The ER-Localized Transmembrane Protein TMEM39A/SUSR2 Regulates Autophagy by Controlling the Trafficking of the PtdIns(4)P Phosphatase SAC1.

TMEM39A, encoding an ER-localized transmembrane protein, is a susceptibility locus for multiple autoimmune diseases. The molecular function of TMEM39A remains completely unknown. Here we demonstrated that TMEM39A, also called SUSR2, modulates autophagy activity by regulating the spatial distribution and levels of PtdIns(4)P. Depletion of SUSR2 elevates late endosomal/lysosomal PtdIns(4)P levels, facilitating recruitment of the HOPS complex to promote assembly of the SNARE complex for autophagosome maturation. SUSR2 knockdown also increases the degradative capability of lysosomes. Mechanistically, SUSR2 interacts with the ER-localized PtdIns(4)P phosphatase SAC1 and also the COPII SEC23/SEC24 subunits to promote the ER-to-Golgi transport of SAC1. Retention of SAC1 on the ER in SUSR2 knockdown cells increases the level of PtdIns(3)P produced by the VPS34 complex, promoting autophagosome formation. Our study reveals that TMEM39A/SUSR2 acts as an adaptor protein for efficient export of SAC1 from the ER and provides insights into the pathogenesis of diseases associated with TMEM39A mutations.

A novel homeostatic mechanism tunes PI(4,5)P2-dependent signaling at the plasma membrane.

The lipid molecule phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] controls all aspects of plasma membrane (PM) function in animal cells, from its selective permeability to the attachment of the cytoskeleton. Although disruption of PI(4,5)P2 is associated with a wide range of diseases, it remains unclear how cells sense and maintain PI(4,5)P2 levels to support various cell functions. Here, we show that the PIP4K family of enzymes, which synthesize PI(4,5)P2 via a minor pathway, also function as sensors of tonic PI(4,5)P2 levels. PIP4Ks are recruited to the PM by elevated PI(4,5)P2 levels, where they inhibit the major PI(4,5)P2-synthesizing PIP5Ks. Perturbation of this simple homeostatic mechanism reveals differential sensitivity of PI(4,5)P2-dependent signaling to elevated PI(4,5)P2 levels. These findings reveal that a subset of PI(4,5)P2-driven functions might drive disease associated with disrupted PI(4,5)P2 homeostasis.

PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P3 Effector Signaling in Colorectal Cancer.

The canonical view of PI3Kα signaling describes phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) generation and activation of downstream effectors at the plasma membrane or at microtubule-bound endosomes. Here, we show that colorectal cancer (CRC) cell lines exhibit a diverse plasma membrane-nuclear distribution of PI3Kα, controlling corresponding levels of subcellular PtdIns(3,4,5)P3 pools. PI3Kα nuclear translocation was mediated by the importin ß-dependent nuclear import pathway. By PtdIns(3,4,5)P3 affinity capture mass spectrometry done in the presence of SDS on CRC cell lines with PI3Kα nuclear localization, we identified 867 potential nuclear PtdIns(3,4,5)P3 effector proteins. Nuclear PtdIns(3,4,5)P3 interactome proteins were characterized by noncanonical PtdIns(3,4,5)P3-binding domains and showed overrepresentation for nuclear membrane, nucleolus, and nuclear speckles. The nuclear PtdIns(3,4,5)P3 interactome was enriched for proteins related to RNA metabolism, with splicing reporter assays and SC-35 foci staining suggesting a role of epidermal growth factor-stimulated nuclear PI3Kα signaling in modulating pre-mRNA splicing. In patient tumors, nuclear p110α staining was associated with lower T stage and mucinous histology. These results indicate that PI3Kα translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in human CRC, modulating signaling responses.

Phosphatidylinositol (3,5)-bisphosphate machinery regulates neurite thickness through neuron-specific endosomal protein NSG1/NEEP21.

Phosphatidylinositol (3,5)-bisphosphate [PtdIns(3,5)P2] is a critical signaling phospholipid involved in endolysosome homeostasis. It is synthesized by a protein complex composed of PIKfyve, Vac14, and Fig4. Defects in PtdIns(3,5)P2 synthesis underlie a number of human neurological disorders, including Charcot-Marie-Tooth disease, child onset progressive dystonia, and others. However, neuron-specific functions of PtdIns(3,5)P2 remain less understood. Here, we show that PtdIns(3,5)P2 pathway is required to maintain neurite thickness. Suppression of PIKfyve activities using either pharmacological inhibitors or RNA silencing resulted in decreased neurite thickness. We further find that the regulation of neurite thickness by PtdIns(3,5)P2 is mediated by NSG1/NEEP21, a neuron-specific endosomal protein. Knockdown of NSG1 expression also led to thinner neurites. mCherry-tagged NSG1 colocalized and interacted with proteins in the PtdIns(3,5)P2 machinery. Perturbation of PtdIns(3,5)P2 dynamics by overexpressing Fig4 or a PtdIns(3,5)P2-binding domain resulted in mislocalization of NSG1 to nonendosomal locations, and suppressing PtdIns(3,5)P2 synthesis resulted in an accumulation of NSG1 in EEA1-positive early endosomes. Importantly, overexpression of NSG1 rescued neurite thinning in PtdIns(3,5)P2-deficient CAD neurons and primary cortical neurons. Our study uncovered the role of PtdIns(3,5)P2 in the morphogenesis of neurons, which revealed a novel aspect of the pathogenesis of PtdIns(3,5)P2-related neuropathies. We also identified NSG1 as an important downstream protein of PtdIns(3,5)P2, which may provide a novel therapeutic target in neurological diseases.

Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity.

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome "fusion-fission" cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P2 to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.

Autophagic and non-autophagic functions of the Saccharomyces cerevisiae PROPPINs Atg18, Atg21 and Hsv2.

Atg18, Atg21 and Hsv2 are homologous ß-propeller proteins binding to PI3P and PI(3,5)P2. Atg18 is thought to organize lipid transferring protein complexes at contact sites of the growing autophagosome (phagophore) with both the ER and the vacuole. Atg21 is restricted to the vacuole phagophore contact, where it organizes part of the Atg8-lipidation machinery. The role of Hsv2 is less understood, it partly affects micronucleophagy. Atg18 is further involved in regulation of PI(3,5)P2 synthesis. Recently, a novel Atg18-retromer complex and its role in vacuole homeostasis and membrane fission was uncovered.

PI(4,5)P2: signaling the plasma membrane.

In the almost 70 years since the first hints of its existence, the phosphoinositide, phosphatidyl-D-myo-inositol 4,5-bisphosphate has been found to be central in the biological regulation of plasma membrane (PM) function. Here, we provide an overview of the signaling, transport and structural roles the lipid plays at the cell surface in animal cells. These include being substrate for second messenger generation, direct modulation of receptors, control of membrane traffic, regulation of ion channels and transporters, and modulation of the cytoskeleton and cell polarity. We conclude by re-evaluating PI(4,5)P2's designation as a signaling molecule, instead proposing a cofactor role, enabling PM-selective function for many proteins.

Roles of Cholesterol and PtdIns(4,5)P2 in the Regulation of STIM1-Orai1 Channel Function.

Calcium is one of the most prominent second messengers. It is involved in a wide range of functions at the single-cell level but also in modulating regulatory mechanisms in the entire organism. One process mediating calcium signaling involves hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by the phospholipase-C (PLC). Thus, calcium and PtdIns(4,5)P2 are intimately intertwined two second-messenger cascades that often depend on each other. Another relevant lipid associated with calcium signaling is cholesterol. Both PtdIns(4,5)P2 and cholesterol play key roles in the formation and maintenance of specialized signaling nanodomains known as lipid rafts. Lipid rafts are particularly important in calcium signaling by concentrating and localizing calcium channels such as the Orai1 channel. Depletion of internal calcium stores is initiated by the production of inositol-1,4,5-trisphosphate (IP3). Calcium depletion from the ER induces the oligomerization of STIM1, which binds Orai1 and initiates calcium influx into the cell. In the present review, we analyzed the complex interactions between cholesterol, PtdIns(4,5)P2, and the complex formed by the Orai1 channel and the signaling molecule STIM1. We explore some of the complex mechanisms governing calcium homeostasis and phospholipid metabolism, as well as the interaction between these two apparently independent signaling cascades.

Phafins Are More Than Phosphoinositide-Binding Proteins.

Phafins are PH (Pleckstrin Homology) and FYVE (Fab1, YOTB, Vac1, and EEA1) domain-containing proteins. The Phafin protein family is classified into two groups based on their sequence homology and functional similarity: Phafin1 and Phafin2. This protein family is unique because both the PH and FYVE domains bind to phosphatidylinositol 3-phosphate [PtdIns(3)P], a phosphoinositide primarily found in endosomal and lysosomal membranes. Phafin proteins act as PtdIns(3)P effectors in apoptosis, endocytic cargo trafficking, and autophagy. Additionally, Phafin2 is recruited to macropinocytic compartments through coincidence detection of PtdIns(3)P and PtdIns(4)P. Membrane-associated Phafins serve as adaptor proteins that recruit other binding partners. In addition to the phosphoinositide-binding domains, Phafin proteins present a poly aspartic acid motif that regulates membrane binding specificity. In this review, we summarize the involvement of Phafins in several cellular pathways and their potential physiological functions while highlighting the similarities and differences between Phafin1 and Phafin2. Besides, we discuss research perspectives for Phafins.

Lysosomal size matters.

Lysosomes are key cellular catabolic centers that also perform fundamental metabolic, signaling and quality control functions. Lysosomes are not static and they respond dynamically to intra- and extracellular stimuli triggering changes in organelle numbers, size and position. Such physical changes have a strong impact on lysosomal activity ultimately influencing cellular homeostasis. In this review, we summarize the current knowledge on lysosomal size regulation, on its physiological role(s) and association to several disease conditions.

Phosphatidylinositol 4-phosphate: a key determinant of plasma membrane identity and function in plants.

Phosphatidylinositol 4-phosphate (PI4P) is an anionic phospholipid which has been described as a master regulator of the Golgi apparatus in eukaryotic cells. However, recent evidence suggests that PI4P mainly accumulates at the plasma membrane in all plant cells analyzed so far. In addition, many functions that are typically attributed to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) in animal and yeast cells are also supported by PI4P in plants. For example, PI4P is the key anionic lipid that powers the strong electrostatic properties of the plasma membrane. Phosphatidylinositol 4-phosphate is also required for the establishment of stable membrane contacts between the endoplasmic reticulum and the plasma membrane, for exocytosis and to support signaling pathways. Thus, we propose that PI4P has a prominent role in specifying the identity of the plasma membrane and in supporting some of its key functions and should be considered a hallmark lipid of this compartment.

Decreased FAK activity and focal adhesion dynamics impair proper neurite formation of medium spiny neurons in Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion in the protein huntingtin (HTT) [55]. While the final pathological consequence of HD is the neuronal cell death in the striatum region of the brain, it is still unclear how mutant HTT (mHTT) causes synaptic dysfunctions at the early stage and during the progression of HD. Here, we discovered that the basal activity of focal adhesion kinase (FAK) is severely reduced in a striatal HD cell line, a mouse model of HD, and the human post-mortem brains of HD patients. In addition, we observed with a FRET-based FAK biosensor [59] that neurotransmitter-induced FAK activation is decreased in HD striatal neurons. Total internal reflection fluorescence (TIRF) imaging revealed that the reduced FAK activity causes the impairment of focal adhesion (FA) dynamics, which further leads to the defect in filopodial dynamics causing the abnormally increased number of immature neurites in HD striatal neurons. Therefore, our results suggest that the decreased FAK and FA dynamics in HD impair the proper formation of neurites, which is crucial for normal synaptic functions [52]. We further investigated the molecular mechanism of FAK inhibition in HD and surprisingly discovered that mHTT strongly associates with phosphatidylinositol 4,5-biphosphate, altering its normal distribution at the plasma membrane, which is crucial for FAK activation [14, 60]. Therefore, our results provide a novel molecular mechanism of FAK inhibition in HD along with its pathological mechanism for synaptic dysfunctions during the progression of HD.

Polarized PtdIns(4,5)P2 distribution mediated by a voltage-sensing phosphatase (VSP) regulates sperm motility.

The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P2 Sperm from VSP-/- mice show more Ca2+ influx upon capacitation than VSP+/- mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P2-sensitive K+ channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca2+ influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P2 in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P2 distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P2 distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P2 distribution plays important roles in ion channel regulation as well as sperm motility.

The Xanthomonas type III effector XopAP prevents stomatal closure by interfering with vacuolar acidification.

Plant stomata close rapidly in response to a rise in the plant hormone abscisic acid (ABA) or salicylic acid (SA) and after recognition of pathogen-associated molecular patterns (PAMPs). Stomatal closure is the result of vacuolar convolution, ion efflux, and changes in turgor pressure in guard cells. Phytopathogenic bacteria secrete type III effectors (T3Es) that interfere with plant defense mechanisms, causing severe plant disease symptoms. Here, we show that the virulence and infection of Xanthomonas oryzae pv. oryzicola (Xoc), which is the causal agent of rice bacterial leaf streak disease, drastically increased in transgenic rice (Oryza sativa L.) plants overexpressing the Xoc T3E gene XopAP, which encodes a protein annotated as a lipase. We discovered that XopAP binds to phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ), a membrane phospholipid that functions in pH control in lysosomes, membrane dynamics, and protein trafficking. XopAP inhibited the acidification of vacuoles by competing with vacuolar H+ -pyrophosphatase (V-PPase) for binding to PtdIns(3,5)P2 , leading to stomatal opening. Transgenic rice overexpressing XopAP also showed inhibition of stomatal closure when challenged by Xoc infection and treatment with the PAMP flg22. Moreover, XopAP suppressed flg22-induced gene expression, reactive oxygen species burst and callose deposition in host plants, demonstrating that XopAP subverts PAMP-triggered immunity during Xoc infection. Taken together, these findings demonstrate that XopAP overcomes stomatal immunity in plants by binding to lipids.

The acyl chains of phosphoinositide PIP3 alter the structure and function of nuclear receptor steroidogenic factor-1.

Nuclear receptors are transcription factors that bind lipids, an event that induces a structural conformation of the receptor that favors interaction with transcriptional coactivators. The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) binds the signaling phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), and our previous crystal structures showed how the phosphoinositide headgroups regulate SF-1 function. However, what role the acyl chains play in regulating SF-1 structure remains unaddressed. Here, we used X-ray crystallography with in vitro binding and functional assays to examine how the acyl chains of PIP3 regulate human SF-1 ligand-binding domain structure and function. Altering acyl chain length and unsaturation regulates apparent binding of all tested phosphoinositides to SF-1. Mass spectrometry-based lipidomics data suggest C16 and C18 phospholipids preferentially associate with SF-1 expressed ectopically in bacteria. We then solved the 2.5 Å crystal structure of SF-1 bound to dioleoyl PIP3(18:1/18:1) to compare it with a matched structure of SF-1 bound to dipalmitoyl PIP3(16:0/16:0). The dioleoyl-bound structure was severely disordered in a specific SF-1 region associated with pathogenic human polymorphisms and within the coactivator-binding region critical for SF-1 function while inducing increased sensitivity to protease digestion in solution. Validating these structural observations, in vitro functional studies showed dioleoyl PIP3 induced 6-fold poorer affinity of a peroxisome proliferator-activated receptor gamma coactivator 1-alpha coactivator peptide for SF-1 compared with dipalmitoyl PIP3. Together, these data suggest the chemical nature of the phosphoinositide acyl chains controls the ordered state of specific, clinically important structural regions in SF-1, regulating SF-1 function in vitro.

Sphingolipid metabolic flow controls phosphoinositide turnover at the trans-Golgi network.

Sphingolipids are membrane lipids globally required for eukaryotic life. The sphingolipid content varies among endomembranes with pre- and post-Golgi compartments being poor and rich in sphingolipids, respectively. Due to this different sphingolipid content, pre- and post-Golgi membranes serve different cellular functions. The basis for maintaining distinct subcellular sphingolipid levels in the presence of membrane trafficking and metabolic fluxes is only partially understood. Here, we describe a homeostatic regulatory circuit that controls sphingolipid levels at the trans-Golgi network (TGN). Specifically, we show that sphingomyelin production at the TGN triggers a signalling pathway leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol and sphingolipid transport to the trans-Golgi network, PtdIns(4)P consumption interrupts this transport in response to excessive sphingomyelin production. Based on this evidence, we envisage a model where this homeostatic circuit maintains a constant lipid composition in the trans-Golgi network and post-Golgi compartments, thus counteracting fluctuations in the sphingolipid biosynthetic flow.

The phosphoinositide phosphatase Sac1 regulates cell shape and microtubule stability in the developing Drosophila eye.

Epithelial patterning in the developing Drosophila melanogaster eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). Here, using a novel temperature-sensitive (ts) allele, we show that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. Sac1ts mutants have rough eyes and retinal patterning defects that resemble rst mutants. Sac1ts retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in Sac1ts mutants. However, Sac1ts mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation ex vivo Together, our data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner.

NCP2/RHD4/SAC7, SAC6 and SAC8 phosphoinositide phosphatases are required for PtdIns4P and PtdIns(4,5)P2 homeostasis and Arabidopsis development.

Phosphoinositides play important roles in plant growth and development. Several SAC domain phosphoinositide phosphatases have been reported to be important for plant development. Here, we show functional analysis of SUPPRESSOR OF ACTIN 6 (SAC6) to SAC8 in Arabidopsis, a subfamily of phosphoinositide phosphatases containing SAC-domain and two transmembrane motifs. We isolated an Arabidopsis mutant ncp2 that lacked cotyledons in seedling and embryo in pid, a background defective in auxin signaling and transport. NCP2 encodes RHD4/SAC7 phosphoinositide phosphatase. SAC6, SAC7 and SAC8 exhibit overlapping and specific expression patterns in seedling and embryo. The sac6 sac7 embryos either fail to develop into seeds, or have three or four cotyledons. The embryo development of sac7 sac8 and sac6 sac7 sac8 mutants is significantly delayed or lethal, and the seedlings are arrested at early stages. Auxin maxima are decreased in double and triple sac mutants. The contents of PtdIns4P and PtdIns(4,5)P2 in sac6 sac7 and sac7 sac8 mutants are dramatically increased. Protein trafficking of the plasma membrane (PM)-localized protein PIN1 and PIN2 from trans-Golgi network/early endosome back to PM is delayed in sac7 sac8 mutants. These results indicate that SAC6-SAC8 are essential for maintaining homeostasis of PtdIns4P and PtdIns(4,5)P2, and auxin-mediated development in Arabidopsis.

Identification of Key Amino Acid Residues Involved in the Localization of Sorting Nexin 10 and Induction of Vacuole Formation.

Sorting nexin 10 (SNX10) induces formation of vacuoles participating in the endosome morphogenesis in mammalian cells, but the key amino acids involved in this function have not been fully identified. In this study, point mutations were introduced to the conserved region of the SNX10 PX domain to elucidate the function of these key amino acid residues. The number of vacuoles in the R53A mutant was partially decreased, while the R52A and R51A mutants completely lacked the vacuoles. All mutant proteins lost the phosphatidylinositol 3-phosphate (PtdIns3P)-binding ability and endosomal localization. Retargeting the mutants to the endosomes rescued partially or fully the vacuole-inducing ability in the R51A and R53A mutants, respectively, but not in the R52A mutant. No vacuoles were induced when the R51A mutant was targeted to other organelles. Structural analysis showed that Arg53 is responsible for the PtdIns(3)P binding, whereas Arg51 and Arg52 contribute to the structural integrity of SNX10. We conclude that the disruption of the key residues affects the structure and function of SNX10 and that induction of vacuole formation by SNX10 depends on its endosomal location.

Deliver on Time or Pay the Fine: Scheduling in Membrane Trafficking.

Membrane trafficking is all about time. Automation in such a biological process is crucial to ensure management and delivery of cellular cargoes with spatiotemporal precision. Shared molecular regulators and differential engagement of trafficking components improve robustness of molecular sorting. Sequential recruitment of low affinity protein complexes ensures directionality of the process and, concomitantly, serves as a kinetic proofreading mechanism to discriminate cargoes from the whole endocytosed material. This strategy helps cells to minimize losses and operating errors in membrane trafficking, thereby matching the appealed deadline. Here, we summarize the molecular pathways of molecular sorting, focusing on their timing and efficacy. We also highlight experimental procedures and genetic approaches to robustly probe these pathways, in order to guide mechanistic studies at the interface between biochemistry and quantitative biology.