Type II phosphatidylinositol 4-kinases promote Listeria monocytogenes entry into target cells

Interaction of the Listeria surface protein InlB with the hepatocyte growth factor receptor Met activates signalling events that trigger bacterial internalization into mammalian epithelial cells. We show here that purified phagosomes containing InlB-coated beads display type II phosphatidylinositol 4-kinase (PI4K) activity. In human epithelial HeLa cells, both PI4KIIalpha and PI4KIIbeta isoforms are corecruited with Met around InlB-coated beads or wild-type Listeria during the early steps of internalization, and phosphatidylinositol 4-phosphate [PI(4)P] is detected at the entry site. We demonstrate that PI4KIIalpha or PI4KIIbeta knockdown, but not type III PI4Kbeta knockdown, inhibits Listeria internalization. Production of PI(4)P derivatives such as phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P(3)] upon InlB stimulation is not affected by PI4KIIalpha or beta knockdown, suggesting that these phosphoinositides are generated by a type III PI4K. Strikingly, knockdown of the PI(4)P ligand and clathrin adaptor AP-1 strongly inhibits bacterial entry. Together, our results reveal a yet non-described role for type II PI4Ks in phagocytosis.     

Distinct Golgi populations of phosphatidylinositol 4-phosphate regulated by phosphatidylinositol 4-kinases

Phosphatidylinositol 4-phosphate (PI4P) regulates biosynthetic membrane traffic at multiple steps and differentially affects the surface delivery of apically and basolaterally destined proteins in polarized cells. Two phosphatidylinositol 4-kinases (PI4Ks) have been localized to the Golgi complex in mammalian cells, type III PI4Kbeta (PI4KIIIbeta) and type II PI4Kalpha (PI4KIIalpha). Here we report that PI4KIIIbeta and PI4KIIalpha localize to discrete subcompartments of the Golgi complex in Madin-Darby canine kidney (MDCK) cells. PI4KIIIbeta was enriched in early Golgi compartments, whereas PI4KIIalpha colocalized with markers of the trans-Golgi network (TGN). To understand the temporal and spatial control of PI4P generation across the Golgi complex, we quantitated the steady state distribution of a fluorescent PI4P-binding domain relative to cis/medial Golgi and TGN markers in transiently transfected MDCK cells. The density of the signal from this PI4P reporter was roughly 2-fold greater in the early Golgi compartments compared with that of the TGN. Furthermore, this ratio could be modulated in vivo by overexpression of catalytically inactive PI4KIIIbeta and PI4KIIalpha or in vitro by the PI4KIIIbeta inhibitor wortmannin. Our data suggest that both PI4KIIIbeta and PI4KIIalpha contribute to the compartmental regulation of PI4P synthesis within the Golgi complex. We discuss our results with respect to the kinetic effects of modulating PI4K activity on polarized biosynthetic traffic in MDCK cells.     

Type II phosphatidylinositol 4-kinase beta is a cytosolic and peripheral membrane protein that is recruited to the plasma membrane and activated by Rac-GTP

Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells.     

Phosphatidylinositol-4-kinase type II alpha contains an AP-3-sorting motif and a kinase domain that are both required for endosome traffic

The adaptor complex 3 (AP-3) targets membrane proteins from endosomes to lysosomes, lysosome-related organelles and synaptic vesicles. Phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha) is one of several proteins possessing catalytic domains that regulate AP-3-dependent sorting. Here we present evidence that PI4KIIalpha uniquely behaves both as a membrane protein cargo as well as an enzymatic regulator of adaptor function. In fact, AP-3 and PI4KIIalpha form a complex that requires a dileucine-sorting motif present in PI4KIIalpha. Mutagenesis of either the PI4KIIalpha-sorting motif or its kinase-active site indicates that both are necessary to interact with AP-3 and properly localize PI4KIIalpha to LAMP-1-positive endosomes. Similarly, both the kinase activity and the sorting signal present in PI4KIIalpha are necessary to rescue endosomal PI4KIIalpha siRNA-induced mutant phenotypes. We propose a mechanism whereby adaptors use canonical sorting motifs to selectively recruit a regulatory enzymatic activity to restricted membrane domains.     

Lipid and peptide control of phosphatidylinositol 4-kinase IIalpha activity on Golgi-endosomal Rafts

The most abundant and widely expressed mammalian phosphoinositide kinase activity is contributed by phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha). In this study we demonstrate that PI4KIIalpha is a novel GTP-independent target of the wasp venom tetradecapeptide mastoparan and that different mechanisms of activation occur in different subcellular membranes. Following cell membrane fractionation mastoparan specifically stimulated a high activity Golgi/endosomal pool of PI4KIIalpha independently of exogenous guanine nucleotides. Conversely, GTPgammaS stimulated a low activity pool of PI4KIIalpha in a separable dense membrane fraction and this response was further enhanced by mastoparan. Overexpression of PI4KIIalpha increased the basal phosphatidylinositol 4-kinase activity of each membrane pool, as well as the mastoparan-dependent activities, thereby demonstrating that mastoparan specifically activates this isozyme. Both mastoparan and M7, at concentrations known to invoke secretion, stimulated PI4KIIalpha with similar efficacies, resulting in an increase in the apparent V(max) and decrease in K(m) for exogenously added PI. Mastoparan also stimulated PI4KIIalpha immunoprecipitated from the raft fraction, indicating that PI4KIIalpha is a direct target of mastoparan. Finally we reveal a striking dependence of both basal and mastoparan-stimulated PI4KIIalpha activity on endogenous cholesterol concentration and therefore conclude that changes in membrane environment can regulate PI4KIIalpha activity.     

Phosphatidylinositol-4-kinase type II alpha is a component of adaptor protein-3-derived vesicles

A membrane fraction enriched in vesicles containing the adaptor protein (AP) -3 cargo zinc transporter 3 was generated from PC12 cells and was used to identify new components of these organelles by mass spectrometry. Proteins prominently represented in the fraction included AP-3 subunits, synaptic vesicle proteins, and lysosomal proteins known to be sorted in an AP-3-dependent way or to interact genetically with AP-3. A protein enriched in this fraction was phosphatidylinositol-4-kinase type IIalpha (PI4KIIalpha). Biochemical, pharmacological, and morphological analyses supported the presence of PI4KIIalpha in AP-3-positive organelles. Furthermore, the subcellular localization of PI4KIIalpha was altered in cells from AP-3-deficient mocha mutant mice. The PI4KIIalpha normally present both in perinuclear and peripheral organelles was substantially decreased in the peripheral membranes of AP-3-deficient mocha fibroblasts. In addition, as is the case for other proteins sorted in an AP-3-dependent way, PI4KIIalpha content was strongly reduced in nerve terminals of mocha hippocampal mossy fibers. The functional relationship between AP-3 and PI4KIIalpha was further explored by PI4KIIalpha knockdown experiments. Reduction of the cellular content of PI4KIIalpha strongly decreased the punctate distribution of AP-3 observed in PC12 cells. These results indicate that PI4KIIalpha is present on AP-3 organelles where it regulates AP-3 function.     

PI4P promotes the recruitment of the GGA adaptor proteins to the trans-Golgi network and regulates their recognition of the ubiquitin sorting signal

Phosphatidylinositol 4 phosphate (PI4P) is highly enriched in the trans-Golgi network (TGN). Here we establish that PI4P is a key regulator of the recruitment of the GGA clathrin adaptor proteins to the TGN and that PI4P has a novel role in promoting their recognition of the ubiquitin (Ub) sorting signal. Knockdown of PI4KIIalpha by RNA interference (RNAi), which depletes the TGN's PI4P, impaired the recruitment of the GGAs to the TGN. GGAs bind PI4P primarily through their GAT domain, in a region called C-GAT, which also binds Ub but not Arf1. We identified two basic residues in the GAT domain that are essential for PI4P binding in vitro and for the recruitment of GGAs to the TGN in vivo. Unlike wild-type GGA, GGA with mutated GATs failed to rescue the abnormal TGN phenotype of the GGA RNAi-depleted cells. These residues partially overlap with those that bind Ub, and PI4P increased the affinity of the GAT domain for Ub. Because the recruitment of clathrin adaptors and their cargoes to the TGN is mediated through a web of low-affinity interactions, our results show that the dual roles of PI4P can promote specific GGA targeting and cargo recognition at the TGN.     

Dual control of membrane targeting by PtdIns(4)P and ARF

In mammalian cells, three types of phosphatidylinositol 4-kinase (PI4K) are associated with the Golgi complex, where their product, phosphatidylinositol 4-phosphate [PtdIns(4)P], is concentrated. The role of PtdIns(4)P in this compartment and how the PtdIns(4)P-positive membrane domain is formed and maintained despite continuous membrane flow are, however, poorly understood. Recent work has shown that PtdIns(4)P and the small GTPase ARF1 function cooperatively in the recruitment of four-phosphate adaptor proteins (FAPPs) to the trans-Golgi network (TGN) and has implicated FAPPs in formation of the membrane domain and in post-Golgi trafficking.     

A plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4-kinase type-III alpha: studies with the PH domains of the oxysterol binding protein and FAPP1

The PH domains of OSBP and FAPP1 fused to GFP were used to monitor PI(4)P distribution in COS-7 cells during manipulations of PI 4-kinase (PI4K) activities. Both domains were associated with the Golgi and small cytoplasmic vesicles, and a small fraction of OSBP-PH was found at the plasma membrane (PM). Inhibition of type-III PI4Ks with 10 microM wortmannin (Wm) significantly reduced but did not abolish Golgi localization of either PH domains. Downregulation of PI4KIIalpha or PI4KIIIbeta by siRNA reduced the localization of the PH domains to the Golgi and in the former case any remaining Golgi localization was eliminated by Wm treatment. PLC activation by Ca2+ ionophores dissociated the domains from all membranes, but after Ca2+ chelation, they rapidly reassociated with the Golgi, the intracellular vesicles and with the PM. PM association of the domains was significantly higher after the Ca2+ transient and was abolished by Wm pretreatment. PM relocalization was not affected by down-regulation of PI4KIIIbeta or -IIalpha, but was inhibited by down-regulation of PI4KIIIalpha, or by 10 microM PAO, which also inhibits PI4KIIIalpha. Our data suggest that these PH domains detect PI(4)P formation in extra-Golgi compartments under dynamic conditions and that various PI4Ks regulate PI(4)P synthesis in distinct cellular compartments.     

A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within mu2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant mu2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant mu2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to mu2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.     

Recruitment of arfaptins to the trans‐Golgi network by PI(4)P and their involvement in cargo export

The BAR (Bin/Amphiphysin/Rvs) domain proteins arfaptin1 and arfaptin2 are localized to the trans‐Golgi network (TGN) and, by virtue of their ability to sense and/or generate membrane curvature, could play an important role in the biogenesis of transport carriers. We report that arfaptins contain an amphipathic helix (AH) preceding the BAR domain, which is essential for their binding to phosphatidylinositol 4‐phosphate (PI(4)P)‐containing liposomes and the TGN of mammalian cells. The binding of arfaptin1, but not arfaptin2, to PI(4)P is regulated by protein kinase D (PKD) mediated phosphorylation at Ser100 within the AH. We also found that only arfaptin1 is required for the PKD‐dependent trafficking of chromogranin A by the regulated secretory pathway. Altogether, these findings reveal the importance of PI(4)P and PKD in the recruitment of arfaptins at the TGN and their requirement in the events leading to the biogenesis of secretory storage granules.     

Arf1 directly recruits the Pik1-Frq1 PI4K complex to regulate the final stages of Golgi maturation

Proper Golgi complex function depends on the activity of Arf1, a GTPase whose effectors assemble and transport outgoing vesicles. Phosphatidylinositol 4-phosphate (PI4P) generated at the Golgi by the conserved PI 4-kinase Pik1 (PI4KIIIβ) is also essential for Golgi function, although its precise roles in vesicle formation are less clear. Arf1 has been reported to regulate PI4P production, but whether Pik1 is a direct Arf1 effector is not established. Using a combination of live-cell time-lapse imaging analyses, acute PI4P depletion experiments, and in vitro protein–protein interaction assays on Golgi-mimetic membranes, we present evidence for a model in which Arf1 initiates the final stages of Golgi maturation by tightly controlling PI4P production through direct recruitment of the Pik1-Frq1 PI4-kinase complex. This PI4P serves as a critical signal for AP-1 and secretory vesicle formation, the final events at maturing Golgi compartments. This work therefore establishes the regulatory and temporal context surrounding Golgi PI4P production and its precise roles in Golgi maturation.     

Spatial Regulation of Golgi Phosphatidylinositol‐4‐Phosphate is Required for Enzyme Localization and Glycosylation Fidelity

The enrichment of phosphatidylinositol‐4‐phosphate (PI(4)P) at the trans Golgi network (TGN) is instrumental for proper protein and lipid sorting, yet how the restricted distribution of PI(4)P is achieved remains unknown. Here, we show that lipid phosphatase Suppressor of actin mutations 1 (SAC1) is crucial for the spatial regulation of Golgi PI(4)P. Ultrastructural analysis revealed that SAC1 is predominantly located at cisternal Golgi membranes but is absent from the TGN, thus confining PI(4)P to the TGN. RNAi‐mediated knockdown of SAC1 caused changes in Golgi morphology and mislocalization of Golgi enzymes. Enzymes involved in glycan processing such as mannosidase‐II (Man‐II) and N‐acetylglucosamine transferase‐I (GnT‐I) redistributed to aberrant intracellular structures and to the cell surface in SAC1 knockdown cells. SAC1 depletion also induced a unique pattern of Golgi‐specific defects in N‐and O‐linked glycosylation. These results indicate that SAC1 organizes PI(4)P distribution between the Golgi complex and the TGN, which is instrumental for resident enzyme partitioning and Golgi morphology.     

NPC1 regulates the distribution of phosphatidylinositol 4‐kinases at Golgi and lysosomal membranes

Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann‐Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4‐phosphate (PtdIns4P) countertransport cycle between Golgi‐endoplasmic reticulum (ER), as well as lysosome‐ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4‐kinases—PI4KIIα and PI4KIIIβ—which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.     

Phosphatidylinositol 4-kinase IIIbeta regulates the transport of ceramide between the endoplasmic reticulum and Golgi

The recently identified ceramide transfer protein, CERT, is responsible for the bulk of ceramide transport from the endoplasmic reticulum (ER) to the Golgi. CERT has a C-terminal START domain for ceramide binding and an N-terminal pleck-strin homology domain that binds phosphatidylinositol 4-phosphate suggesting that phosphatidylinositol (PI) 4-kinases are involved in the regulation of CERT-mediated ceramide transport. In the present study fluorescent analogues were used to follow the ER to Golgi transport of ceramide to determine which of the four mammalian PI 4-kinases are involved in this process. Overexpression of pleckstrin homology domains that bind phosphatidylinositol 4-phosphate strongly inhibited the transport of C5-BODIPY-ceramide to the Golgi. A newly identified PI 3-kinase inhibitor, PIK93 that selectively inhibits the type III PI 4-kinase beta enzyme, and small interfering RNA-mediated down-regulation of the individual PI 4-kinase enzymes, revealed that PI 4-kinase beta has a dominant role in ceramide transport between the ER and Golgi. Accordingly, inhibition of PI 4-kinase III beta either by wortmannin or PIK93 inhibited the conversion of [3H]serine-labeled endogenous ceramide to sphingomyelin. Therefore, PI 4-kinase beta is a key enzyme in the control of spingomyelin synthesis by controlling the flow of ceramide from the ER to the Golgi compartment.     

Rag GTPases and phosphatidylinositol 3-phosphate mediate recruitment of the AP-5/SPG11/SPG15 complex

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation.     

Multiple roles for phosphatidylinositol 4-kinase in biosynthetic transport in polarized Madin-Darby canine kidney cells.

Phosphatidylinositols (PI) play important roles in regulating numerous cellular processes including cytoskeletal organization and membrane trafficking. The control of PI metabolism by phosphatidylinositol kinases has been the subject of extensive investigation; however, little is known about how phosphatidylinositol kinases regulate traffic in polarized epithelial cells. Because phosphatidylinositol 4-kinase (PI4K)-mediated phosphatidylinositol 4-phosphate (PI(4)P) production has been suggested to regulate biosynthetic traffic in yeast and mammalian cells, we have examined the role of PI4Kbeta in protein delivery in polarized MDCK cells, at different levels of the biosynthetic pathway. Expression of wild type PI4Kbeta had no effect on the rate of transport of influenza hemagglutinin (HA) through the Golgi complex, but inhibited the rate of trans-Golgi network (TGN)-to-cell surface delivery of this protein. By contrast, expression of dominant-negative, kinase-dead PI4Kbeta (PI4Kbeta(D656A)) inhibited intra-Golgi transport but stimulated TGN-to-cell surface delivery of HA. Moreover, expression of PI4Kbeta(D656A) significantly increased the solubility in cold Triton X-100 of HA staged in the TGN, suggesting that altered association of HA with lipid rafts may be responsible for the enhanced transport rate. Both wild type and kinase-dead PI4Kbeta inhibited basolateral delivery of vesicular stomatitis virus G protein, suggesting an effector function for PI4Kbeta in the regulation of basolateral traffic. Thus, by contrast with the observed requirement for PI4Kbeta activity and PI(4)P for efficient transport in yeast, our data suggest that changes in PI(4)P levels can stimulate and inhibit Golgi to cell surface delivery in mammalian cells.     

Interaction of neuronal calcium sensor-1 and ADP-ribosylation factor 1 allows bidirectional control of phosphatidylinositol 4-kinase beta and trans-Golgi network-plasma membrane traffic

We have identified a novel Ca(2+)-dependent interaction between neuronal calcium sensor-1 (NCS-1) and the GTPase ARF1. Both of these proteins are localized to the Golgi complex, and both regulate phosphatidylinositol 4-kinase IIIbeta (PI(4)Kbeta). Spatial and temporal control of phosphatidylinositol 4-phosphate levels through activation of PI(4)Kbeta is important for the recruitment of trafficking complexes to the trans-Golgi network (TGN) and vesicular traffic from this organelle. The NCS-1-ARF1 interaction and its specificity have been demonstrated through in vitro binding assays, in vitro enzyme assay, and through functional cellular assays. We show that NCS-1 can exert bidirectional effects to activate PI(4)Kbeta on its own or inhibit the activation by ARF1. NCS-1 was shown to modulate the effects of expression of ARF mutants that disrupt Golgi morphology and to recruit GDP-loaded ARF to the Golgi complex in a Ca(2+)-dependent manner. We demonstrate antagonist effects of NCS-1 and ARF on constitutive and regulated exocytosis. The NCS-1-ARF1 interaction provides evidence for functional cross-talk between Ca(2+)-dependent and ARF-dependent pathways in TGN to plasma membrane traffic.     

Neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta regulate IgE receptor-triggered exocytosis in cultured mast cells

We examined the possible occurrence and function of neuronal Ca(2+) sensor 1 (NCS-1/frequenin) in the mast cell line rat basophilic leukemia, RBL-2H3. This protein has been implicated in the control of neurosecretion from dense core granules in neuronal cells as well as in the control of constitutive secretory pathways in both yeast and mammalian cells. We show that RBL-2H3 cells, secretory cells of the immune system, endogenously express the 22-kDa NCS-1 protein as well as an immune-related 50-kDa protein. Both proteins associate in vivo with phosphatidylinositol 4-kinase beta (PI4Kbeta) and colocalize with the enzyme in the Golgi region. We show further that overexpression of NCS-1 in RBL-2H3 cells stimulates the catalytic activity of PI4Kbeta, increases IgE receptor (FcepsilonRI)-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), and stimulates FcepsilonRI-triggered, but not Ca(2+) ionophore-triggered, exocytosis. Conversely, expression of a kinase-dead mutant of PI4Kbeta reduces PI4Kbeta activity, decreases FcepsilonRI-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, and blocks FcepsilonRI-triggered, but not Ca(2+) ionophore-triggered, exocytosis. Our results indicate that PI(4)P, produced by the Golgi-localized PI4Kbeta, is the rate-limiting factor in the synthesis of the pool of PI(4,5)P(2) that serves as substrate for the generation of lipid-derived second messengers in FcepsilonRI-triggered cells. We conclude that NCS-1 is involved in the control of regulated exocytosis in nonneural cells, where it contributes to stimulus-secretion coupling by interacting with PI4Kbeta and positive regulation of its activity.     

Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain–containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.