CRISPRa Analysis of Phosphoinositide Phosphatases Shows That TMEM55A Is a Positive Regulator of Autophagy.

Transcriptional activation, based on Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) and known as CRISPR activation (CRISPRa), is a specific and safe tool to upregulate endogenous genes. Therefore, CRISPRa is valuable not only for analysis of molecular mechanisms of cellular events, but also for treatment of various diseases. Regulating autophagy has been proposed to enhance effects of some therapies. In this study, we upregulated genes for phosphoinositide phosphatases, SACM1L, PIP4P1, and PIP4P2, using CRISPRa, and their effects on autophagy were examined. Our results suggested that TMEM55A/PIP4P2, a phosphatidylinositol-4,5-bisphosphate 4-phosphatase, positively regulates basal autophagy in 293A cells. Furthermore, it was also suggested that SAC1, a phosphatidylinositol 4-phosphatase, negatively regulates basal autophagic degradation.

Increased Phospholipid Flux Bypasses Overlapping Essential Requirements for the Yeast Sac1p Phosphoinositide Phosphatase and ER-PM Membrane Contact Sites.

In budding yeast cells, much of the inner surface of the plasma membrane (PM) is covered with the endoplasmic reticulum (ER). This association is mediated by seven ER membrane proteins that confer cortical ER-PM association at membrane contact sites (MCSs). Several of these membrane "tether" proteins are known to physically interact with the phosphoinositide phosphatase Sac1p. However, it is unclear how or if these interactions are necessary for their interdependent functions. We find that SAC1 inactivation in cells lacking the homologous synaptojanin-like genes INP52 and INP53 results in a significant increase in cortical ER-PM MCSs. We show in sac1Δ, sac1tsinp52Δ inp53Δ, or Δ-super-tether (Δ-s-tether) cells lacking all seven ER-PM tethering genes that phospholipid biosynthesis is disrupted and phosphoinositide distribution is altered. Furthermore, SAC1 deletion in Δ-s-tether cells results in lethality, indicating a functional overlap between SAC1 and ER-PM tethering genes. Transcriptomic profiling indicates that SAC1 inactivation in either Δ-s-tether or inp52Δ inp53Δ cells induces an ER membrane stress response and elicits phosphoinositide-dependent changes in expression of autophagy genes. In addition, by isolating high-copy suppressors that rescue sac1Δ Δ-s-tether lethality, we find that key phospholipid biosynthesis genes bypass the overlapping function of SAC1 and ER-PM tethers and that overexpression of the phosphatidylserine/phosphatidylinositol-4-phosphate transfer protein Osh6 also provides limited suppression. Combined with lipidomic analysis and determinations of intracellular phospholipid distributions, these results suggest that Sac1p and ER phospholipid flux controls lipid distribution to drive Osh6p-dependent phosphatidylserine/phosphatidylinositol-4-phosphate counter-exchange at ER-PM MCSs.

The chloride antiporter CLCN7 is a modifier of lysosome dysfunction in FIG4 and VAC14 mutants.

The phosphatase FIG4 and the scaffold protein VAC14 function in the biosynthesis of PI(3,5)P2, a signaling lipid that inhibits the lysosomal chloride transporter ClC-7. Loss-of-function mutations of FIG4 and VAC14 reduce PI(3,5)P2 and result in lysosomal disorders characterized by accumulation of enlarged lysosomes and neurodegeneration. Similarly, a gain of function mutation of CLCN7 encoding ClC-7 also results in enlarged lysosomes. We therefore tested the ability of reduced CLCN7 expression to compensate for loss of FIG4 or VAC14. Knock-out of CLCN7 corrected lysosomal swelling and partially corrected lysosomal hyperacidification in FIG4 null cell cultures. Knockout of the related transporter CLCN6 (ClC-6) in FIG4 null cells did not affect the lysosome phenotype. In the Fig4 null mouse, reduction of ClC-7 by expression of the dominant negative CLCN7 variant p.Gly215Arg improved growth and neurological function and increased lifespan by 20%. These observations demonstrate a role for the CLCN7 chloride transporter in pathogenesis of FIG4 and VAC14 disorders. Reduction of CLCN7 provides a new target for treatment of FIG4 and VAC14 deficiencies that lack specific therapies, such as Charcot-Marie-Tooth Type 4J and Yunis-Varón syndrome.

Altered phenotypes due to genetic interaction between the mouse phosphoinositide biosynthesis genes Fig4 and Pip4k2c.

Loss-of-function mutations of FIG4 are responsible for neurological disorders in human and mouse that result from reduced abundance of the signaling lipid PI(3,5)P2. In contrast, loss-of-function mutations of the phosphoinositide kinase PIP4K2C result in elevated abundance of PI(3,5)P2. These opposing effects on PI(3,5)P2 suggested that we might be able to compensate for deficiency of FIG4 by reducing expression of PIP4K2C. To test this hypothesis in a whole animal model, we generated triallelic mice with genotype Fig 4-/-, Pip4k2c+/-; these mice are null for Fig 4 and haploinsufficient for Pip4k2c. The neonatal lethality of Fig 4 null mice in the C57BL/6J strain background was rescued by reduced expression of Pip4k2c. The lysosome enlargement characteristic of Fig 4 null cells was also reduced by heterozygous loss of Pip4k2c. The data demonstrate interaction between these two genes, and suggest that inhibition of the kinase PIPK4C2 could be a target for treatment of FIG4 deficiency disorders such as Charcot-Marie-Tooth Type 4J and Yunis-Varón Syndrome.

Chloroquine corrects enlarged lysosomes in FIG4 null cells and reduces neurodegeneration in Fig4 null mice.

Loss-of-function mutations of FIG4 impair the biosynthesis of PI(3,5)P2 and are responsible for rare genetic disorders including Yunis-Varón Syndrome and Charcot-Marie-Tooth Disease Type 4 J. Cultured cells deficient in FIG4 accumulate enlarged lysosomes with hyperacidic pH, due in part to impaired regulation of lysosomal ion channels and elevated intra-lysosomal osmotic pressure. We evaluated the effects of the FDA approved drug chloroquine, which is known to reduce lysosome acidity, on FIG4 deficient cell culture and on a mouse model. Chloroquine corrected the enlarged lysosomes in FIG4 null cells. In null mice, addition of chloroquine to the drinking water slowed progression of the disorder. Growth and mobility were dramatically improved during the first month of life, and spongiform degeneration of the nervous system was reduced. The median survival of Fig4 null mice was increased from 4 weeks for untreated mutants to 8 weeks with chloroquine treatment (p < 0.009). Chloroquine thus corrects the lysosomal swelling in cultured cells and ameliorates Fig4 deficiency in vivo. The improved phenotype of mice with complete loss of Fig4 suggests that chloroquine could be beneficial FIG2 in partial loss-of-function disorders such as Charcot-Marie-Tooth Type 4 J.

AAV9-mediated FIG4 delivery prolongs life span in Charcot-Marie-Tooth disease type 4J mouse model.

Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive, loss-of-function mutations in FIG4, encoding a phosphoinositol(3,5)P2-phosphatase. CMT4J patients have both neuron loss and demyelination in the peripheral nervous system, with vacuolization indicative of endosome/lysosome trafficking defects. Although the disease is highly variable, the onset is often in childhood and FIG4 mutations can dramatically shorten life span. There is currently no treatment for CMT4J. Here, we present the results of preclinical studies testing a gene-therapy approach to restoring FIG4 expression. A mouse model of CMT4J, the Fig4-pale tremor (plt) allele, was dosed with a single-stranded adeno-associated virus serotype 9 (AAV9) to deliver a codon-optimized human FIG4 sequence. Untreated, Fig4plt/plt mice have a median survival of approximately 5 weeks. When treated with the AAV9-FIG4 vector at P1 or P4, mice survived at least 1 year, with largely normal gross motor performance and little sign of neuropathy by neurophysiological or histopathological evaluation. When mice were treated at P7 or P11, life span was still significantly prolonged and peripheral nerve function was improved, but rescue was less complete. No unanticipated adverse effects were observed. Therefore, AAV9-mediated delivery of FIG4 is a well-tolerated and efficacious strategy in a mouse model of CMT4J.

Regulation of lysosomal positioning via TMEM55B phosphorylation.

Lysosomes are dynamic organelles that are transported along microtubules bidirectionally via kinesin and dynein motor proteins. Lysosomal positioning, which is determined by the balance of the bidirectional lysosomal movement, changes under various conditions and affects lysosomal functions such as autophagy and antigen presentation. A recent study by Takemasu et al. (Phosphorylation of TMEM55B by Erk/MAPK regulates lysosomal positioning. J. Biochem. 2019; 166:175-185) has shown that phosphorylation of the transmembrane protein TMEM55B is involved in the retrograde lysosomal trafficking towards the perinuclear region. They found that TMEM55B is phosphorylated upon stimulation with various ligands and that Erk/MAPK mediates the TMEM55B phosphorylation. They have also revealed that a phosphorylation mimic mutant of TMEM55B enhances perinuclear lysosomal clustering compared to the wild-type TMEM55B. These findings suggest that TMEM55B phosphorylation by Erk/MAPK is responsible for regulating lysosomal positioning in response to external stimuli.

Phosphatidylinositol-(4,5)-Bisphosphate Regulates Plasma Cholesterol Through LDL (Low-Density Lipoprotein) Receptor Lysosomal Degradation.

OBJECTIVE: TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of Tmem55b knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P2) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against Tmem55b or a nontargeting control for 3 to 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ≈70%, and plasma non-HDL (high-density lipoprotein) cholesterol was increased ≈1.8-fold (P<0.0001). Immunoblot analysis of fast protein liquid chromatography (FPLC) fractions revealed enrichment of ApoE-containing particles in the LDL size range. In contrast, Tmem55b knockdown had no effect on plasma cholesterol in Ldlr-/- mice. In primary hepatocytes and liver tissues from Tmem55b knockdown mice, there was decreased LDLR protein. In the hepatocytes, there was increased lysosome staining and increased LDLR-lysosome colocalization. Impairment of lysosome function (incubation with NH4Cl or knockdown of the lysosomal proteins LAMP1 or RAB7) abolished the effect of TMEM55B knockdown on LDLR in HepG2 (human hepatoma) cells. Colocalization of the recycling endosome marker RAB11 (Ras-related protein 11) with LDLR in HepG2 cells was reduced by 50% upon TMEM55B knockdown. Finally, knockdown increased hepatic PI(4,5)P2 levels in vivo and in HepG2 cells, while TMEM55B overexpression in vitro decreased PI(4,5)P2. TMEM55B knockdown decreased, whereas overexpression increased, LDL uptake in HepG2 cells. Notably, the TMEM55B overexpression effect was reversed by incubation with PI(4,5)P2. Conclusions: These findings indicate a role for TMEM55B in regulating plasma cholesterol levels by affecting PI(4,5)P2-mediated LDLR lysosomal degradation.

A SAC Phosphoinositide Phosphatase Controls Rice Development via Hydrolyzing PI4P and PI(4,5)P2.

Phosphoinositides (PIs) as regulatory membrane lipids play essential roles in multiple cellular processes. Although the exact molecular targets of PI-dependent modulation remain largely elusive, the effects of disturbed PI metabolism could be employed to identify regulatory modules associated with particular downstream targets of PIs. Here, we identified the role of GRAIN NUMBER AND PLANT HEIGHT1 (GH1), which encodes a suppressor of actin (SAC) domain-containing phosphatase with unknown function in rice (Oryza sativa). Endoplasmic reticulum-localized GH1 specifically dephosphorylated and hydrolyzed phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Inactivation of GH1 resulted in massive accumulation of both PI4P and PI(4,5)P2, while excessive GH1 caused their depletion. Notably, superabundant PI4P and PI(4,5)P2 could both disrupt actin cytoskeleton organization and suppress cell elongation. Interestingly, both PI4P and PI(4,5)P2 inhibited actin-related protein2 and -3 (Arp2/3) complex-nucleated actin-branching networks in vitro, whereas PI(4,5)P2 showed more dramatic effects in a dose-dependent manner. Overall, the overaccumulation of PI(4,5)P2 resulting from dysfunction of SAC phosphatase possibly perturbs Arp2/3 complex-mediated actin polymerization, thereby disordering cell development. These findings imply that the Arp2/3 complex might be the potential molecular target of PI(4,5)P2-dependent modulation in eukaryotes, thereby providing insights into the relationship between PI homeostasis and plant growth and development.

Investigation of candidate genes of non-syndromic cleft lip with or without cleft palate, using both case-control and family-based association studies.

OBJECTIVE: Non-syndromic cleft of the lip and/or palate (NSCL/P) is one of the most common polygenic diseases. In this study, both case-control and family-based association study were used to confirm whether the Single Nucleotide Polymorphisms (SNPs) were associated with NSCL/P. METHODS: A total of 37 nuclear families and 189 controls were recruited, whose blood DNA was extracted and subjected to genotyping of SNPs of 27 candidate genes by polymerase chain reaction-improved multiple ligase detection reaction technology (PCR-iMLDR). Case-control statistical analysis was performed using the SPSS 19.0. Haplotype Relative Risk (HRR), transmission disequilibrium test (TDT), and Family-Based Association Test (FBAT) were used to test for over-transmission of the target alleles in case-parent trios. The gene-gene interactions on NSCL/P were analyzed by Unphased-3.1.4. RESULTS: In case-control statistical analysis, only C14orf49 chr14_95932477 had statistically significant on genotype model (P = .03) and allele model (P = .03). Seven SNPs had statistically significant on TDT. None of 26 alleles has association with NSCL/P on FBAT. Some SNPs had haplotype-haplotype interactions and genotype-genotype interactions. CONCLUSION: C14orf49 chr14_95932477 was significantly different between cases and controls on genotype model and allele model by case-control design. Seven SNPs were significantly different on HRR. Four SNPs were significantly different on TDT.

CRISPR knockout screen implicates three genes in lysosome function.

Defective biosynthesis of the phospholipid PI(3,5)P2 underlies neurological disorders characterized by cytoplasmic accumulation of large lysosome-derived vacuoles. To identify novel genetic causes of lysosomal vacuolization, we developed an assay for enlargement of the lysosome compartment that is amenable to cell sorting and pooled screens. We first demonstrated that the enlarged vacuoles that accumulate in fibroblasts lacking FIG4, a PI(3,5)P2 biosynthetic factor, have a hyperacidic pH compared to normal cells'. We then carried out a genome-wide knockout screen in human HAP1 cells for accumulation of acidic vesicles by FACS sorting. A pilot screen captured fifteen genes, including VAC14, a previously identified cause of endolysosomal vacuolization. Three genes not previously associated with lysosome dysfunction were selected to validate the screen: C10orf35, LRRC8A, and MARCH7. We analyzed two clonal knockout cell lines for each gene. All of the knockout lines contained enlarged acidic vesicles that were positive for LAMP2, confirming their endolysosomal origin. This assay will be useful in the future for functional evaluation of patient variants in these genes, and for a more extensive genome-wide screen for genes required for endolysosome function. This approach may also be adapted for drug screens to identify small molecules that rescue endolysosomal vacuolization.

Sac1 Phosphoinositide Phosphatase Regulates Foam Cell Formation by Modulating SR-A Expression in Macrophages.

Macrophages endocytose modified low-density lipoproteins (LDL) vigorously via scavenger receptor A (SR-A) to become foam cells. In the present study, we found that Sac1, a member of the Sac family of phosphoinositide phosphatases, increases the protein level of SR-A and upregulates foam cell formation. Mouse macrophages (RAW264.7) were transfected with short hairpin RNAs (shRNAs) against Sac1. Sac1 knockdown decreased cell surface SR-A levels and impaired acetylated LDL-induced foam cell formation. Transfection of Sac1-knockdown cells with shRNA-resistant flag-Sac1 effectively rescued the expression of SR-A. Glycosylation of SR-A was largely attenuated by Sac1 knockdown, but neither mRNA expression nor protein degradation of SR-A were affected. These results suggest that Sac1 maintains SR-A protein levels by modulating SR-A glycosylation.

Phosphoinositide phosphatases in cancer cell dynamics-Beyond PI3K and PTEN.

Phosphoinositides are a group of lipids that regulate intracellular signaling and subcellular biological events. The signaling by phosphatidylinositol-3,4,5-trisphosphate and Akt mediates the action of growth factors that are essential for cell proliferation, gene transcription, cell migration, and polarity. The hyperactivation of this signaling has been identified in different cancer cells; and, it has been implicated in oncogenic transformation and cancer cell malignancy. Recent studies have argued the role of phosphoinositides in cancer cell dynamics, including actin cytoskeletal rearrangement at the plasma membrane and the organization of intracellular compartments. The focus of this review is to summarize the impact of the activities of phosphoinositide phosphatases on intracellular signaling related to cancer cell dynamics and to discuss how the abnormalities in the activities of the enzymes alter the levels of phosphoinositides in cancer cells.

The PIKfyve complex regulates the early melanosome homeostasis required for physiological amyloid formation.

The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import-export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity.This article has an associated First Person interview with the first author of the paper.

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.

Protective role of the lipid phosphatase Fig4 in the adult nervous system.

The signaling lipid phosphatidylinositol 3,5-bisphosphate, PI(3,5)P2, functions in vesicular trafficking through the endo-lysosomal compartment. Cellular levels of PI(3,5)P2 are regulated by an enzyme complex comprised of the kinase PIKFYVE, the phosphatase FIG4, and the scaffold protein VAC14. Mutations of human FIG4 cause inherited disorders including Charcot-Marie-Tooth disease type 4J, polymicrogyria with epilepsy, and Yunis-Varón syndrome. Constitutive Fig4-/- mice exhibit intention tremor, spongiform degeneration of neural tissue, hypomyelination, and juvenile lethality. To determine whether PI(3,5)P2 is required in the adult, we generated Fig4flox/-; CAG-creER mice and carried out tamoxifen-induced gene ablation. Global ablation in adulthood leads to wasting, tremor, and motor impairment. Death follows within 2 months of tamoxifen treatment, demonstrating a life-long requirement for Fig4. Histological examinations of the sciatic nerve revealed profound Wallerian degeneration of myelinated fibers, but not C-fiber axons in Remak bundles. In optic nerve sections, myelinated fibers appear morphologically intact and carry compound action potentials at normal velocity and amplitude. However, when iKO mice are challenged with a chemical white matter lesion, repair of damaged CNS myelin is significantly delayed, demonstrating a novel role for Fig4 in remyelination. Thus, in the adult PNS Fig4 is required to protect myelinated axons from Wallerian degeneration. In the adult CNS, Fig4 is dispensable for fiber stability and nerve conduction, but is required for the timely repair of damaged white matter. The greater vulnerability of the PNS to Fig4 deficiency in the mouse is consistent with clinical observations in patients with Charcot-Marie-Tooth disease.

Beyond voltage-gated ion channels: Voltage-operated membrane proteins and cellular processes.

Voltage-gated ion channels were believed to be the only voltage-sensitive proteins in excitable (and some non-excitable) cells for a long time. Emerging evidence indicates that the voltage-operated model is shared by some other transmembrane proteins expressed in both excitable and non-excitable cells. In this review, we summarize current knowledge about voltage-operated proteins, which are not classic voltage-gated ion channels as well as the voltage-dependent processes in cells for which single voltage-sensitive proteins have yet to be identified. Particularly, we will focus on the following. (1) Voltage-sensitive phosphoinositide phosphatases (VSP) with four transmembrane segments homologous to the voltage sensor domain (VSD) of voltage-gated ion channels; VSPs are the first family of proteins, other than the voltage-gated ion channels, for which there is sufficient evidence for the existence of the VSD domain; (2) Voltage-gated proton channels comprising of a single voltage-sensing domain and lacking an identified pore domain; (3) G protein coupled receptors (GPCRs) that mediate the depolarization-evoked potentiation of Ca2+ mobilization; (4) Plasma membrane (PM) depolarization-induced but Ca2+ -independent exocytosis in neurons. (5) Voltage-dependent metabolism of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2 , PIP2 ) in the PM. These recent discoveries expand our understanding of voltage-operated processes within cellular membranes.

TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes.

Lysosomal distribution is linked to the role of lysosomes in many cellular functions, including autophagosome degradation, cholesterol homeostasis, antigen presentation, and cell invasion. Alterations in lysosomal positioning contribute to different human pathologies, such as cancer, neurodegeneration, and lysosomal storage diseases. Here we report the identification of a novel mechanism of lysosomal trafficking regulation. We found that the lysosomal transmembrane protein TMEM55B recruits JIP4 to the lysosomal surface, inducing dynein-dependent transport of lysosomes toward the microtubules minus-end. TMEM55B overexpression causes lysosomes to collapse into the cell center, whereas depletion of either TMEM55B or JIP4 results in dispersion toward the cell periphery. TMEM55B levels are transcriptionally upregulated following TFEB and TFE3 activation by starvation or cholesterol-induced lysosomal stress. TMEM55B or JIP4 depletion abolishes starvation-induced retrograde lysosomal transport and prevents autophagosome-lysosome fusion. Overall our data suggest that the TFEB/TMEM55B/JIP4 pathway coordinates lysosome movement in response to a variety of stress conditions.

Phosphoinositide Diversity, Distribution, and Effector Function: Stepping Out of the Box.

Phosphoinositides (PtdInsPs) modulate a plethora of functions including signal transduction and membrane trafficking. PtdInsPs are thought to consist of seven interconvertible species that localize to a specific organelle, to which they recruit a set of cognate effector proteins. Here, in reviewing the literature, we argue that this model needs revision. First, PtdInsPs can carry a variety of acyl chains, greatly boosting their molecular diversity. Second, PtdInsPs are more promiscuous in their localization than is usually acknowledged. Third, PtdInsP interconversion is likely achieved through kinase-phosphatase enzyme complexes that coordinate their activities and channel substrates without affecting bulk substrate population. Additionally, we contend that despite hundreds of PtdInsP effectors, our attention is biased toward few proteins. Lastly, we recognize that PtdInsPs can act to nucleate coincidence detection at the effector level, as in PDK1 and Akt. Overall, better integrated models of PtdInsP regulation and function are not only possible but needed.

Characterization of a putative Plasmodium falciparum SAC1 phosphoinositide-phosphatase homologue potentially required for survival during the asexual erythrocytic stages.

Despite marked reductions in morbidity and mortality in the last ten years, malaria still takes a tremendous toll on human populations throughout tropical and sub-tropical regions of the world. The absence of an effective vaccine and resistance to most antimalarial drugs available demonstrate the urgent need for new intervention strategies. Phosphoinositides are a class of lipids with critical roles in numerous processes and their specific subcellular distribution, generated through the action of kinases and phosphatases, define organelle identity in a wide range of eukaryotic cells. Recent studies have highlighted important functions of phosphoinositide kinases in several parts of the Plasmodium lifecycle such as hemoglobin endocytosis and cytokinesis during the erythrocytic stage however, nothing is known with regards to the parasite's putative phosphoinositide phosphatases. We present the identification and initial characterization of a putative homologue of the SAC1 phosphoinositide phosphatase family. Our results show that the protein is expressed throughout the asexual blood stages and that it localises to the endoplasmic reticulum and potentially to the Golgi apparatus. Furthermore, conditional knockdown and knockout studies suggest that a minimal amount of the protein are likely required for survival during the erythrocytic cycle.