The lipid flippase ATP8A1 regulates the recruitment of ARF effectors to the trans-Golgi Network.

Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.

Direct evidence of lipid transport by the Drs2-Cdc50 flippase upon truncation of its terminal regions.

P4-ATPases in complex with Cdc50 subunits are lipid flippases that couple ATP hydrolysis with lipid transport to the cytoplasmic leaflet of membranes to create lipid asymmetry. Such vectorial transport has been shown to contribute to vesicle formation in the late secretory pathway. Some flippases are regulated by autoinhibitory regions that can be destabilized by protein kinase-mediated phosphorylation and possibly by binding of cytosolic proteins. In addition, the binding of lipids to flippases may also induce conformational changes required for the activity of these transporters. Here, we address the role of phosphatidylinositol-4-phosphate (PI4P) and the terminal autoinhibitory tails on the lipid flipping activity of the yeast lipid flippase Drs2-Cdc50. By functionally reconstituting the full-length and truncated forms of Drs2 in a 1:1 complex with the Cdc50 subunit, we provide compelling evidence that lipid flippase activity is exclusively detected for the truncated Drs2 variant and is dependent on the presence of the phosphoinositide PI4P. These findings highlight the critical role of phosphoinositides as lipid co-factors in the regulation of lipid transport by the Drs2-Cdc50 flippase.

The Alternating Access Mechanism in Mammalian Multidrug Resistance Transporters and Their Bacterial Homologs.

Multidrug resistance (MDR) proteins belonging to the ATP-Binding Cassette (ABC) transporter group play a crucial role in the export of cytotoxic drugs across cell membranes. These proteins are particularly fascinating due to their ability to confer drug resistance, which subsequently leads to the failure of therapeutic interventions and hinders successful treatments. One key mechanism by which multidrug resistance (MDR) proteins carry out their transport function is through alternating access. This mechanism involves intricate conformational changes that enable the binding and transport of substrates across cellular membranes. In this extensive review, we provide an overview of ABC transporters, including their classifications and structural similarities. We focus specifically on well-known mammalian multidrug resistance proteins such as MRP1 and Pgp (MDR1), as well as bacterial counterparts such as Sav1866 and lipid flippase MsbA. By exploring the structural and functional features of these MDR proteins, we shed light on the roles of their nucleotide-binding domains (NBDs) and transmembrane domains (TMDs) in the transport process. Notably, while the structures of NBDs in prokaryotic ABC proteins, such as Sav1866, MsbA, and mammalian Pgp, are identical, MRP1 exhibits distinct characteristics in its NBDs. Our review also emphasizes the importance of two ATP molecules for the formation of an interface between the two binding sites of NBD domains across all these transporters. ATP hydrolysis occurs following substrate transport and is vital for recycling the transporters in subsequent cycles of substrate transportation. Specifically, among the studied transporters, only NBD2 in MRP1 possesses the ability to hydrolyze ATP, while both NBDs of Pgp, Sav1866, and MsbA are capable of carrying out this reaction. Furthermore, we highlight recent advancements in the study of MDR proteins and the alternating access mechanism. We discuss the experimental and computational approaches utilized to investigate the structure and dynamics of MDR proteins, providing valuable insights into their conformational changes and substrate transport. This review not only contributes to an enhanced understanding of multidrug resistance proteins but also holds immense potential for guiding future research and facilitating the development of effective strategies to overcome multidrug resistance, thus improving therapeutic interventions.

P4-ATPase subunit Cdc50 plays a role in yeast budding and cell wall integrity in Candida glabrata.

BACKGROUND: As highly-conserved types of lipid flippases among fungi, P4-ATPases play a significant role in various cellular processes. Cdc50 acts as the regulatory subunit of flippases, forming heterodimers with Drs2 to translocate aminophospholipids. Cdc50 homologs have been reported to be implicated in protein trafficking, drug susceptibility, and virulence in Saccharomyces cerevisiae, Candida albicans and Cryptococcus neoformans. It is likely that Cdc50 has an extensive influence on fungal cellular processes. The present study aimed to determine the function of Cdc50 in Candida glabrata by constructing a Δcdc50 null mutant and its complemented strain. RESULTS: In Candida glabrata, the loss of Cdc50 led to difficulty in yeast budding, probably caused by actin depolarization. The Δcdc50 mutant also showed hypersensitivity to azoles, caspofungin, and cell wall stressors. Further experiments indicated hyperactivation of the cell wall integrity pathway in the Δcdc50 mutant, which elevated the major cell wall contents. An increase in exposure of ß-(1,3)-glucan and chitin on the cell surface was also observed through flow cytometry. Interestingly, we observed a decrease in the phagocytosis rate when the Δcdc50 mutant was co-incubated with THP-1 macrophages. The Δcdc50 mutant also exhibited weakened virulence in nematode survival tests. CONCLUSION: The results suggested that the lipid flippase subunit Cdc50 is implicated in yeast budding and cell wall integrity in C. glabrata, and thus have a broad influence on drug susceptibility and virulence. This work highlights the importance of lipid flippase, and offers potential targets for new drug research.

Functional Analysis of the P-Type ATPases Apt2-4 from Cryptococcus neoformans by Heterologous Expression in Saccharomyces cerevisiae.

Lipid flippases of the P4-ATPase family actively transport phospholipids across cell membranes, an activity essential for key cellular processes such as vesicle budding and membrane trafficking. Members of this transporter family have also been implicated in the development of drug resistance in fungi. The encapsulated fungal pathogen Cryptococcus neoformans contains four P4-ATPases, among which Apt2-4p are poorly characterized. Using heterologous expression in the flippase-deficient S. cerevisiae strain dnf1Δdnf2Δdrs2Δ, we tested their lipid flippase activity in comparison to Apt1p using complementation tests and fluorescent lipid uptake assays. Apt2p and Apt3p required the co-expression of the C. neoformans Cdc50 protein for activity. Apt2p/Cdc50p displayed a narrow substrate specificity, limited to phosphatidylethanolamine and -choline. Despite its inability to transport fluorescent lipids, the Apt3p/Cdc50p complex still rescued the cold-sensitive phenotype of dnf1Δdnf2Δdrs2Δ, suggesting a functional role for the flippase in the secretory pathway. Apt4p, the closest homolog to Saccharomyces Neo1p, which does not require a Cdc50 protein, was unable to complement several flippase-deficient mutant phenotypes, neither in the presence nor absence of a ß-subunit. These results identify C. neoformans Cdc50 as an essential subunit for Apt1-3p and provide a first insight into the molecular mechanisms underlying their physiological functions.

Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders.

P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with severe diseases, for example in ATP8B1 causing progressive familial intrahepatic cholestasis, a rare inherited disorder progressing toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report functional studies and the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 Å resolution. We find that ATP8B1 is autoinhibited by its N- and C-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the C-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminal segment further suggests molecular communication between N- and C-termini in the autoinhibition and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A recurring (G/A)(Y/F)AFS motif of the C-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.

High-Throughput Mutagenesis and Cross-Complementation Experiments Reveal Substrate Preference and Critical Residues of the Capsule Transporters in Streptococcus pneumoniae.

MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family transporters are found in almost all life forms. They are responsible for transporting lipid-linked precursors across the cell membrane to support the synthesis of various glycoconjugates. While significant progress has been made in elucidating their transport mechanism, how these transporters select their substrates remains unclear. Here, we systematically tested the MOP transporters in the Streptococcus pneumoniae capsule pathway for their ability to translocate noncognate capsule precursors. Sequence similarity cannot predict whether these transporters are interchangeable. We showed that subtle changes in the central aqueous cavity of the transporter are sufficient to accommodate a different cargo. These changes can occur naturally, suggesting a potential mechanism of expanding substrate selectivity. A directed evolution experiment was performed to identify gain-of-function variants that translocate a noncognate cargo. Coupled with a high-throughput mutagenesis and sequencing (Mut-seq) experiment, residues that are functionally important for the capsule transporter were revealed. Lastly, we showed that the expression of a flippase that can transport unfinished precursors resulted in an increased susceptibility to bacitracin and mild cell shape defects, which may be a driving force to maintain transporter specificity. IMPORTANCE All licensed pneumococcal vaccines target the capsular polysaccharide (CPS). This layer is highly variable and is important for virulence in many bacterial pathogens. Most of the CPSs are produced by the Wzx/Wzy mechanism. In this pathway, CPS repeating units are synthesized in the cytoplasm, which must be flipped across the cytoplasmic membrane before polymerization. This step is mediated by the widely conserved MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family transporters. Here, we systematically evaluated the interchangeability of these transporters and identified the residues important for substrate specificity and function. Understanding how CPS is synthesized will inform glycoengineering, vaccine development, and antimicrobial discovery.

Lipid flippases in polarized growth.

Polarized growth is required in eukaryotic cells for processes such as cell division, morphogenesis and motility, which involve conserved and interconnected signalling pathways controlling cell cycle progression, cytoskeleton reorganization and secretory pathway functioning. While many of the factors involved in polarized growth are known, it is not yet clear how they are coordinated both spatially and temporally. Several lines of evidence point to the important role of lipid flippases in polarized growth events. Lipid flippases, which mainly belong to the P4 subfamily of P-type ATPases, are active transporters that move different lipids to the cytosolic side of biological membranes at the expense of ATP. The involvement of the Saccharomyces cerevisiae plasma membrane P4 ATPases Dnf1p and Dnf2p in polarized growth and their activation by kinase phosphorylation were established some years ago. However, these two proteins do not seem to be responsible for the phosphatidylserine internalization required for early recruitment of proteins to the plasma membrane during yeast mating and budding. In a recent publication, we demonstrated that the Golgi-localized P4 ATPase Dnf3p has a preference for PS as a substrate, can reach the plasma membrane in a cell cycle-dependent manner, and is regulated by the same kinases that activate Dnf1p and Dnf2p. This finding solves a long-lasting enigma in the field of lipid flippases and suggests that tight and heavily coordinated spatiotemporal control of lipid translocation at the plasma membrane is important for proper polarized growth.

ATP2, The essential P4-ATPase of malaria parasites, catalyzes lipid-stimulated ATP hydrolysis in complex with a Cdc50 ß-subunit.

Gene targeting approaches have demonstrated the essential role for the malaria parasite of membrane transport proteins involved in lipid transport and in the maintenance of membrane lipid asymmetry, representing emerging oportunites for therapeutical intervention. This is the case of ATP2, a Plasmodium-encoded 4 P-type ATPase (P4-ATPase or lipid flippase), whose activity is completely irreplaceable during the asexual stages of the parasite. Moreover, a recent chemogenomic study has situated ATP2 as the possible target of two antimalarial drug candidates. In eukaryotes, P4-ATPases assure the asymmetric phospholipid distribution in membranes by translocating phospholipids from the outer to the inner leaflet. In this work, we have used a recombinantly-produced P. chabaudi ATP2 (PcATP2), to gain insights into the function and structural organization of this essential transporter. Our work demonstrates that PcATP2 associates with two of the three Plasmodium-encoded Cdc50 proteins: PcCdc50B and PcCdc50A. Purified PcATP2/PcCdc50B complex displays ATPase activity in the presence of either phosphatidylserine or phosphatidylethanolamine. In addition, this activity is upregulated by phosphatidylinositol 4-phosphate. Overall, our work describes the first biochemical characterization of a Plasmodium lipid flippase, a first step towards the understanding of the essential physiological role of this transporter and towards its validation as a potential antimalarial drug target.

Transport mechanism of P4 ATPase phosphatidylcholine flippases.

The P4 ATPases use ATP hydrolysis to transport large lipid substrates across lipid bilayers. The structures of the endosome- and Golgi-localized phosphatidylserine flippases-such as the yeast Drs2 and human ATP8A1-have recently been reported. However, a substrate-binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases with other substrates are unknown. Here, we report structures of the S. cerevisiae Dnf1-Lem3 and Dnf2-Lem3 complexes. We captured substrate phosphatidylcholine molecules on both the exoplasmic and cytosolic sides and found that they have similar structures. Unexpectedly, Lem3 contributes to substrate binding. The conformational transitions of these phosphatidylcholine transporters match those of the phosphatidylserine transporters, suggesting a conserved mechanism among P4 ATPases. Dnf1/Dnf2 have a unique P domain helix-turn-helix insertion that is important for function. Therefore, P4 ATPases may have retained an overall transport mechanism while evolving distinct features for different lipid substrates.

Pseudohyphal growth in Saccharomyces cerevisiae involves protein kinase-regulated lipid flippases.

Lipid flippases of the P4 ATPase family establish phospholipid asymmetry in eukaryotic cell membranes and are involved in many essential cellular processes. The yeast Saccharomyces cerevisiae contains five P4 ATPases, among which Dnf3p is poorly characterized. Here, we demonstrate that Dnf3p is a flippase that catalyzes translocation of major glycerophospholipids, including phosphatidylserine, towards the cytosolic membrane leaflet. Deletion of the genes encoding Dnf3p and the distantly related P4 ATPases Dnf1p and Dnf2p results in yeast mutants with aberrant formation of pseudohyphae, suggesting that the Dnf1p-Dnf3p proteins have partly redundant functions in the control of this specialized form of polarized growth. Furthermore, as previously demonstrated for Dnf1 and Dnf2p, the phospholipid flipping activity of Dnf3p is positively regulated by flippase kinase 1 (Fpk1p) and Fpk2p. Phylogenetic analyses demonstrate that Dnf3p belongs to a subfamily of P4 ATPases specific for fungi and are likely to represent a hallmark of fungal evolution.

More than flipping the lid: Cdc50 contributes to echinocandin resistance by regulating calcium homeostasis in Cryptococcus neoformans.

Echinocandins are the newest fungicidal drug class approved for clinical use against common invasive mycoses. Yet, they are ineffective against cryptococcosis, predominantly caused by Cryptococcus neoformans. The underlying mechanisms of innate echinocandin resistance in C. neoformans remain unclear. We know that Cdc50, the ß-subunit of the lipid translocase (flippase), mediates echinocandin resistance, as loss of the CDC50 gene sensitizes C. neoformans to caspofungin, a member of the echinocandins class. We sought to elucidate how Cdc50 facilitates caspofungin resistance by performing a forward genetic screen for cdc50Δ suppressor mutations that are caspofungin resistant. We identified a novel mechanosensitive calcium channel protein Crm1 that correlates with Cdc50 function (Cao et al., 2019). In addition to regulating phospholipid translocation, Cdc50 also interacts with Crm1 to regulate intracellular calcium homeostasis and calcium/calcineurin signaling that likely drives caspofungin resistance in C. neoformans. Our study revealed a novel dual function of Cdc50 that connects lipid flippase with calcium signaling. These unexpected findings provide new insights into the mechanisms of echinocandin resistance in C. neoformans that may drive future drug design.

Mutated ATP10B increases Parkinson's disease risk by compromising lysosomal glucosylceramide export.

Parkinson's disease (PD) is a progressive neurodegenerative brain disease presenting with a variety of motor and non-motor symptoms, loss of midbrain dopaminergic neurons in the substantia nigra pars compacta and the occurrence of α-synuclein-positive Lewy bodies in surviving neurons. Here, we performed whole exome sequencing in 52 early-onset PD patients and identified 3 carriers of compound heterozygous mutations in the ATP10B P4-type ATPase gene. Genetic screening of a Belgian PD and dementia with Lewy bodies (DLB) cohort identified 4 additional compound heterozygous mutation carriers (6/617 PD patients, 0.97%; 1/226 DLB patients, 0.44%). We established that ATP10B encodes a late endo-lysosomal lipid flippase that translocates the lipids glucosylceramide (GluCer) and phosphatidylcholine (PC) towards the cytosolic membrane leaflet. The PD associated ATP10B mutants are catalytically inactive and fail to provide cellular protection against the environmental PD risk factors rotenone and manganese. In isolated cortical neurons, loss of ATP10B leads to general lysosomal dysfunction and cell death. Impaired lysosomal functionality and integrity is well known to be implicated in PD pathology and linked to multiple causal PD genes and genetic risk factors. Our results indicate that recessive loss of function mutations in ATP10B increase risk for PD by disturbed lysosomal export of GluCer and PC. Both ATP10B and glucocerebrosidase 1, encoded by the PD risk gene GBA1, reduce lysosomal GluCer levels, emerging lysosomal GluCer accumulation as a potential PD driver.

A Mechanosensitive Channel Governs Lipid Flippase-Mediated Echinocandin Resistance in Cryptococcus neoformans.

Echinocandins show fungicidal activity against common invasive mycoses but are ineffective against cryptococcosis. The underlying mechanism for echinocandin resistance in Cryptococcus neoformans remains poorly understood but has been shown to involve Cdc50, the regulatory subunit of lipid flippase. In a forward genetic screen for cdc50Δ suppressor mutations that are caspofungin resistant, we identified Crm1 (caspofungin resistant mutation 1), a homolog of mechanosensitive channel proteins, and showed that crm1Δ restored caspofungin resistance in cdc50Δ cells. Caspofungin-treated cdc50Δ cells exhibited abnormally high intracellular calcium levels ([Ca2+]c) and heightened activation of the calcineurin pathway. Deletion of CRM1 in the cdc50Δ background normalized the abnormally high [Ca2+]c. Cdc50 interacts with Crm1 to maintain cellular calcium homeostasis. Analysis of chitin/chitosan content showed that deleting CRM1 reversed the decreased chitosan production of cdc50Δ cells. Together, these results demonstrate that Cdc50 and Crm1 regulation of the calcineurin pathway and cytoplasmic calcium homeostasis may underlie caspofungin resistance in C. neoformansIMPORTANCECryptococcus neoformans is the leading cause of fungal meningitis, accounting for ∼15% of HIV/AIDS-related deaths, but treatment options for cryptococcosis are limited. Echinocandins are the newest fungicidal drug class introduced but are ineffective in treating cryptococcosis. Our previous study identified the lipid flippase subunit Cdc50 as a contributor to echinocandin resistance in C. neoformans Here, we further elucidated the mechanism of Cdc50-mediated caspofungin drug resistance. We discovered that Cdc50 interacts with the mechanosensitive calcium channel protein Crm1 to regulate calcium homeostasis and caspofungin resistance via calcium/calcineurin signaling. These results provide novel insights into echinocandin resistance in this pathogen, which may lead to new treatment options, as well as inform echinocandin resistance mechanisms in other fungal organisms and, hence, advance our understanding of modes of antifungal drug susceptibility and resistance.

Structural Insights into the Lipid A Transport Pathway in MsbA.

MsbA is an essential ATP-binding cassette transporter in Gram-negative bacteria that transports lipid A and lipopolysaccharide from the cytoplasmic leaflet to the periplasmic leaflet of the inner membrane. Here we report the X-ray structure of MsbA from Salmonella typhimurium at 2.8-Å resolution in an inward-facing conformation after cocrystallization with lipid A and using a stabilizing facial amphiphile. The structure displays a large amplitude opening in the transmembrane portal, which is likely required for lipid A to pass from its site of synthesis into the protein-enclosed transport pathway. Putative lipid A density is observed further inside the transmembrane cavity, consistent with a trap and flip model. Additional electron density attributed to lipid A is observed near an outer surface cleft at the periplasmic ends of the transmembrane helices. These findings provide new structural insights into the lipid A transport pathway through comparative analysis with existing MsbA structures.

The lipid head group is the key element for substrate recognition by the P4 ATPase ALA2: a phosphatidylserine flippase.

Type IV P-type ATPases (P4 ATPases) are lipid flippases that catalyze phospholipid transport from the exoplasmic to the cytoplasmic leaflet of cellular membranes, but the mechanism by which they recognize and transport phospholipids through the lipid bilayer remains unknown. In the present study, we succeeded in purifying recombinant aminophospholipid ATPase 2 (ALA2), a member of the P4 ATPase subfamily in Arabidopsis thaliana, in complex with the ALA-interacting subunit 5 (ALIS5). The ATP hydrolytic activity of the ALA2-ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine. Small changes in the stereochemistry or the functional groups of the phosphatidylserine head group affected enzymatic activity, whereas alteration in the length and composition of the acyl chains only had minor effects. Likewise, the enzymatic activity of the ALA2-ALIS5 complex was stimulated by both mono- and di-acyl phosphatidylserines. Taken together, the results identify the lipid head group as the key structural element for substrate recognition by the P4 ATPase.

Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli.

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophile Thermosipho africanus Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

ALA6, a P4-type ATPase, Is Involved in Heat Stress Responses in Arabidopsis thaliana.

Maintaining lipid membrane integrity is an essential aspect of plant tolerance to high temperature. P4-type ATPases are responsible for flipping and stabilizing asymmetric phospholipids in membrane systems, though their functions in stress tolerance are not entirely clear. Aminophospholipid ATPase6 (ALA6) is a member of the P4-type ATPase family, which has 12 members in Arabidopsis thaliana. Here, we show that a loss-of-function mutant of ALA6 (ala6) exhibits clear sensitivity to heat stress, including both basal and acquired thermotolerance treatments. Overexpression of ALA6 improves seedling resistance to heat stress, while mutated ALA6 transgenic plants, in which the conserved functional site of the ALA family has a point mutation, are still susceptible to heat stress like ala6 loss-of-function mutant. In addition, ala6 displays higher ion-leakage during heat treatment, suggesting that the lipid flippase activity of ALA6 plays a vital role in heat stress responses. Transcriptome analysis reveals differences in gene expression between ala6 and wild-type plants with or without heat stress. The differentially expressed genes are involved primarily in the physiological processes of stress response, cellular compartment maintenance, macromolecule stability and energy production. Our results suggest that ALA6 is crucial for the stability of membrane when plants suffer from high temperature stress.

Lipid flippases promote antiviral silencing and the biogenesis of viral and host siRNAs in Arabidopsis.

Dicer-mediated processing of virus-specific dsRNA into short interfering RNAs (siRNAs) in plants and animals initiates a specific antiviral defense by RNA interference (RNAi). In this study, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in Arabidopsis thaliana Using whole-genome sequencing and a computational pipeline, we identified aminophospholipid transporting ATPase 2 (ALA2) and the related ALA1 in the type IV subfamily of P-type ATPases as key components of antiviral RNAi. ALA1 and ALA2 are flippases, which are transmembrane lipid transporter proteins that transport phospholipids across cellular membranes. We found that the ala1/ala2 single- and double-mutant plants exhibited enhanced disease susceptibility to cucumber mosaic virus when the virus-encoded function to suppress RNAi was disrupted. Notably, the antiviral activity of both ALA1 and ALA2 was abolished by a single amino acid substitution known to inactivate the flippase activity. Genetic analysis revealed that ALA1 and ALA2 acted to enhance the amplification of the viral siRNAs by RNA-dependent RNA polymerase (RdRP) 1 (RDR1) and RDR6 and of the endogenous virus-activated siRNAs by RDR1. RNA virus replication by plant viral RdRPs occurs inside vesicle-like membrane invaginations induced by the recruitment of the viral RdRP and host factors to subcellular membrane microdomains enriched with specific phospholipids. Our results suggest that the phospholipid transporter activity of ALA1/ALA2 may be necessary for the formation of similar invaginations for the synthesis of dsRNA precursors of highly abundant viral and host siRNAs by the cellular RdRPs.

Cryptococcus flips its lid - membrane phospholipid asymmetry modulates antifungal drug resistance and virulence.

Human fungal infections are increasing in prevalence and acquisition of antifungal drug resistance, while our antifungal drug armamentarium remains very limited, constituting a significant public health problem. Despite the fact that prominent antifungal drugs target the fungal cell membrane, very little is known about how fungal membrane biology regulates drug-target interactions. Asymmetrical phospholipid distribution is an essential property of biological membranes, which is maintained by a group of transporters that dynamically translocate specific phospholipid groups across the membrane bilayer. Lipid flippase is the enzyme responsible for translocation of certain phospholipids, including phosphatidylserine (PS), across the plasma membrane from the exocytoplasmic to the cytoplasmic leaflet. Loss of lipid flippase leads to abnormal phospholipid distribution and impaired intracellular vesicular trafficking. The recent research article by Huang et al. reported that in pathogenic fungus Cryptococcus neoformans loss of lipid flippase activity sensitized cryptococcal cells to multiple classes of antifungal drugs, including the cell wall active echinocandins, and abolished fungal virulence in murine models. This finding demonstrates that lipid flippase may promote fungal drug resistance and virulence and indicates that this enzyme may represent a novel antifungal drug target.