The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)] is a key second messenger that regulates actin and membrane dynamics, as well as other cellular processes. Many of the effects of PtdIns(4,5)P(2) are mediated by binding to effector proteins that contain a pleckstrin homology (PH) domain. Here, we identify two novel effectors of PtdIns(4,5)P(2) in the budding yeast Saccharomyces cerevisiae: the PH domain containing protein Slm1 and its homolog Slm2. Slm1 and Slm2 serve redundant roles essential for cell growth and actin cytoskeleton polarization. Slm1 and Slm2 bind PtdIns(4,5)P(2) through their PH domains. In addition, Slm1 and Slm2 physically interact with Avo2 and Bit61, two components of the TORC2 signaling complex, which mediates Tor2 signaling to the actin cytoskeleton. Together, these interactions coordinately regulate Slm1 targeting to the plasma membrane. Our results thus identify two novel effectors of PtdIns(4,5)P(2) regulating cell growth and actin organization and suggest that Slm1 and Slm2 integrate inputs from the PtdIns(4,5)P(2) and TORC2 to modulate polarized actin assembly and growth.     

The yeast PH domain proteins Slm1 and Slm2 are targets of sphingolipid signaling during the response to heat stress

The PH domain-containing proteins Slm1 and Slm2 were previously identified as effectors of the phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)) and TORC2 signaling pathways. Here, we demonstrate that Slm1 and Slm2 are also targets of sphingolipid signaling during the heat shock response. We show that upon depletion of cellular sphingolipid levels, Slm1 function becomes essential for survival under heat stress. We further demonstrate that Slm proteins are regulated by a phosphorylation/dephosphorylation cycle involving the sphingolipid-activated protein kinases Pkh1 and Pkh2 and the calcium/calmodulin-dependent protein phosphatase calcineurin. By using a combination of mass spectrometry and mutational analysis, we identified serine residue 659 in Slm1 as a site of phosphorylation. Characterization of Slm1 mutants that mimic dephosphorylated and phosphorylated states demonstrated that phosphorylation at serine 659 is vital for survival under heat stress and promotes the proper polarization of the actin cytoskeleton. Finally, we present evidence that Slm proteins are also required for the trafficking of the raft-associated arginine permease Can1 to the plasma membrane, a process that requires sphingolipid synthesis and actin polymerization. Together with previous work, our findings suggest that Slm proteins are subject to regulation by multiple signals, including PI4,5P(2), TORC2, and sphingolipids, and may thus integrate inputs from different signaling pathways to temporally and spatially control actin polarization.     

Genome-wide lethality screen identifies new PI4,5P2 effectors that regulate the actin cytoskeleton

To further understand the roles played by the essential phosphoinositide PI4,5P(2), we have used a synthetic lethal analysis, which systematically combined the mss4(ts) mutation, partially defective in PI4P 5-kinase activity, with each of approximately 4700 deletion mutations. This genomic screening technique uncovered numerous new candidate effectors and regulators of PI4,5P(2) in yeast. In particular, we identified Slm1 (Yil105c), a previously uncharacterized PI4,5P(2) binding protein. Like Mss4, Slm1 and its homolog Slm2 (Ynl047c) were required for actin cytoskeleton polarization and viability. Co-immunoprecipitation experiments revealed that Slm1 interacts with a component of TORC2, a Tor2 kinase-containing complex, which also regulates the actin cytoskeleton. Consistent with these findings, phosphorylation of Slm1 and Slm2 was dependent on TORC2 protein kinase activity, both in vivo and in vitro, and Slm1 localization required both PI4,5P(2) and functional TORC2. Together, these data suggest that Slm1 and Slm2 function downstream of PI4,5P(2) and the TORC2 kinase pathway to control actin cytoskeleton organization.     

Stt4 PI 4-kinase localizes to the plasma membrane and functions in the Pkc1-mediated MAP kinase cascade

Production of the essential phospholipid PI4P at the Golgi by the Pik1 kinase is required for protein secretion, while a distinct pool of PI4P generated by the Stt4 kinase is critical for normal actin cytoskeleton organization. We identify a transmembrane protein that stabilizes Stt4 at the plasma membrane where it directs synthesis of PI4P, which is required for activation of the Rho1/Pkc1-mediated MAP kinase cascade. Inactivation of Stt4 or the PI4P 5-kinase Mss4 results in mislocalization of the Rho-GTPase GEF Rom2. Rom2 binds PI4,5P(2) through its PH domain and represents the first identified effector in the Stt4-Mss4 pathway. Based on these results, we propose that Stt4-Mss4 generates PI4,5P(2) at the plasma membrane, required to recruit/activate effector proteins such as Rom2.     

Assembly of the PtdIns 4-kinase Stt4 complex at the plasma membrane requires Ypp1 and Efr3

The phosphoinositide phosphatidylinositol 4-phosphate (PtdIns4P) is an essential signaling lipid that regulates secretion and polarization of the actin cytoskeleton. In Saccharomyces cerevisiae, the PtdIns 4-kinase Stt4 catalyzes the synthesis of PtdIns4P at the plasma membrane (PM). In this paper, we identify and characterize two novel regulatory components of the Stt4 kinase complex, Ypp1 and Efr3. The essential gene YPP1 encodes a conserved protein that colocalizes with Stt4 at cortical punctate structures and regulates the stability of this lipid kinase. Accordingly, Ypp1 interacts with distinct regions on Stt4 that are necessary for the assembly and recruitment of multiple copies of the kinase into phosphoinositide kinase (PIK) patches. We identify the membrane protein Efr3 as an additional component of Stt4 PIK patches. Efr3 is essential for assembly of both Ypp1 and Stt4 at PIK patches. We conclude that Ypp1 and Efr3 are required for the formation and architecture of Stt4 PIK patches and ultimately PM-based PtdIns4P signaling.     

Phosphatidylinositol 4-phosphate is required for translation initiation in Saccharomyces cerevisiae

The small natural product wortmannin inhibits protein synthesis by modulating several phosphatidylinositol (PI) metabolic pathways. A primary target of wortmannin in yeast is the plasma membrane-associated PI 4-kinase (PI4K) Stt4p, which is required for actin cytoskeleton organization. Here we show that wortmannin treatment or inactivation of Stt4p, but not disorganization of the actin cytoskeleton per se, leads to a rapid attenuation of translation initiation. Interestingly, inactivation of Pik1p, a wortmannin-insensitive, functionally distinct PI4K, implicated in the regulation of Golgi functions and secretion, also results in severe translation initiation defects with a marked increase of the phosphorylation of the translation initiation factor eIF2alpha. Because wortmannin largely phenocopies the effects of rapamycin (e.g. it triggers nuclear accumulation of Gln3p), it likely also inhibits the PI kinase-related, target of rapamycin (TOR) kinases. Importantly, however, neither inactivation of Stt4p nor Pik1p significantly affects TOR-controlled readouts other than translation initiation, indicating that these PI4Ks do not simply function upstream of TOR. Together, our results reveal the existence of a novel translation initiation control mechanism in yeast that is tightly coupled to the synthesis of distinct PI4P pools.     

Interruption of inositol sphingolipid synthesis triggers Stt4p-dependent protein kinase C signaling

The protein kinase C (PKC)-MAPK signaling cascade is activated and is essential for viability when cells are starved for the phospholipid precursor inositol. In this study, we report that inhibiting inositol-containing sphingolipid metabolism, either by inositol starvation or treatment with agents that block sphingolipid synthesis, triggers PKC signaling independent of sphingoid base accumulation. Under these same growth conditions, a fluorescent biosensor that detects the necessary PKC signaling intermediate, phosphatidylinositol (PI)-4-phosphate (PI4P), is enriched on the plasma membrane. The appearance of the PI4P biosensor on the plasma membrane correlates with PKC activation and requires the PI 4-kinase Stt4p. Like other mutations in the PKC-MAPK pathway, mutants defective in Stt4p and the PI4P 5-kinase Mss4p, which generates phosphatidylinositol 4,5-bisphosphate, exhibit inositol auxotrophy, yet fully derepress INO1, encoding inositol-3-phosphate synthase. These observations suggest that inositol-containing sphingolipid metabolism controls PKC signaling by regulating access of the signaling lipids PI4P and phosphatidylinositol 4,5-bisphosphate to effector proteins on the plasma membrane.     

Plasma membrane proteins Slm1 and Slm2 mediate activation of the AGC kinase Ypk1 by TORC2 and sphingolipids in S. cerevisiae

The PH domain-containing proteins Slm1 and Slm2 were originally identified as substrates of the rapamycin-insensitive TOR complex 2 (TORC2) and as mediators of signaling by the lipid second messenger phosphatidyl-inositol-4,5-bisphosphate (PI4,5P2) in budding yeast S. cerevisiae. More recently, these proteins have been identified as critical effectors that facilitate phosphorylation and activation of the AGC kinases Ypk1 and Ypk2 by TORC2.¹ Here, we review the molecular basis for this regulation as well as place it within the context of recent findings that have revealed Slm1/2 and TORC2-dependent phosphorylation of Ypk1 is coupled to the biosynthesis of complex sphingolipids and to their levels within the plasma membrane (PM) as well as other forms of PM stress. Together, these studies reveal the existence of an intricate homeostatic feedback mechanism, whereby the activity of these signaling components is linked to the biosynthesis of PM lipids according to cellular need.     

Plasma membrane proteins Slm1 and Slm2 mediate activation of the AGC kinase Ypk1 by TORC2 and sphingolipids in S. cerevisiae

The PH domain-containing proteins Slm1 and Slm2 were originally identified as substrates of the rapamycin-insensitive TOR complex 2 (TORC2) and as mediators of signaling by the lipid second messenger phosphatidyl-inositol-4,5-bisphosphate (PI4,5P2) in budding yeast S. cerevisiae. More recently, these proteins have been identified as critical effectors that facilitate phosphorylation and activation of the AGC kinases Ypk1 and Ypk2 by TORC2.¹ Here, we review the molecular basis for this regulation as well as place it within the context of recent findings that have revealed Slm1/2 and TORC2-dependent phosphorylation of Ypk1 is coupled to the biosynthesis of complex sphingolipids and to their levels within the plasma membrane (PM) as well as other forms of PM stress. Together, these studies reveal the existence of an intricate homeostatic feedback mechanism, whereby the activity of these signaling components is linked to the biosynthesis of PM lipids according to cellular need.     

Slm1 and slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease

The Ca2+/calmodulin-dependent phosphatase calcineurin promotes yeast survival during environmental stress. We identified Slm1 and Slm2 as calcineurin substrates required for sphingolipid-dependent processes. Slm1 and Slm2 bind to calcineurin via docking sites that are required for their dephosphorylation by calcineurin and are related to the PXIXIT motif identified in NFAT. In vivo, calcineurin mediates prolonged dephosphorylation of Slm1 and Slm2 during heat stress, and this response can be mimicked by exogenous addition of the sphingoid base phytosphingosine. Slm proteins also promote the growth of yeast cells in the presence of myriocin, an inhibitor of sphingolipid biosynthesis, and regulation of Slm proteins by calcineurin is required for their full activity under these conditions. During heat stress, sphingolipids signal turnover of the uracil permease, Fur4. In cells lacking Slm protein activity, stress-induced endocytosis of Fur4 is blocked, and Fur4 accumulates at the cell surface in a ubiquitinated form. Furthermore, cells expressing a version of Slm2 that cannot be dephosphorylated by calcineurin display an increased rate of Fur4 turnover during heat stress. Thus, calcineurin may modulate sphingolipid-dependent events through regulation of Slm1 and Slm2. These findings, in combination with previous work identifying Slm1 and Slm2 as targets of Mss4/phosphatidylinositol 4,5-bisphosphate and TORC2 signaling, suggest that Slm proteins integrate information from a variety of signaling pathways to coordinate the cellular response to heat stress.     

Plasma membrane stress induces relocalization of Slm proteins and activation of TORC2 to promote sphingolipid synthesis

The plasma membrane delimits the cell, and its integrity is essential for cell survival. Lipids and proteins form domains of distinct composition within the plasma membrane. How changes in plasma membrane composition are perceived, and how the abundance of lipids in the plasma membrane is regulated to balance changing needs remains largely unknown. Here, we show that the Slm1/2 paralogues and the target of rapamycin kinase complex 2 (TORC2) play a central role in this regulation. Membrane stress, induced by either inhibition of sphingolipid metabolism or by mechanically stretching the plasma membrane, redistributes Slm proteins between distinct plasma membrane domains. This increases Slm protein association with and activation of TORC2, which is restricted to the domain known as the membrane compartment containing TORC2 (MCT; ref.?). As TORC2 regulates sphingolipid metabolism, our discoveries reveal a homeostasis mechanism in which TORC2 responds to plasma membrane stress to mediate compensatory changes in cellular lipid synthesis and hence modulates the composition of the plasma membrane. The components of this pathway and their involvement in signalling after membrane stretch are evolutionarily conserved.     

Ypp1/YGR198w plays an essential role in phosphoinositide signalling at the plasma membrane

Phosphoinositide signalling through the eukaryotic plasma membrane makes essential contributions to many processes, including remodelling of the actin cytoskeleton, vesicle trafficking and signalling from the cell surface. A proteome-wide screen performed in Saccharomyces cerevisiae revealed that Ypp1 interacts physically with the plasma-membrane-associated phosphoinositide 4-kinase, Stt4. In the present study, we demonstrate that phenotypes of ypp1 and stt4 conditional mutants are identical, namely osmoremedial temperature sensitivity, hypersensitivity to cell wall destabilizers and defective organization of actin. We go on to show that overexpression of STT4 suppresses the temperature-sensitive growth defect of ypp1 mutants. In contrast, overexpression of genes encoding the other two phosphoinositide 4-kinases in yeast, Pik1 and Lsb6, do not suppress this phenotype. This implies a role for Ypp1 in Stt4-dependent events at the plasma membrane, as opposed to a general role in overall metabolism of phosphatidylinositol 4-phosphate. Use of a pleckstrin homology domain sensor reveals that there are substantially fewer plasma-membrane-associated 4-phosphorylated phosphoinositides in ypp1 mutants in comparison with wild-type cells. Furthermore, in vivo labelling with [(3)H]inositol indicates a dramatic reduction in the level of phosphatidylinositol 4-phosphate in ypp1 mutants. This is the principal cause of lethality under non-permissive conditions in ypp1 mutants, as limiting the activity of the Sac1 phosphoinositide 4-phosphate phosphatase leads to restoration of viability. Additionally, the endocytic defect associated with elevated levels of PtdIns4P in sac1Delta cells is restored in combination with a ypp1 mutant, consistent with the opposing effects that these two mutations have on levels of this phosphoinositide.     

Requirements of Slm proteins for proper eisosome organization,endocytic trafficking and recycling in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Eisosomes are large immobile assemblies at the cortex of a cell under the membrane compartment of Can1 (MCC) in yeast. Slm1 has recently been identified as an MCC component that acts downstream of Mss4 in a pathway that regulates actin cytoskeleton organization in response to stress. In this study, we showed that inactivation of Slm proteins disrupts proper localization of the primary eisosome marker Pil1, providing evidence that Slm proteins play a role in eisosome organization. Furthermore, we found that slm ^ts mutant cells exhibit actin defects in both the ability to polarize cortical F-actin and the formation of cytoplasmic actin cables even at the permissive temperature (30°C). We further demonstrated that the actin defect accounts for the slow traffic of FM4-64-labelled endosome in the cytoplasm, supporting the notion that intact actin is essential for endosome trafficking. However, our real-time microscopic analysis of Abp1-RFP revealed that the actin defect in slm ^ts cells was not accompanied by a noticeable defect in actin patch internalization during receptor-mediated endocytosis. In addition, we found that slm ^ts cells displayed impaired membrane recycling and that recycling occurred in an actin-independent manner. Our data provide evidence for the requirement of Slm proteins in eisosome organization and endosome trafficking and recycling.     

Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells

Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).     

Modulation of phosphoinositide metabolism by pathogenic bacteria

Phosphoinositide metabolism plays a pivotal role in the regulation of receptor-mediated signal transduction, actin remodelling and membrane dynamics. Phosphoinositides co-ordinate these processes by recruiting protein effectors to distinct cellular membranes in a time- and organelle-dependent manner. Intracellular bacterial pathogens interfere with phosphoinositide metabolism to direct their entry into eukaryotic cells, form replication-permissive vacuoles, modulate apoptosis, or trigger fluid secretion. Gram-negative pathogens such as Legionella pneumophila, Shigella flexneri, or Salmonella enterica employ secretion systems to invade host cells by 'pathogen-triggered phagocytosis' and thereby bypass a requirement for phosphatidylinositol 3-kinases [PI(3)Ks]. Contrarily, 'receptor-mediated phagocytosis' of Yersinia spp., Listeria monocytogenes and other pathogenic bacteria depends on PI(3)Ks. Secreted effector proteins have been found to directly bind to and modify host cell phosphoinositides, thus modulating phagocytosis and intracellular survival of the pathogens. These effectors include L. pneumophila proteins that specifically attach to phosphatidylinositol 4-phosphate [PI(4)P] on the Legionella-containing vacuole, and phosphoinositide phosphatases produced by S. flexneri, S. enterica or Mycobacterium tuberculosis. This review covers current knowledge about subversion of host cell phosphoinositide metabolism by intracellular bacterial pathogens with an emphasis on recently identified secreted effector proteins directly engaging phosphoinositides.     

Sac1 lipid phosphatase and Stt4 phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol 4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology

Synthesis and turnover of phosphoinositides are tightly regulated processes mediated by a set of recently identified kinases and phosphatases. We analyzed the primary role of the phosphoinositide phosphatase Sac1p in Saccharomyces cerevisiae with the use of a temperature-sensitive allele of this gene. Our analysis demonstrates that inactivation of Sac1p leads to a specific increase in the cellular levels of phosphatidylinositol 4-phosphate (PtdIns(4)P), accompanied by changes in vacuole morphology and an accumulation of lipid droplets. We have found that the majority of Sac1p localizes to the endoplasmic reticulum, and this localization is crucial for the efficient turnover of PtdIns(4)P. By generating double mutant strains harboring the sac1(ts) allele and one of two temperature-sensitive PtdIns 4-kinase genes, stt4(ts) or pik1(ts), we have demonstrated that the bulk of PtdIns(4)P that accumulates in sac1 mutant cells is generated by the Stt4 PtdIns 4-kinase, and not Pik1p. Consistent with these findings, inactivation of Sac1p partially rescued defects associated with stt4(ts) but not pik1(ts) mutant cells. To analyze potential overlapping functions between Sac1p and other homologous phosphoinositide phosphatases, sac1(ts) mutant cells lacking various other synaptojanin-like phosphatases were generated. These double and triple mutants exacerbated the accumulation of intracellular phosphoinositides and caused defects in Golgi function. Together, our results demonstrate that Sac1p primarily turns over Stt4p-generated PtdIns(4)P and that the membrane localization of Sac1p is important for its function in vivo. Regulation of this PtdIns(4)P pool appears to be crucial for the maintenance of vacuole morphology, regulation of lipid storage, Golgi function, and actin cytoskeleton organization.     

SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases

The SAC1 gene product has been implicated in the regulation of actin cytoskeleton, secretion from the Golgi, and microsomal ATP transport; yet its function is unknown. Within SAC1 is an evolutionarily conserved 300-amino acid region, designated a SAC1-like domain, that is also present at the amino termini of the inositol polyphosphate 5-phosphatases, mammalian synaptojanin, and certain yeast INP5 gene products. Here we report that SAC1-like domains have intrinsic enzymatic activity that defines a new class of polyphosphoinositide phosphatase (PPIPase). Purified recombinant SAC1-like domains convert yeast lipids phosphatidylinositol (PI) 3-phosphate, PI 4-phosphate, and PI 3,5-bisphosphate to PI, whereas PI 4,5-bisphosphate is not a substrate. Yeast lacking Sac1p exhibit 10-, 2.5-, and 2-fold increases in the cellular levels of PI 4-phosphate, PI 3,5-bisphosphate, and PI 3-phosphate, respectively. The 5-phosphatase domains of synaptojanin, Inp52p, and Inp53p are also catalytic, thus representing the first examples of an inositol signaling protein with two distinct lipid phosphatase active sites within a single polypeptide chain. Together, our data provide a long sought mechanism as to how defects in Sac1p overcome certain actin mutants and bypass the requirement for yeast phosphatidylinositol/phosphatidylcholine transfer protein, Sec14p. We demonstrate that PPIPase activity is a key regulator of membrane trafficking and actin cytoskeleton organization and suggest signaling roles for phosphoinositides other than PI 4,5-bisphosphate in these processes. Additionally, the tethering of PPIPase and 5-phosphatase activities indicate a novel mechanism by which concerted phosphoinositide hydrolysis participates in membrane trafficking.     

Homer3 regulates the establishment of neutrophil polarity

Most chemoattractants rely on activation of the heterotrimeric G-protein Gαi to regulate directional cell migration, but few links from Gαi to chemotactic effectors are known. Through affinity chromatography using primary neutrophil lysate, we identify Homer3 as a novel Gαi2-binding protein. RNA interference–mediated knockdown of Homer3 in neutrophil-like HL-60 cells impairs chemotaxis and the establishment of polarity of phosphatidylinositol 3,4,5-triphosphate (PIP₃) and the actin cytoskeleton, as well as the persistence of the WAVE2 complex. Most previously characterized proteins that are required for cell polarity are needed for actin assembly or activation of core chemotactic effectors such as the Rac GTPase. In contrast, Homer3-knockdown cells show normal magnitude and kinetics of chemoattractant-induced activation of phosphoinositide 3-kinase and Rac effectors. Chemoattractant-stimulated Homer3-knockdown cells also exhibit a normal initial magnitude of actin polymerization but fail to polarize actin assembly and intracellular PIP₃ and are defective in the initiation of cell polarity and motility. Our data suggest that Homer3 acts as a scaffold that spatially organizes actin assembly to support neutrophil polarity and motility downstream of GPCR activation.     

Regulation of PI4,5P2 synthesis by nuclear-cytoplasmic shuttling of the Mss4 lipid kinase

The essential phospholipid PI4,5P(2) is generated by a well conserved PI4P 5-kinase, Mss4, in yeast. Balanced production and turnover of PI4,5P(2) is important for normal organization of the actin cytoskeleton and cell viability. Previous studies have shown that multiple PI phosphatases can regulate PI4,5P(2) levels. We report a new, unexpected regulatory mechanism for PI4,5P(2) homeostasis, directed by nuclear-cytoplasmic shuttling of the lipid kinase. We show that Mss4 is a phosphoprotein, which contains a functional nuclear localization signal (NLS) and can shuttle between the cytoplasm and the nucleus. Temperature-conditional mss4 cells that accumulate Mss4 protein in the nucleus exhibit reduced levels of PI4,5P(2), depolarization of the actin cytoskeleton and a block in Mss4 phosphorylation, suggesting an essential role for phosphorylated Mss4 at the plasma membrane. Through the isolation of gene dosage-dependent suppressors of mss4 mutants, we identified Bcp1, a protein enriched in the nucleus, which is required for Mss4 nuclear export and is related to the mammalian BRCA2-interacting protein BCCIP. Together, these studies suggest a new mechanism for lipid kinase regulation through regulated nuclear-cytoplasmic shuttling.     

Coordination of Golgi functions by phosphatidylinositol 4-kinases

Phosphatidylinositol 4-kinases (PI4Ks) regulate vesicle-mediated export from the Golgi apparatus via phosphatidylinositol 4-phosphate (PtdIns4P) binding effector proteins that control vesicle budding reactions and regulate membrane dynamics. Evidence has emerged from the characterization of Golgi PI4K effectors that vesicle budding and lipid dynamics are tightly coupled via a regulatory network that ensures that the appropriate membrane composition is established before a transport vesicle buds from the Golgi. An important hub of this network is protein kinase D, which regulates the activity of PI4K and several PtdIns4P effectors that control sphingolipid and sterol content of Golgi membranes. Other newly identified PtdIns4P effectors include Vps74/GOLPH3, a phospholipid flippase called Drs2 and Sec2, a Rab guanine nucleotide exchange factor (GEF). These effectors orchestrate membrane transformation events facilitating vesicle formation and targeting. In this review, we discuss how PtdIns4P signaling is integrated with membrane biosynthetic and vesicle budding machineries to potentially coordinate these crucial functions of the Golgi apparatus.