxysporum f. sp. lycopersici (Fol), a fungal pathogen of tomato. FolVam7 contains SNARE and PX (Phox homology) domains that are indispensable for normal localization and function of FolVam7. Targeted gene deletion showed that FolVam7‐mediated vesicle trafficking is important for vegetative growth, asexual development, conidial morphology and plant infection. Further cytological examinations revealed that FolVam7 is localized to vesicles and vacuole membranes in the hyphae stage. Moreover, the ΔFolvam7 mutant is insensitive to salt and osmotic stresses and hypersensitive to cell wall stressors. Taken together, our results suggested that FolVam7‐mediated vesicle trafficking promotes vegetative growth, conidiogenesis and pathogenicity of Fol.

Structure and function of the Fgd family of divergent FYVE domain proteins 1.

FYVE domains are highly conserved protein modules that typically bind phosphatidylinositol 3-phosphate (PI3P) on the surface of early endosomes. Along with pleckstrin homology (PH) and phox homology (PX) domains, FYVE domains are the principal readers of the phosphoinositide (PI) code that mediate specific recognition of eukaryotic organelles. Of all the human FYVE domain containing proteins, those within the faciogenital dysplasia (Fgd) subfamily are particularly divergent and couple with GTPases to exert unique cellular functions. The subcellular distributions and functions of these evolutionarily conserved signal transducers, which also include Dbl homology (DH) and two PH domains, are discussed here to better understand the biological range of processes that such multidomain proteins engage in. Determinants of their various functions include specific multidomain architectures, posttranslational modifications including PIP stops that have been discovered in sorting nexins, PI recognition motifs, and phospholipid-binding surfaces as defined by the Membrane Optimal Docking Area (MODA) program. How these orchestrate Fgd function remains unclear but has implications for developmental diseases including Aarskog-Scott syndrome, which is also known as faciogenital dysplasia, and forms of cancer that are associated with mutations and amplifications of Fgd genes.

Molecular Basis for Membrane Recruitment by the PX and C2 Domains of Class II Phosphoinositide 3-Kinase-C2α

Phosphorylation of phosphoinositides by the classIIphosphatidylinositol 3-kinase (PI3K) PI3K-C2α isessential for many processes, including neuroexocytosis and formation of clathrin-coated vesicles. Adefining feature of the class II PI3Ks is a C-terminal module composed of phox-homology (PX) and C2 membrane interacting domains; however, the mechanisms that control their specific cellular localization remain poorly understood. Here we report the crystal structure of the C2 domain of PI3K-C2α in complex with the phosphoinositide head-group mimic inositol hexaphosphate, revealing two distinct pockets for membrane binding. The C2 domain preferentially binds to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate, and low-resolution structures of the combined PX-C2 module by small-angle X-ray scattering reveal a compact conformation in which cooperative lipid binding by each domain binding can occur. Finally, we demonstrate an unexpected role for calcium in perturbing the membrane interactions of the PX-C2 module, which we speculate may be important for regulating the activity of PI3K-C2α.

SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20.

Mutations in SNX14 cause the autosomal recessive cerebellar ataxia 20 (SCAR20). Mutations generally result in loss of protein although several coding region deletions have also been reported. Patient-derived fibroblasts show disrupted autophagy, but the precise function of SNX14 is unknown. The yeast homolog, Mdm1, functions in endoplasmic reticulum (ER)-lysosome/vacuole inter-organelle tethering, but functional conservation in mammals is still required. Here, we show that loss of SNX14 alters but does not block autophagic flux. In addition, we find that SNX14 is an ER-associated protein that functions in neutral lipid homeostasis and inter-organelle crosstalk. SNX14 requires its N-terminal transmembrane helices for ER localization, while the Phox homology (PX) domain is dispensable for subcellular localization. Both SNX14-mutant fibroblasts and SNX14KO HEK293 cells accumulate aberrant cytoplasmic vacuoles, suggesting defects in endolysosomal homeostasis. However, ER-late endosome/lysosome contact sites are maintained in SNX14KO cells, indicating that it is not a prerequisite for ER-endolysosomal tethering. Further investigation of SNX14- deficiency indicates general defects in neutral lipid metabolism. SNX14KO cells display distinct perinuclear accumulation of filipin in LAMP1-positive lysosomal structures indicating cholesterol accumulation. Consistent with this, SNX14KO cells display a slight but detectable decrease in cholesterol ester levels, which is exacerbated with U18666A. Finally, SNX14 associates with ER-derived lipid droplets (LD) following oleate treatment, indicating a role in ER-LD crosstalk. We therefore identify an important role for SNX14 in neutral lipid homeostasis between the ER, lysosomes and LDs that may provide an early intervention target to alleviate the clinical symptoms of SCAR20.

Phosphorylation of conserved phosphoinositide binding pocket regulates sorting nexin membrane targeting

Sorting nexins anchor trafficking machines to membranes by binding phospholipids. The paradigm of the superfamily is sorting nexin 3 (SNX3), which localizes to early endosomes by recognizing phosphatidylinositol 3-phosphate (PI3P) to initiate retromer-mediated segregation of cargoes to the trans-Golgi network (TGN). Here we report the solution structure of full length human SNX3, and show that PI3P recognition is accompanied by bilayer insertion of a proximal loop in its extended Phox homology (PX) domain. Phosphoinositide (PIP) binding is completely blocked by cancer-linked phosphorylation of a conserved serine beside the stereospecific PI3P pocket. This “PIP-stop” releases endosomal SNX3 to the cytosol, and reveals how protein kinases control membrane assemblies. It constitutes a widespread regulatory element found across the PX superfamily and throughout evolution including of fungi and plants. This illuminates the mechanism of a biological switch whereby structured PIP sites are phosphorylated to liberate protein machines from organelle surfaces.

Role of Cationic Groove and Hydrophobic Residues in Phosphatidylinositol‐Dependent Membrane‐Binding Properties of Tks5‐Phox Homology Domain

AbstractTyrosine kinase substrate with 5 Src homology 3 domains (Tks5) regulates several important cellular functions and is crucial for invadosome formation. The phox homology (PX) domain of Tks5 interacts with phosphatidylinositols (PIPs) and essential for Tks5‐induced invadosome formation. However, PIP‐binding specificity of the Tks5‐PX domain is not clear and its membrane recruitment mechanism also not known. To understand how PX domain deferentially interacts with the PIPs, we quantitatively determined the PIP‐binding properties. These measurements revealed that PX domain has strong PIP‐binding selectivity for PI(3,4)P2 and PI(3)P lipids. Mutational studies showed that the presence of hydrophobic residues at the putative membrane binding surface and basic residues within the PIP‐binding pocket are essential for its membrane penetration and binding properties. In vitro PIP‐selectivity correlates well with the cellular localization patterns of the wild‐type and mutant PX domains in A549 cells. The hydrophobic residues play a crucial role in cellular localization and translocation studies. The mutational analysis allowed for understanding these cellular activities and results are in accordance with their membrane binding properties. Based on our findings, we describe characteristic membrane binding properties of Tks5‐PX domain, in which the protein first interacts with specific PIP‐containing membrane and then its hydrophobic residues partially penetrate into the membrane.

Mechanism of activation of SGK3 by growth factors via the Class 1 and Class 3 PI3Ks.

Derailment of the PI3K-AGC protein kinase signalling network contributes to many human diseases including cancer. Recent work has revealed that the poorly studied AGC kinase family member, SGK3, promotes resistance to cancer therapies that target the Class 1 PI3K pathway, by substituting for loss of Akt kinase activity. SGK3 is recruited and activated at endosomes, by virtue of its phox homology domain binding to PtdIns(3)P. Here, we demonstrate that endogenous SGK3 is rapidly activated by growth factors such as IGF1, through pathways involving both Class 1 and Class 3 PI3Ks. We provide evidence that IGF1 enhances endosomal PtdIns(3)P levels via a pathway involving the UV-RAG complex of hVPS34 Class 3 PI3K. Our data point towards IGF1-induced activation of Class 1 PI3K stimulating SGK3 through enhanced production of PtdIns(3)P resulting from the dephosphorylation of PtdIns(3,4,5)P3. Our findings are also consistent with activation of Class 1 PI3K promoting mTORC2 phosphorylation of SGK3 and with oncogenic Ras-activating SGK3 solely through the Class 1 PI3K pathway. Our results highlight the versatility of upstream pathways that activate SGK3 and help explain how SGK3 substitutes for Akt following inhibition of Class 1 PI3K/Akt pathways. They also illustrate robustness of SGK3 activity that can remain active and counteract physiological conditions or stresses where either Class 1 or Class 3 PI3K pathways are inhibited.

The Phox Homology (PX) Domain.

The phox-homology (PX) domain is a phosphoinositide-binding domain conserved in all eukaryotes and present in 49 human proteins. Proteins containing PX domains, many of which are also known as sorting nexins (SNXs), have a large variety of functions in membrane trafficking, cell signaling, and lipid metabolism in association with membranes of the secretory and endocytic system. In this review we discuss the structural basis for both canonical lipid interactions with the endosome-enriched lipid phosphatidylinositol-3-phosphate (PtdIns3P) as well as non-canonical lipids that promote membrane association. We also describe recent advances in defining the diverse mechanisms by which PX domains interact with other proteins including the retromer trafficking complex and proteins secreted by bacterial pathogens. Like other membrane interacting domains, the attachment of PX domain proteins to specific membranes is often facilitated by additional interactions that contribute to binding avidity, and we discuss this coincidence detection for several known examples.

Phospholipases AtPLDζ1 and AtPLDζ2 function differently in hypoxia.

Besides hydrolyzing different membrane phospholipids, plant phospholipases D and molecular species of their byproducts phosphatidic acids (PLDs/PAs) are involved in diverse cellular events such as membrane-cytoskeleton dynamics, hormone regulation and biotic and/or abiotic stress responses at cellular or subcellular levels. Among the 12 Arabidopsis PLD genes, PLDζ1 and PLDζ2 uniquely possess Ca2+ -independent phox (PX) and pleckstrin (PH) homology domains. Here, we report that mutants deficient in these PLDs, pldζ1 and pldζ2, show differential sensitivities to hypoxia stimulus. In the present study, we used protoplasts of wild type and mutants and compared the hypoxia-induced changes in the levels of three major signaling mediators such as cytoplasmic free calcium [Ca2+cyt. ], hydrogen peroxide (H2 O2 ) and PA. The concentrations of cytosolic Ca2+ and H2 O2 were determined by fluorescence microscopy and the fluorescent dyes Fura 2-AM and CM-H2 DCFDA, specific for calcium and H2 O2 , respectively, while PA production was analyzed by an enzymatic method. The study reveals that AtPLDζ1 is involved in reactive oxygen species (ROS) signaling, whereas AtPLDζ2 is involved in cytosolic Ca2+ signaling pathways during hypoxic stress. Hypoxia induces an elevation of PA level both in Wt and pldζ1, while the PA level is unchanged in pldζ2. Thus, it is likely that AtPLDζ2 is involved in PA production by a calcium signaling pathway, while AtPLDζ1 is more important in ROS signaling.

Characteristics of a Regulator of G-Protein Signaling (RGS) rgsC in Aspergillus fumigatus.

The regulator of G-protein signaling (RGS) proteins have a conserved RGS domain that facilitates the intrinsic GTPase activity of an activated Gα subunit of heterotrimeric G protein, thereby attenuating signal transduction. Among six predicted RGS proteins in the opportunistic human pathogenic fungus Aspergillus fumigatus, only three (FlbA, GprK, and Rax1) have been studied. The unexplored RgsC composed of the Phox-associated (PXA), RGS, Phox homology (PX), and Nexin_C superfamily domains is highly conserved in many ascomycete fungi, suggesting a crucial role of RgsC in fungal biology. To address this, we have investigated functions of the rgsC gene. The deletion (Δ) of rgsC causes impaired vegetative growth and asexual development coupled with reduced expression of key developmental regulators. Moreover, ΔrgsC results in accelerated and elevated conidial germination regardless of the presence or absence of an external carbon source. Furthermore, ΔrgsC causes reduced conidial tolerance to oxidative stress. In addition, activities and expression of catalases and superoxide dismutases (SODs) are severely decreased in the ΔrgsC mutant. The deletion of rgsC results in a slight reduction in conidial tolerance to cell wall damaging agents, yet significantly lowered mRNA levels of cell wall integrity/biogenesis transcription factors, indicating that RgsC may function in proper activation of cell wall stress response. The ΔrgsC mutant exhibits defective gliotoxin (GT) production and decreased virulence in the wax moth larvae, Galleria mellonella. Transcriptomic studies reveal that a majority of transporters is down-regulated by ΔrgsC and growth of the ΔrgsC mutant is reduced on inorganic and simple nitrogen medium, suggesting that RgsC may function in external nitrogen source sensing and/or transport. In summary, RgsC is necessary for proper growth, development, stress response, GT production, and external nutrients sensing.