Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy.

Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK-dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK-dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation-independent accumulation of ULK substrates and kinase activity-regulated recruitment of autophagy proteins to ubiquitin-positive structures.

Development of selective inhibitors of phosphatidylinositol 3-kinase C2α.

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer.

Arabidopsis PLDζ1 and PLDζ2 localize to post-Golgi membrane compartments in a partially overlapping manner.

KEY MESSAGE: Arabidopsis PLDζ1 and PLDζ2 localize to the trans-Golgi network and to compartments including the trans-Golgi network, multi-vesicular bodies, and the tonoplast, respectively, depending on their N-terminal regions containing PX-PH domains. Phospholipase D (PLD) is involved in dynamic cellular processes, including membrane trafficking, cytoskeletal reorganization, and signal transduction for gene expression, through the production of phosphatidic acid in membrane compartments specific to each process. Although PLD plays crucial roles in various plant phenomena, the underlying processes involving PLD for each phenomenon remain largely elusive, partly because the subcellular localization of PLD remains obscure. In this study, we performed comparative subcellular localization analyses of the Arabidopsis thaliana PX-PH-PLDs PLDζ1 and PLDζ2. In mature lateral root cap cells, own promoter-driven fluorescence protein fusions of PLDζ1 localized to the entire trans-Golgi network (TGN) while that of PLDζ2 localized to punctate structures including part of the TGN and multi-vesicular bodies as well as the tonoplast. These localization patterns were reproduced using N-terminal partial proteins, which contain PX-PH domains. An inducibly overexpressed fluorescence protein fusion of the PLDζ2 partial protein first localized to punctate structures, and then accumulated predominantly on the tonoplast. Further domain dissection analysis revealed that the N-terminal moiety preceding the PX-PH domain of PLDζ2 was required for the tonoplast-predominant accumulation. These findings suggest that PLDζ1 and PLDζ2 play partially overlapping but nonetheless distinctive roles in post-Golgi compartments along the membrane trafficking pathway from the TGN to the tonoplast.

Impact on visual acuity and psychological outcomes of ranibizumab and subsequent treatment for diabetic macular oedema in Japan (MERCURY).

PURPOSE: The MERCURY study aimed to evaluate the effects on visual acuity and psychological symptoms, and safety, of ranibizumab and subsequent treatment in patients with diabetic macular oedema (DME) and impaired visual acuity (VA). We report data from the prespecified 12-month interim analysis. METHODS: This was a 24-month, phase 4, open-label, single-arm, prospective, observational study conducted at 20 specialised retinal centres in Japan. Participants were 209 patients with DME and impaired VA, not previously treated with either intravitreal or systemic anti-vascular endothelial growth factor (anti-VEGF) agents, who initiated ranibizumab 0.5 mg per investigator discretion. Following ranibizumab administration, patients were treated per routine clinical practice. Other treatments were allowed. The main outcome measure was the mean change in best-corrected VA (BCVA) in logarithmic minimum angle of resolution (logMAR) from baseline to month 12. An exploratory objective was to assess patients' psychological status using the Hospital Anxiety and Depression Scale (HADS). RESULTS: The mean ± standard deviation BCVA at baseline was 0.43 ± 0.39 logMAR. The mean number of injections of ranibizumab and anti-VEGF agents from baseline to month 11 was 3.2 ± 2.0 and 3.6 ± 2.4, respectively. The BCVA change from baseline to 12 months was - 0.08 ± 0.34 logMAR (p = 0.011), showing a significant improvement; the HADS-anxiety score also decreased significantly (p = 0.001) and the depression score decreased numerically (p = 0.080). CONCLUSION: MERCURY study data confirm the effectiveness of real-world treatment initiated with ranibizumab in Japanese patients with DME. In addition, treatment was able to positively influence anxiety via VA improvement.

The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism.

VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.

VPS34 complexes from a structural perspective.

VPS34 phosphorylates phosphatidylinositol to produce PtdIns3P and is the progenitor of the phosphoinositide 3-kinase (PI3K) family. VPS34 has a simpler domain organization than class I PI3Ks, which belies the complexity of its quaternary organization, with the enzyme always functioning within larger assemblies. PtdIns3P recruits specific recognition modules that are common in protein-sorting pathways, such as autophagy and endocytic sorting. It is best characterized in two heterotetramers, complexes I and II. Complex I is composed of VPS34, VPS15, Beclin 1, and autophagy-related gene (ATG)14L, whereas complex II replaces ATG14L with UVRAG. Because VPS34 can form a component of several distinct complexes, it enables independent regulation of various pathways that are controlled by PtdIns3P. Complexes I and II are critical for early events in autophagy and endocytic sorting, respectively. Autophagy has a complex association with cancer. In early stages, it inhibits tumorigenesis, but in later stages, it acts as a survival factor for tumors. Recently, various disease-associated somatic mutations were found in genes encoding complex I and II subunits. Lipid kinase activities of the complexes are also influenced by posttranslational modifications (PTMs). Mapping PTMs and somatic mutations on three-dimensional models of the complexes suggests mechanisms for how these affect VPS34 activity.

GLABRA2 Directly Suppresses Basic Helix-Loop-Helix Transcription Factor Genes with Diverse Functions in Root Hair Development.

The Arabidopsis thaliana GLABRA2 (GL2) gene encodes a transcription factor involved in the cell differentiation of various epidermal tissues. During root hair pattern formation, GL2 suppresses root hair development in non-hair cells, acting as a node between the gene regulatory networks for cell fate determination and cell differentiation. Despite the importance of GL2 function, its molecular basis remains obscure because the GL2 target genes leading to the network for cell differentiation are unknown. We identified five basic helix-loop-helix (bHLH) transcription factor genes (ROOT HAIR DEFECTIVE6 [RHD6], RHD6-LIKE1 [RSL1], RSL2, Lj-RHL1-LIKE1 [LRL1], and LRL2) as GL2 direct targets using transcriptional and posttranslational induction systems. Chromatin immunoprecipitation analysis confirmed GL2 binding to upstream regions of these genes in planta. Reporter gene analyses showed that these genes are expressed in various stages of root hair development and are suppressed by GL2 in non-hair cells. GL2 promoter-driven GFP fusions of LRL1 and LRL2, but not those of the other bHLH proteins, conferred root hair development on non-hair cells. These results indicate that GL2 directly suppresses bHLH genes with diverse functions in root hair development.

Cofilin1 controls transcolumnar plasticity in dendritic spines in adult barrel cortex.

During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure in vitro, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of neocortical circuits that have more sophisticated columnar organization than the rodent neocortex, such as the primate neocortex.

Avian sarcoma leukosis virus receptor-envelope system for simultaneous dissection of multiple neural circuits in mammalian brain.

Pathway-specific gene delivery is requisite for understanding complex neuronal systems in which neurons that project to different target regions are locally intermingled. However, conventional genetic tools cannot achieve simultaneous, independent gene delivery into multiple target cells with high efficiency and low cross-reactivity. In this study, we systematically screened all receptor-envelope pairs resulting from the combination of four avian sarcoma leukosis virus (ASLV) envelopes (EnvA, EnvB, EnvC, and EnvE) and five engineered avian-derived receptors (TVA950, TVB(S3), TVC, TVB(T), and DR-46TVB) in vitro. Four of the 20 pairs exhibited both high infection rates (TVA-EnvA, 99.6%; TVB(S3)-EnvB, 97.7%; TVC-EnvC, 98.2%; and DR-46TVB-EnvE, 98.8%) and low cross-reactivity (<2.5%). Next, we tested these four receptor-envelope pairs in vivo in a pathway-specific gene-transfer method. Neurons projecting into a limited somatosensory area were labeled with each receptor by retrograde gene transfer. Three of the four pairs exhibited selective transduction into thalamocortical neurons expressing the paired receptor (>98%), with no observed cross-reaction. Finally, by expressing three receptor types in a single animal, we achieved pathway-specific, differential fluorescent labeling of three thalamic neuronal populations, each projecting into different somatosensory areas. Thus, we identified three orthogonal pairs from the list of ASLV subgroups and established a new vector system that provides a simultaneous, independent, and highly specific genetic tool for transferring genes into multiple target cells in vivo. Our approach is broadly applicable to pathway-specific labeling and functional analysis of diverse neuronal systems.

FoxP2 is a parvocellular-specific transcription factor in the visual thalamus of monkeys and ferrets.

Although the parallel visual pathways are a fundamental basis of visual processing, our knowledge of their molecular properties is still limited. Here, we uncovered a parvocellular-specific molecule in the dorsal lateral geniculate nucleus (dLGN) of higher mammals. We found that FoxP2 transcription factor was specifically expressed in X cells of the adult ferret dLGN. Interestingly, FoxP2 was also specifically expressed in parvocellular layers 3-6 of the dLGN of adult old world monkeys, providing new evidence for a homology between X cells in the ferret dLGN and parvocellular cells in the monkey dLGN. Furthermore, this expression pattern was established as early as gestation day 140 in the embryonic monkey dLGN, suggesting that parvocellular specification has already occurred when the cytoarchitectonic dLGN layers are formed. Our results should help in gaining a fundamental understanding of the development, evolution, and function of the parallel visual pathways, which are especially prominent in higher mammals.

A bicistronic lentiviral vector-based method for differential transsynaptic tracing of neural circuits.

We developed a bicistronic HIV1-derived lentiviral vector system co-expressing green fluorescent protein (AcGFP1) and wheat germ agglutinin (WGA) mediated by picornaviral 2A peptide. This system was first applied to the analysis of the rat cerebellar efferent pathways. When the lentiviral vector was injected into a specific lobule, the local Purkinje cell population (first-order neurons) was efficiently infected and co-expressed both AcGFP1 and WGA protein. In the second-order neurons in the cerebellar and vestibular nuclei, WGA but not AcGFP1 protein was differentially detected, demonstrating that the presence of AcGFP1 protein enables discrimination of first-order neurons from second-order neurons. Furthermore, WGA protein was detected in the contralateral ventrolateral thalamic nucleus (third-order nucleus). This system also successfully labeled rat cortical pathways from the primary somatosensory cortex and monkey cerebellar efferent pathways. Thus, this bicistronic lentiviral vector system is a useful tool for differential transsynaptic tracing of neural pathways originating from local brain regions.

In vivo visualization of single-unit recording sites using MRI-detectable elgiloy deposit marking.

Precise localization of single-neuron activity has elucidated functional architectures of the primate cerebral cortex, related to vertically stacked layers and horizontally aligned columns. The traditional "gold standard" method for localizing recorded neuron is histological examination of electrolytic lesion marks at recording sites. Although this method can localize recorded neurons with fine neuroanatomy, the necessity for postmortem analysis prohibits its use in long-term chronic experiments. To localize recorded single-neuron positions in vivo, we introduced MRI-detectable elgiloy deposit marks, which can be created by electrolysis of an elgiloy microelectrode tip and visualized on highly contrasted magnetic resonance (MR) images. Histological analysis validated that the deposit mark centers could be localized relative to neuroanatomy in vivo with single-voxel accuracy, at an in-plane resolution of 200 µm. To demonstrate practical applications of the technique, we recorded single-neuron activity from a monkey performing a cognitive task and localized it in vivo using deposit marks (deposition: 2 µA for 3 min; scanning: fast-spin-echo sequence with 0.15 × 0.15 × 0.8 mm(3) resolution, 120/4,500 ms of echo-time/repetition-time and 8 echo-train-length), as is usually performed with conventional postmortem methods using electrolytic lesion marks. Two localization procedures were demonstrated: 1) deposit marks within a microelectrode track were used to reconstruct a dozen recorded neuron positions along the track directly on MR images; 2) combination with X-ray imaging allowed estimation of hundreds of neuron positions on MR images. This new in vivo method is feasible for chronic experiments with nonhuman primates, enabling analysis of the functional architecture of the cerebral cortex underlying cognitive processes.

Functional analysis of HOXD9 in human gliomas and glioma cancer stem cells.

BACKGROUND: HOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies. RESULTS: In this study, we found high expression of the HOXD9 gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of HOXD9 in gliomas, we silenced its expression in the glioma cell line U87 using HOXD9-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that HOXD9 contributes to both cell proliferation and/or cell survival. The HOXD9 gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. HOXD9 siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of HOXD9 in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs). CONCLUSIONS: Our results suggest that HOXD9 may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.

Class III phosphatidylinositol 3-kinase complex I subunit NRBF2/Atg38 - from cell and structural biology to health and disease.

Macroautophagy/autophagy is triggered by various starvation and stress conditions. The phospholipid phosphatidylinositol-3-phosphate (PtdIns3P) is essential for the formation of the autophagosome both in yeast and mammals. The class III phosphatidylinositol 3-kinase, PIK3C3C in humans or Vps34 in yeast, produces PtdIns3P by phosphorylating the 3'-OH position of phosphatidylinositol (PtdIns). In order to synthesize PtdIns3P for the initiation of autophagy, PIK3C3/Vps34 has a heterotetrameric core, the PIK3C3 complex I (hereafter complex I) composed of PIK3C3/Vps34, PIK3R4/Vps15, BECN1/Vps30, and ATG14/Atg14. A fifth component of complex I, NRBF2 in mammals and Atg38 in yeast, was found and has been characterized in the past decade. The field has been expanding from cell and structural biology to mouse model and cohort studies. Here I will summarize the structures and models of complex I binding NRBF2/Atg38, its intracellular roles, and its involvement in health and disease. Along with this expansion of the field, different conclusions have been drawn in several topics. I will clarify what has and has not been agreed, and what is to be clarified in the future.

Activation Mechanisms of the VPS34 Complexes.

Phosphatidylinositol-3-phosphate (PtdIns(3)P) is essential for cell survival, and its intracellular synthesis is spatially and temporally regulated. It has major roles in two distinctive cellular pathways, namely, the autophagy and endocytic pathways. PtdIns(3)P is synthesized from phosphatidylinositol (PtdIns) by PIK3C3C/VPS34 in mammals or Vps34 in yeast. Pathway-specific VPS34/Vps34 activity is the consequence of the enzyme being incorporated into two mutually exclusive complexes: complex I for autophagy, composed of VPS34/Vps34-Vps15/Vps15-Beclin 1/Vps30-ATG14L/Atg14 (mammals/yeast), and complex II for endocytic pathways, in which ATG14L/Atg14 is replaced with UVRAG/Vps38 (mammals/yeast). Because of its involvement in autophagy, defects in which are closely associated with human diseases such as cancer and neurodegenerative diseases, developing highly selective drugs that target specific VPS34/Vps34 complexes is an essential goal in the autophagy field. Recent studies on the activation mechanisms of VPS34/Vps34 complexes have revealed that a variety of factors, including conformational changes, lipid physicochemical parameters, upstream regulators, and downstream effectors, greatly influence the activity of these complexes. This review summarizes and highlights each of these influences as well as clarifying key questions remaining in the field and outlining future perspectives.

Unsaturation, curvature and charge: effects of membrane parameters on PIK3C3/VPS34-containing complexes.

Phosphatidylinositol-3-phosphate (PtdIns3P) is essential for generating autophagosomes and regulating endocytic trafficking. Recently, we have shown that the activities of human PIK3C3/VPS34-containing complexes I and II, which synthesize PtdIns3P, are greatly affected by three membrane physicochemical parameters: lipid unsaturation, membrane curvature, and negative charge. Both complexes are more active on membranes composed of unsaturated lipids than saturated lipids, and high membrane curvature can compensate for the negative effect of high lipid saturation. Negatively charged phosphatidylserine (PS) activates the complexes, as well as PIK3C3/VPS34 alone. The kinase activity of complex I depends critically on the ATG14 BATS domain, whereas complex II relies on the BECN1 BARA domain. Our findings highlight the importance of the membrane character as sensed by the unique membrane binding motifs/domain of the complexes for regulating PIK3C3/VPS34 activity.

The randomized ZIPANGU trial of ranibizumab and adjunct laser for macular edema following branch retinal vein occlusion in treatment-naïve patients.

The ZIPANGU study assessed the efficacy and safety of ranibizumab as a one loading dose + pro re nata (one + PRN) regimen with/without focal/grid laser among treatment-naïve patients suffering from macular edema (ME) following branch retinal vein occlusion (BRVO). ZIPANGU was a phase IV, prospective, randomized, open-label, active-controlled, 12-month, two-arm, multicenter study. Treatment-naïve patients with visual impairment (19-73 letters) caused by ME, defined as central subfield thickness (CSFT) > 300 µm, due to BRVO were randomly assigned to ranibizumab monotherapy (n = 29) or combination therapy (ranibizumab + focal/grid short-pulse laser, n = 30). The primary endpoint was the number of ranibizumab injections. Secondary endpoints were mean changes in best-corrected visual acuity (BCVA) and CSFT, and safety. There were no statistically significant differences in the mean number of ranibizumab injections between monotherapy (4.3 injections) vs. combination (4.1 injections) therapy, or in CSFT. BCVA improvement in the monotherapy arm (22.0 letters) was better than the combination therapy arm (15.0 letters) (p = 0.035). Overall, both regimens appeared to be safe and well tolerated. One + PRN ranibizumab is safe and efficacious in treatment-naïve patients with ME secondary to BRVO. A conjunctive laser treatment did not lead to better functional outcomes or fewer ranibizumab injections.

Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes.

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.

Membrane characteristics tune activities of endosomal and autophagic human VPS34 complexes.

The lipid kinase VPS34 orchestrates diverse processes, including autophagy, endocytic sorting, phagocytosis, anabolic responses and cell division. VPS34 forms various complexes that help adapt it to specific pathways, with complexes I and II being the most prominent ones. We found that physicochemical properties of membranes strongly modulate VPS34 activity. Greater unsaturation of both substrate and non-substrate lipids, negative charge and curvature activate VPS34 complexes, adapting them to their cellular compartments. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of complexes I and II on membranes elucidated structural determinants that enable them to bind membranes. Among these are the Barkor/ATG14L autophagosome targeting sequence (BATS), which makes autophagy-specific complex I more active than the endocytic complex II, and the Beclin1 BARA domain. Interestingly, even though Beclin1 BARA is common to both complexes, its membrane-interacting loops are critical for complex II, but have only a minor role for complex I.