The erlin1/erlin2 (E1/E2) complex is an endoplasmic reticulum membrane-located assemblage of the proteins erlin1 and erlin2. Here, we demonstrate direct and selective binding of phosphatidylinositol 3-phosphate (PI(3)P) to recombinant erlins and that disruption or deletion of the E1/E2 complex reduces HeLa cell PI(3)P levels by ∼50 %. This reduction correlated with a decrease in autophagic flux, with no effect on the endocytic pathway, and was not due to reduced VPS34 kinase activity, which is critical for maintaining steady-state PI(3)P levels. Pharmacological inhibition of VPS34 and suppression of PI(3)P levels caused a similar reduction in autophagic flux. Overall, these data indicate that by binding to PI(3)P, the E1/E2 complex plays an important role in maintaining the steady-state levels of PI(3)P and, thus, sustains some key PI(3)P-dependent processes, e.g., autophagy.

KRAS is a small GTPase, ubiquitously expressed in mammalian cells, that functions as a molecular switch to regulate cell proliferation and differentiation. Oncogenic mutations that render KRAS constitutively active occur frequently in human cancers. KRAS must localize to the plasma membrane (PM) for biological activity. KRAS PM binding is mediated by interactions of the KRAS membrane anchor with phosphatidylserine (PtdSer), therefore, depleting PM PtdSer content abrogates KRAS PM binding and oncogenic function. From a genome-wide siRNA screen to search for genes that regulate KRAS PM localization, we identified a set of phosphatidylinositol (PI) 3-phosphatase family members: myotubularin-related (MTMR) proteins 2, 3, 4 and 7. Here we show that knockdown of MTMR 2/3/4/7 expression disrupts KRAS PM interactions. The molecular mechanism involves depletion of PM PI 4-phosphate (PI4P) levels, which in turn disrupts the subcellular localization and operation of oxysterol-binding protein related protein (ORP) 5, a PtdSer lipid transfer protein that maintains PM PtdSer content. Concomitantly, silencing MTMR 2/3/4/7 expression elevates PM levels of PI3P and reduces PM and total cellular levels of PtdSer. In summary we propose that the PI 3-phosphatase activity provided by MTMR proteins is required to generate PM PI for the synthesis of PM PI4P, which in turn, promotes the PM localization of PtdSer and KRAS.

Breast cancer (BC) is considered the leading cause of death among females worldwide. Various risk factors contribute to BC development, such as age, genetics, reproductive factors, obesity, alcohol intake, and lifestyle. Obesity is considered to be a pandemic health problem globally, affecting millions of people worldwide. Obesity has been associated with a high risk of BC development. Determining the impact of obesity on BC development risk in women by demonstrating the molecular and genetic association in pre- and post-menopause females and risk to BC initiation is crucial in order to improve the diagnosis and prognosis of BC disease. In epidemiological studies, BC in premenopausal women was shown to be protective in a certain pattern. These altered effects between the two phases could be due to various physiological changes, such as estrogen/progesterone fluctuating levels. In addition, the relationship between BC risk and obesity is indicated by different molecular alterations as metabolic pathways and genetic mutation or epigenetic DNA changes supporting a strong connection between obesity and BC risk. However, these molecular and genetic alteration remain incompletely understood. The aim of this review is to highlight and elucidate the different molecular mechanisms and genetic changes occurring in obese women and their association with BC risk and development.

Iron homeostasis, which is pivotal to virulence, is regulated by the phosphatidylinositol 3-kinase CgVps34 in the human fungal pathogen Candida glabrata. Here, we identify CgPil1 as a phosphatidylinositol 3-phosphate (PI3P)-binding protein and unveil its role in retaining the high-affinity iron transporter CgFtr1 at the plasma membrane (PM), with PI3P negatively regulating CgFtr1-CgPil1 interaction. PI3P production and its PM localization are elevated in the high-iron environment. Surplus iron also leads to intracellular distribution and vacuolar delivery of CgPil1 and CgFtr1, respectively, from the PM. Loss of CgPil1 or CgFtr1 ubiquitination at lysines 391 and 401 results in CgFtr1 trafficking to the endoplasmic reticulum and a decrease in vacuole-localized CgFtr1. The E3-ubiquitin ligase CgRsp5 interacts with CgFtr1 and forms distinct CgRsp5-CgFtr1 puncta at the PM, with high iron resulting in their internalization. Finally, PI3P controls retrograde transport of many PM proteins. Altogether, we establish PI3P as a key regulator of membrane transport in C. glabrata.

The mechanisms underlying autophagic defects in nonalcoholic steatohepatitis (NASH) remain largely unknown. We aimed to elucidate the roles of hepatic cyclooxygenase 1 (COX1) in autophagy and the pathogenesis of diet-induced steatohepatitis in mice. Human nonalcoholic fatty liver disease (NAFLD) liver samples were used to examine the protein expression of COX1 and the level of autophagy. Cox1Δhepa mice and their wildtype littermates were generated and fed with 3 different NASH models. We found that hepatic COX1 expression was increased in patients with NASH and diet-induced NASH mice models accompanied by impaired autophagy. COX1 was required for basal autophagy in hepatocytes and liver specific COX1 deletion exacerbated steatohepatitis by inhibiting autophagy. Mechanistically, COX1 directly interacted with WD repeat domain, phosphoinositide interacting 2 (WIPI2), which was crucial for autophagosome maturation. Adeno-associated virus (AAV)-mediated rescue of WIPI2 reversed the impaired autophagic flux and improved NASH phenotypes in Cox1Δhepa mice, indicating that COX1 deletion-mediated steatohepatitis was partially dependent on WIPI2-mediated autophagy. In conclusion, we demonstrated a novel role of COX1 in hepatic autophagy that protected against NASH by interacting with WIPI2. Targeting the COX1-WIPI2 axis may be a novel therapeutic strategy for NASH.

Phosphatidylinositol 3-phosphate (PI3P) is a signaling phospholipid that play a key role in endomembrane trafficking, specifically autophagy and endosomal trafficking. However, the mechanisms underlying the contribution of PI3P downstream effectors to plant autophagy remain unknown. Known PI3P effectors for autophagy in Arabidopsis thaliana include ATG18A (Autophagy-related 18A) and FYVE2 (Fab1p, YOTB, Vac1p, and EEA1 2), which are implicated in autophagosome biogenesis. Here, we report that FYVE3, a paralog of plant-specific FYVE2, plays a role in FYVE2-dependent autophagy. Using yeast two-hybrid and bimolecular fluorescence complementation assays, we determined that the FYVE3 protein was associated with autophagic machinery containing ATG18A and FYVE2, by interacting with ATG8 isoforms. The FYVE3 protein was transported to the vacuole, and the vacuolar delivery of FYVE3 relies on PI3P biosynthesis and the canonical autophagic machinery. Whereas the fyve3 mutation alone barely affects autophagic flux, it suppresses defective autophagy in fyve2 mutants. Based on the molecular genetics and cell biological data, we propose that FYVE3 specifically regulates FYVE2-dependent autophagy.

Rapid phagosomal escape mediated by listeriolysin O (LLO) is a prerequisite for Listeria monocytogenes intracellular replication and pathogenesis. Escape takes place within minutes after internalization from vacuoles that are negative to the early endosomal Rab5 GTPase and positive to the late endosomal Rab7. Using mutant analysis, we found that the listerial invasin InlB was required for optimal intracellular proliferation of L. monocytogenes. Starting from this observation, we determined in HeLa cells that InlB promotes early phagosomal escape and efficient Rab7 acquisition by the Listeria-containing vacuole (LCV). Recruitment of the class III phosphoinositide 3-kinase (PI3K) Vps34 to the LCV and accumulation of its lipid product, phosphatidylinositol 3-phosphate (PI3P), two key endosomal maturation mediators, were also dependent on InlB. Small interfering RNA (siRNA) knockdown experiments showed that Vps34 was required for Rab7 recruitment and early (LLO-mediated) escape and supported InlB-dependent intracellular proliferation. Together, our data indicate that InlB accelerates LCV conversion into an escape-favorable Rab7 late phagosome via subversion of class III PI3K/Vps34 signaling. Our findings uncover a new function for the InlB invasin in Listeria pathogenesis as an intracellular proliferation-promoting virulence factor. IMPORTANCE Avoidance of lysosomal killing by manipulation of the endosomal compartment is a virulence mechanism assumed to be largely restricted to intravacuolar intracellular pathogens. Our findings are important because they show that cytosolic pathogens like L. monocytogenes, which rapidly escape the phagosome after internalization, can also extensively subvert endocytic trafficking as part of their survival strategy. They also clarify that, instead of delaying phagosome maturation (to allow time for LLO-dependent disruption, as currently thought), via InlB L. monocytogenes appears to facilitate the rapid conversion of the phagocytic vacuole into an escape-conducive late phagosome. Our data highlight the multifunctionality of bacterial virulence factors. At the cell surface, the InlB invasin induces receptor-mediated phagocytosis via class I PI3K activation, whereas after internalization it exploits class III PI3K (Vsp34) to promote intracellular survival. Systematically elucidating the mechanisms by which Listeria interferes with PI3K signaling all along the endocytic pathway may lead to novel anti-infective therapies.

Type I inositol polyphosphate-4-phosphatase (INPP4A) belongs to the group of phosphoinositide phosphatases controlling proliferation, apoptosis, and endosome function by hydrolyzing phosphatidylinositol 3,4-bisphosphate. INPP4A produces multiple transcripts encoding shorter and longer INPP4A isoforms with hydrophilic or hydrophobic C-terminus. Biallelic INPP4A truncating variants cause a spectrum of neurodevelopmental disorders ranging from moderate intellectual disability to postnatal microcephaly with developmental and epileptic encephalopathy and (ponto)cerebellar hypoplasia. We report a girl with the novel homozygous INPP4A variant NM_001134224.2:c.2840del/p.(Gly947Glufs*12) (isoform d). She presented with postnatal microcephaly, global developmental delay, visual impairment, myoclonic seizures, and pontocerebellar hypoplasia and died at the age of 27 months. The level of mutant INPP4A mRNAs in proband-derived leukocytes was comparable to controls suggesting production of C-terminally altered INPP4A isoforms. We transiently expressed eGFP-tagged INPP4A isoform a (NM_004027.3) wildtype and p.(Gly908Glufs*12) mutant [p.(Gly947Glufs*12) according to NM_001134224.2] as well as INPP4A isoform b (NM_001566.2) wildtype and p.(Asp915Alafs*2) mutant, previously reported in family members with moderate intellectual disability, in HeLa cells and determined their subcellular distributions. While INPP4A isoform a was preferentially found in perinuclear clusters co-localizing with the GTPase Rab5, isoform b showed a net-like distribution, possibly localizing near and/or on microtubules. Quantification of intracellular localization patterns of the two INPP4A mutants revealed significant differences compared with the respective wildtype and similarity with each other. Our data suggests an important non-redundant function of INPP4A isoforms with hydrophobic or hydrophilic C-terminus in the brain.

Phosphatidylinositol 3-phosphate (PI(3)P) serves important functions in endocytosis, phagocytosis, and autophagy. PI(3)P is generated by Vps34 of the class III phosphatidylinositol 3-kinase (PI3K) complex. The Vps34-PI3K complex can be divided into Vps34-PI3K class II (containing Vps38, endosomal) and Vps34-PI3K class I (containing Atg14, autophagosomal). Most PI(3)Ps are associated with endosomal membranes. In yeast, the endosomal localization of Vps34 and PI(3)P is tightly regulated by Vps21-module proteins. At yeast phagophore assembly site (PAS) or mammalian omegasomes, PI(3)P binds to WD-repeat protein interacting with phosphoinositide (WIPI) proteins to further recruit two conjugation systems, Atg5-Atg12·Atg16 and Atg8-PE (LC3-II), to initiate autophagy. However, the spatiotemporal regulation of PI(3)P during autophagy remains obscure. Therefore, in this study, we determined the effect of Vps21 on localization and interactions of Vps8, Vps34, Atg21, Atg8, and Atg16 upon autophagy induction. The results showed that Vps21 was required for successive colocalizations and interactions of Vps8-Vps34 and Vps34-Atg21 on endosomes, and Atg21-Atg8/Atg16 on the PAS. In addition to disrupted localization of the PI3K complex II subunits Vps34 and Vps38 on endosomes, the localization of the PI3K complex I subunits Vps34 and Atg14, as well as Atg21, was partly disrupted from the PAS in vps21∆ cells. The impaired PI3K-PI(3)P-Atg21-Atg16 axis in vps21∆ cells might delay autophagy, which is consistent with the delay of early autophagy when Atg21 was absent. This study provides the first insight into the upstream sequential regulation of the PI3K-PI(3)P-Atg21-Atg16 module by Vps21 in autophagy.

Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3-phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2-mediated hyperactivation of Cdk5 downstream of receptor- and activity-dependent calcium influx. Our results unravel an unexpected function for PI(3)P-containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P-producing VPS34 kinase in neurological disease and neurodegeneration.