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Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15. Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle’s balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild type

Inherited cutis laxa, or inelastic, sagging skin is a genetic condition of premature and generalised connective tissue ageing, affecting various elastic components of the extracellular matrix. Several cutis laxa syndromes are inborn errors of metabolism and lead to severe neurological symptoms. In a patient with cutis laxa, a choreoathetoid movement disorder, dysmorphic features and intellectual disability we performed exome sequencing to elucidate the underlying genetic defect. We identified the amino acid substitution R275W in phosphatidylinositol 4‐kinase type IIα, caused by a homozygous missense mutation in the PI4K2A gene. We used lipidomics, complexome profiling and functional studies to measure phosphatidylinositol 4‐phosphate synthesis in the patient and evaluated PI4K2A deficient mice to define a novel metabolic disorder. The R275W residue, located on the surface of the protein, is involved in forming electrostatic interactions with the membrane. The catalytic activity of PI4K2A in patient fibroblasts was severely reduced and lipid mass spectrometry showed that particular acyl‐chain pools of PI4P and PI(4,5)P2 were decreased. Phosphoinositide lipids play a major role in intracellular signalling and trafficking and regulate the balance between proliferation and apoptosis. Phosphatidylinositol 4‐kinases such as PI4K2A mediate the first step in the main metabolic pathway that generates PI4P, PI(4,5)P2 and PI(3,4,5)P3. Although neurologic involvement is common, cutis laxa has not been reported previously in metabolic defects affecting signalling. Here we describe a patient with a complex neurological phenotype, premature ageing and a mutation in PI4K2A, illustrating the importance of this enzyme in the generation of inositol lipids with particular acylation characteristics.

We previously reported that recruitment of the Type IIA phosphatidylinositol 4-kinase (PI4K2A) to autophagosomes by GABARAP, a member of the Atg8 family of autophagy-related proteins, is important for autophagosome-lysosome fusion. Because both PI4K2A and GABARAP have also been implicated in the intracellular trafficking of plasma membrane receptors in the secretory/endocytic pathway, we characterized their interaction in cells under non-autophagic conditions. Fluorescence fluctuation spectroscopy measurements revealed that GABARAP exists predominantly as a cytosolic monomer in live cells, but is recruited to small cytoplasmic vesicles upon overexpression of PI4K2A. C-terminal lipidation of GABARAP, which is essential for its autophagic activities, is not necessary for its recruitment to these PI4K2A-containing transport vesicles. However, a GABARAP truncation mutant lacking C-terminal residues 103–117 fails to bind to PI4K2A, is not recruited to cytoplasmic vesicles, and does not co-distribute with PI4K2A on subcellular organelles. These observations suggest that that the PI4K2A-GABARAP interaction plays a role in membrane trafficking both under autophagic and non-autophagic conditions.

The small GTPase Rab7 is a key organizer of receptor sorting and lysosomal degradation by recruiting of a variety of effectors depending on its GDP/GTP‐bound state. However, molecular mechanisms that trigger Rab7 inactivation remain elusive. Here we find that, among the endosomal pools, Rab7‐positive compartments possess the highest level of PI4P, which is primarily produced by PI4K2A kinase. Acute conversion of this endosomal PI4P to PI(4,5)P(2) causes Rab7 dissociation from late endosomes and releases a regulator of autophagosome–lysosome fusion, PLEKHM1, from the membrane. Rab7 effectors Vps35 and RILP are not affected by acute PI(4,5)P(2) production. Deletion of PI4K2A greatly reduces PIP5Kγ‐mediated PI(4,5)P(2) production in Rab7‐positive endosomes leading to impaired Rab7 inactivation and increased number of LC3‐positive structures with defective autophagosome–lysosome fusion. These results reveal a late endosomal PI4P‐PI(4,5)P(2)‐dependent regulatory loop that impacts autophagosome flux by affecting Rab7 cycling and PLEKHM1 association.

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

Phosphoinositides are a minor class of short-lived membrane phospholipids that serve crucial functions in cell physiology ranging from cell signalling and motility to their role as signposts of compartmental membrane identity. Phosphoinositide 4-phosphates such as phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are concentrated at the plasma membrane, on secretory organelles, and on lysosomes, whereas phosphoinositide 3-phosphates, most notably phosphatidylinositol 3-phosphate (PI(3)P), are a hallmark of the endosomal system. Directional membrane traffic between endosomal and secretory compartments, although inherently complex, therefore requires regulated phosphoinositide conversion. The molecular mechanism underlying this conversion of phosphoinositide identity during cargo exit from endosomes by exocytosis is unknown. Here we report that surface delivery of endosomal cargo requires hydrolysis of PI(3)P by the phosphatidylinositol 3-phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy (also called myotubular myopathy) in humans. Removal of endosomal PI(3)P by MTM1 is accompanied by phosphatidylinositol 4-kinase-2α (PI4K2α)-dependent generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. Our data establish a mechanism for phosphoinositide conversion from PI(3)P to PI(4)P at endosomes en route to the plasma membrane and suggest that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans.

Nucleoredoxin (NXN) is a redox-regulating protein potentially targeted by reactive oxygen species (ROS). It regulates molecular pathways that participate in several key cellular processes. However, the role of NXN in the alcohol liver disease (ALD) redox regulation has not been fully understood. Here, we investigated the effects of ethanol and ethanol plus lipopolysaccharide, a two-hit liver injury model (Ethanol/LPS), on NXN/dishevelled (DVL) interaction and on DVL-dependent phosphoinositides production both in mouse liver and in a co-culture system consisting of human hepatic stellate cells (HSC) and ethanol metabolizing-VL17A human hepatocyte cells. Ethanol and two-hit model increased Nxn protein and mRNA expression, and 4-hydroxynonenal adducts. Two-hit model promoted Nxn nuclear translocation and Dvl/Phosphatidylinositol 4-kinase type-IIα (Pi4k2a) interaction ratio but surprisingly decreased Dvl protein and mRNA levels and reverted ethanol-induced Nxn/Dvl and Dvl/frizzled (Fzd) interaction ratios. Ethanol resulted in a significant increase of Dvl protein and mRNA expression, and decreased Nxn/Dvl interaction ratio but promoted the interaction of Dvl with Fzd and Pi4k2a; formation of this complex induced phosphatidylinositol 4-phosphate [PI(4)P] production. Ethanol and LPS treatments provoked similar alterations on NXN/DVL interaction and its downstream effect in HSC/VL17A co-culture system. Interestingly, ROS and glutathione levels as well as most of ethanol-induced alterations were modified by NXN overexpression in the co-culture system. In conclusion, two-hit model of ethanol exposure disrupts NXN/DVL homeostatic status to allow DVL/FZD/PI4K2A complex formation and stimulates PI(4)P production. These results provide a new mechanism showing that NXN also participates in the regulation of phosphoinositides production that is altered by ethanol during alcoholic liver disease progression.

For decades, phosphatidylinositol 4-phosphate (PtdIns4P) was considered primarily as a precursor in the synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2). More recently, specific functions for PtdIns4P itself have been identified, particularly in the regulation of intracellular membrane trafficking. PI4K2A/PI4KIIα (phosphatidylinositol 4-kinase type 2 α), one of the 4 enzymes that catalyze PtdIns4P production in mammalian cells, promotes vesicle formation from the trans-Golgi network (TGN) and endosomes. We recently identified a novel function for PI4K2A-derived PtdIns4P, as a facilitator of autophagosome-lysosome (A-L) fusion. We further showed that that this function requires the presence of the autophagic adaptor protein GABARAP (GABA[A] receptor-associated protein), which binds to PI4K2A and recruits it to autophagosomes. The mechanism whereby GABARAP-PI4K2A-PtdIns4P promotes A-L fusion remains to be defined. Based on other examples of phosphoinositide involvement in membrane trafficking, we speculate that it acts by recruiting elements of the membrane docking and fusion machinery.

I Table of Content 5 II Zusammenfassung 8 III Summary 10 1 Introduction 11 1.1 Defective PI homeostasis and membrane remodeling might cause X-linked centronuclear myopathy 11 1.1.1 Exocytic membrane transport is counterbalanced by retrograde traffic 11 1.1.2 Phosphoinositides determine membrane identity 12 1.2 Membrane traffic is regulated by phosphoinositides 15 1.2.1 Phosphoinositides serve as spatio-temporal landmarks 15 1.2.2 PI(4,5)P2-to- PI(3)P conversion in endocytic membrane traffic 16 1.2.3 Endosomal sorting occurs via tubular cargo-enriched subdomains 17 1.2.4 Exocyst tethering in endosomal exocytosis is regulated by endosomal and cell surface determinants 20 1.3 Myotubularins are a family of PI 3-phosphatases 22 1.3.1 MTM1 regulates endosomal PI(3)P turnover and membrane dynamics 23 1.3.2 Other myotubularin family members contribute to endosomal PI(3)P turnover ………………………………………………………………………………...24 1.4 Endosomal PI(4)P is synthesized by type II PI 4-kinases 26 1.5 Aims of this study 27 2 Material and methods 28 2.1 Materials 28 2.1.1 Chemicals 28 2.1.2 Buffers, Media and Solutions 28 2.1.3 DNA Oligonucleotides 31 2.1.4 Small interference RNA Oligos 31 2.1.5 Plasmid vectors 32 2.1.6 Molecular weight standards 34 2.1.7 Antibodies 34 2.1.8 Bacterial strains 36 2.1.9 Eukaryotic cell lines 36 2.1.10 Enzymes 37 2.1.11 Molecular biology kits 37 2.1.12 Supplier 37 2.2 Molecular biology methods 39 2.2.1 Plasmid construction 39 2.2.2 Preparation of complementary DNA (cDNA) libraries 39 2.2.3 Polymerase chain reaction (PCR) 40 2.2.4 Analytical agarose gel electrophoresis 42 2.2.5 Purification of DNA from agarose gels and PCR 42 2.2.6 Restriction digest 42 2.2.7 Integration of PCR products into linearized vector backbones 43 2.2.8 Preparation of chemically competent E.coli 43 2.2.9 Transformation of chemically competent E.coli 44 2.2.10 Isolation of DNA from bacterial cultures 44 2.2.11 Measurement of DNA concentrations 44 2.2.12 Sequencing 45 2.3 Cell biological methods 45 2.3.1 Cell culture 45 2.3.2 Plasmid Transfection 46 2.3.3 siRNA Transfection 46 2.3.4 Immunocytochemistry 47 2.3.5 Transferrin and EGF uptake assays 48 2.3.6 Autophagy assay 50 2.3.7 Cholera toxin uptake 51 2.3.8 Consecutive secretion assay 51 2.3.9 Elevation of PI(3)P and PI(4)P by PIP/AM treatment 51 2.3.10 Inhibition of Vps34 by VPS34-IN1 treatment 52 2.3.11 Fluorescence microscopy 53 2.3.12 Flow cytometry 55 2.3.13 Statistical analysis 55 2.4 Biochemical methods 56 2.4.1 Expression of recombinant proteins in E. coli 56 2.4.2 Purification of His6-tagged MTM1 fragment expressed in E. coli for subsequent antibody purification 56 2.4.3 Affinity purification of polyclonal MTM1antibody from serum 57 2.4.4 Preparation of cell extracts from mammalian cell cultures 58 2.4.5 Determination of protein concentration using Bradford assay 59 2.4.6 Sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS- PAGE) 59 2.4.7 Immunoblotting 60 2.4.8 Membrane fractionation 61 2.4.9 Co- Immunoprecipitation 61 3 Results 62 3.1 Exocytosis from endosomes is impaired in XLCNM patient cells 62 3.1.1 β1-integrin accumulates in late and recycling endosomes upon loss of MTM1 ………………………………………………………………………………...62 3.1.2 Impaired cargo exit from endosomes due to defective exocytosis 64 3.2 Tf exocytosis from endosomes requires MTM1-mediated PI(3)P hydrolysis 66 3.2.1 The subcellular distribution of TfR is altered upon loss of MTM1 68 3.2.2 Endosomal trafficking and cargo sorting in MTM1-depleted cells 71 3.2.3 Other Myotubularin family members in TfR exocytosis 73 3.3 Endosomal accumulation of PI(3)P and PI(3)P effector proteins inhibits exocytosis from endosomes 75 3.3.1 Sub-plasma membrane early and recycling endosomes accumulate upon loss of MTM1 ………………………………………………………………………………...75 3.3.2 MTM1 localizes to early and recycling endosomes 79 3.3.3 PI(3)P accumulates on endosomes in MTM1-depleted cells 80 3.3.4 PI(3)P levels can be manipulated by genetic and pharmacological tools 83 3.4 PI(3)P-to-PI(4)P conversion is required for exocyst-dependent endosomal exocytosis 88 3.4.1 Exocytic vesicles acquire PI(4)P before plasma membrane fusion 88 3.4.2 PI4K2α and exocyst associate and are required for exocytosis 90 3.4.3 Rab11 is required for endosomal exocyst localization 93 3.4.4 Exocyst recruitment to endosomes depends on PI4K2α and PI(4)P 95 3.4.5 PI4K2α-mediated MTM1 recruitment to endosomes initiates PI(3)P-dephosphorylation 95 4 Discussion 100 4.1 Regulation of endosomal PI(3)P and PI(4)P is tightly coupled 101 4.1.1 Endosomal PI(3)P levels are balanced by 3’-metabolizing enzymes 101 4.1.2 Membrane recruitment of MTM1 is regulated by lipid and protein content 103 4.1.3 PI(3)P and PI(3,5)P2 are lipid substrates of MTM1 104 4.1.4 PI4K2α-dependent PI(4)P synthesis defines endosomal subdomains for cargo sorting ……………………………………………………………………………….105 4.1.5 PI(3)P turnover regulates endosomal tubulation and cargo sorting 108 4.2 PI(3)P-to-PI(4)P conversion regulates endosomal recycling 109 4.2.1 PI conversion in endosomal exocytosis parallels switching of the Rab identity ……………………………………………………………………………….109 4.2.2 Regulation of the exocyst complex by endosomal and PM determinants 111 4.3 Common features of MTM1 deficiency cause XLCNM pathologies 111 4.3.1 Late endosomal recycling contributes to β1-integrin traffic 113 4.4 Outlook 115 5 Bibliography 117 6 Appendix 130 6.1 Appendix A: Abbreviations 130 6.2 Appendix B: Mass spectrometry results 133 6.2.1 MTM1 binding partners identified by mass spectrometry 133 6.2.2 PI4K2α binding partners identified by mass spectrometry 139 6.3 Appendix C: List of Figures and Tables 142 6.3.1 Tables 142 6.3.2 Figures 142 6.4 Appendix D: Primers (DNA oligonucleotides) 143 6.5 Appendix E: Publications 145, Phosphoinositides (PIs) are a minor class of short-lived phospholipids that serve as crucial signposts of membrane identity. Thereby, PIs full fill important functions in cell signaling and membrane transport. PI 4-phosphates such as phosphatitylinositol-4-phosphate (PI(4)P) and phosphatitylinositol-4,5-bisphosphate (PI(4,5)P2) are enriched at the plasma membrane (PM), on secretory organelles and lysosomes, while PI 3-phosphates, i.e. phosphatitylinositol-3-phosphate (PI(3)P), are a hallmark of the endosomal system. Directional transport between these compartments, thus, requires regulated PI conversion. However, PI conversion in exit from PI(3)P-enriched endosomes en route to the PI(4)P- and PI(4,5)P2-positive PM in endosomal recycling remained unknown. Here, we report that cargo exit from endosomes requires removal of PI(3)P by the PI(3)P 3-phosphatase myotubularin 1 (MTM1), and concomitant PI(4)P synthesis by PI 4-kinase type II α (PI4K2α). Loss of MTM1 causes endosomal accumulation of PI(3)P and PI(3)P effector proteins, i.e. sorting nexins, Kif16b-mediated outward traffic of PI(3)P containing endosomes and sub-plasmalemmal accumulation of exocytosis-deficient endosomes. As PI4K2α associates with MTM1 and thereby facilitates membrane recruitment of MTM1, these phenotypic changes are mimicked by loss of PI4K2α. The conversion of PI(3)P-to-PI(4)P is paralleled by a switch in Rab GTPase- identity from early endosomal Rab5 to recycling endosomal Rab11. This conversion mechanism is driven by coincident detection of Rab GTPase and PI content as well as by reciprocal interactions among its components, including the association of exocyst with PI4K2α and PI(4)P, Rab11 and with MTM1. Thus, distinct recycling endosomal domains, characterized by PI4K2α, its lipid product PI(4)P, MTM1 and Rab11, are created destined for exocyst recruitment and subsequent exocyst-dependent fusion with the PM. A similar PI conversion mechanism involving myotubularin family members may underlie cargo exit from endosomes towards late endosomes. Importantly, MTM1 depletion and XLCNM- patient mutations are functionally correlated as patient fibroblast are exocytosis deficient and accumulate endosomal β1-integrin. Our data, thus, suggest that defective PI conversion at endosomes may account for the disturbed muscle morphology and impaired integrin-dependent muscle attachment in developing muscle fibres in XLCNM-patients. These integrin localization defects can be reversed by application of a Vps34-specific inhibitor and thus offer a potential avenue to combat this devastating disease., Phosphoinositide (PI) sind eine seltene Gruppe von kurzlebigen Phospholipiden, die als Organisationsprinzip von Membranen hervorgetreten sind und Zellmembranen ihre Identität verleihen. Hierdurch erfüllen sie essentielle Funktionen in der Signaltransduktion und dem Membrantransport. PI 4-Phosphate wie Phosphatidylinositol-4-Phosphat (PI(4)P) und Phosphatidylinositol-4,5-Bisphosphat (PI(4,5)P2) sind an der Plasmamembran (PM), auf sekretorischen Organellen und Lysosomen angereichert, während PI 3-Phosphate, z.B. Phosphatidylinositol-3-Phosphat (PI(3)P), das Endomembransystem kennzeichnen. Gerichteter Transport zwischen diesen Kompartimenten benötigt daher die kontrollierte Konversion von PIs. Dennoch ist die PI Konversion auf dem Weg von PI(3)P-reichen Endosomen hin zur PI(4)P- und PI(4,5)P2-angereicherten PM in endosomalem Recycling nur unzureichend verstanden. In dieser Studie legen wir dar, dass der Austritt von Cargoproteinen aus Endosomen die Entfernung von PI(3)P durch die PI(3)P 3-Phosphatase Myotubularin 1 (MTM1) benötigt sowie die anschließende Synthese von PI(4)P durch PI 4-Kinase Typ II α (PI4K2α). Der Verlust von MTM1 führt zur endosomalen Akkumulation von PI(3)P und PI(3)P-Effektoren, zum Kif16b- vermittelten auswärtsgerichteten Transport von PI(3)P-reichen Endosomen und der Akkumulation von Exozytose-defizienten Endosomen unterhalb der PM. Da PI4K2α mit MTM1 assoziiert und hierdurch die Rekrutierung von MTM1 auf endosomale Membranen ermöglicht, spiegelt der Verlust von PI4K2α diese phenotypischen Veränderungen wider. Die Konversion von PI(3)P zu PI(4)P wird begleited durch einen Wechsel in der Identität der Rab GTPasen. Hierbei wird der frühe endosomale Marker Rab5 durch den Marker für Recycling-Endosomen, Rab11, ersetzt. Dieser Konversionsmechanismus wird angetrieben durch die Koinzidenz-Detektion von Rab GTPasen und PIs sowie durch reziproke Interaktionen zwischen den Komponenten. Diese beinhalten die Assoziation des Exozyst-Komplexes mit PI4K2α und PI(4)P, Rab11 und mit MTM1. Hierdurch werden endosomale Subdomänen für endosomales Recycling geschaffen, die durch PI4K2α, dessen Lipidprodukt PI(4)P, Rab11 und MTM1 gekennzeichnet sind. Auf diesen Subdomänen kann der Exozyst-Komplex assembliert werden, in dessen Abhängigkeit exozytische Vesikel mit der PM fusionieren. Ein ähnlicher PI Konversionsmechanismus, der Myotubularine beinhaltet, könnte den Austritt von Cargoproteinen aus frühen Endosomen hin zu späten Endosomen kontrollieren. Wesentlich hierbei ist, dass der Verlust von MTM1 und XLCNM-assoziierte Mutationen funktionell stark korrelieren. Fibroblasten von XLCNM-Patienten zeigen Exozytosedefekte und akkumulieren β1-Integrin in Endosomen. Unsere Daten legen daher nahe, dass defekte PI Konversion auf Endosomen für die gestörte Muskelmorphologie und Defizite in der Integrin-basierten Muskeladhäsion in sich entwickelnden Muskeln von XLCNM Patienten verantwortlich ist. Diese Misslokalisation von Integrinen kann in Zellkultur durch die Behandlung mit einem Vps34-spezifischen Inhibitor aufgehoben werden und stellt dadurch eine Möglichkeit dar, dieser unheilbaren Krankheit entgegen zu wirken.

Phosphatidylinositol 4-kinases (PI 4-kinases) catalyze the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate (PtdIns4P). The four known mammalian PI 4-kinases, PI4KA, PI4KB, PI4K2A, and PI4K2B have roles in intracellular lipid and protein trafficking. PI4KA and PI4KB also assist in the replication of several positive-sense RNA viruses. The identification of selective inhibitors of these kinases would be facilitated by assays suitable for high-throughput screening. We describe a homogeneous and nonisotopic assay for PI 4-kinase activity based on the bioluminescent detection of the ADP produced by kinase reactions. We have evaluated this assay with known nonselective inhibitors of PI 4-kinases and show that it performs similar to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of >0.7 for PI4KA in a 384-well format, demonstrating its suitability for high-throughput screening applications.

Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, substantial specificity is achieved in cells owing to the spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study, we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) on endosomes significantly delayed VAMP3 trafficking. Modulation of SNARE function by phospholipids had previously been proposed based on in vitro studies, and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells.

Numerous genes causing autosomal recessive hereditary spastic paraplegia (AR HSP) have been described. Despite this, in many families the causative gene and mutation are unknown. In this study we sequenced the Pi4k2a gene, whose knockout has been shown to cause a typical HSP model in mice, in 24 index cases of autosomal recessive HSP not known to be linked to any other HSP locus. No pathogenic changes were identified in exons or splice sites, suggesting the Pi4k2a gene may not be a cause of AR HSP in humans.