Biology subjects

Jain, B. K.

Publications and source records attributed to Jain, B. K..

5 recordsLinked to original sources

bioRxiv2025

P4-ATPase control over phosphoinositide membrane asymmetry and neomycin resistance

Neomycin, an aminoglycoside antibiotic, has robust antibacterial properties, yet its clinical utility is curtailed by its nephrotoxicity and ototoxicity. The mechanism by which the polycationic neomycin enters specific eukaryotic cell types remains poorly understood. In budding yeast, NEO1 is required for neomycin resistance and encodes a phospholipid flippase that establishes membrane asymmetry. Here, we show that mutations altering Neo1 substrate recognition cause neomycin hypersensitivity by exposing phosphatidylinositol-4-phosphate (PI4P) in the plasma membrane extracellular leaflet. Human cells also expose extracellular PI4P upon knockdown of ATP9A, a Neo1 ortholog and ATP9A expression level correlates to neomycin sensitivity. In yeast, the extracellular PI4P is initially produced in the cytosolic leaflet of the plasma membrane and then delivered by Osh6-dependent nonvesicular transport to the endoplasmic reticulum (ER). Here, a portion of PI4P escapes degradation by the Sac1 phosphatase by entering the ER lumenal leaflet. COPII vesicles transport lumenal PI4P to the Golgi where Neo1 flips this substrate back to the cytosolic leaflet. Cryo-EM reveals that PI4P binds Neo1 within the substrate translocation pathway. Loss of Neo1 activity in the Golgi allows secretion of extracellular PI4P, which serves as a neomycin receptor and facilitates its endocytic uptake. These findings unveil novel mechanisms of aminoglycoside sensitivity and phosphoinositide homeostasis, with important implications for signaling by extracellular phosphoinositides.

cell biology↗

bioRxiv2022

Lipid transport by Candida albicans Dnf2 is required for hyphal growth and virulence

Candida albicans is a common cause of human mucosal yeast infections, and invasive candidiasis can be fatal. Antifungal medications are limited, but those targeting the pathogen cell wall or plasma membrane have been effective. Therefore, virulence factors controlling membrane biogenesis are potential targets for drug development. P4-ATPases contribute to membrane biogenesis by selecting and transporting specific lipids from the extracellular leaflet to the cytoplasmic leaflet of the bilayer to generate lipid asymmetry. A subset of heterodimeric P4-ATPases, including Dnf1-Lem3 and Dnf2-Lem3 from Saccharomyces cerevisiae, transport phosphatidylcholine (PC), phosphatidylethanolamine (PE), and the sphingolipid glucosylceramide (GlcCer). GlcCer is a critical lipid for Candida albicans polarized growth and virulence, but the role of GlcCer transporters in virulence has not been explored. Here we show that the Candida albicans Dnf2 (CaDnf2) requires association with CaLem3 to form a functional transporter and flip fluorescent derivatives of GlcCer, PC and PE across the plasma membrane. Mutation of conserved substrate-selective residues in the membrane domain strongly abrogates GlcCer transport and partially disrupts PC transport by CaDnf2. Candida strains harboring dnf2 null alleles (dnf2{Delta}{Delta}) or point mutations that disrupt substrate recognition exhibit defects in the yeast to hyphal growth transition, filamentous growth and virulence in systemically infected mice. The influence of CaDNF1 deletion on the morphological phenotypes is negligible although the dnf1{Delta}{Delta} dnf2{Delta}{Delta} strain was less virulent than the dnf2{Delta}{Delta} strain. These results indicate that the transport of GlcCer and/or PC by plasma membrane P4-ATPases is important for pathogenicity of Candida albicans.

microbiology↗

bioRxiv2021

Structural basis of the P4B ATPase lipid flippase activity

P4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic -subunit and accessory {beta}-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a {beta}-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the first structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1s ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.

biochemistry↗

bioRxiv2020

Transport mechanism of class-3 P4 ATPase lipid flippases

The P4 ATPases are a large family of membrane-embedded enzymes that use ATP hydrolysis to transport large lipid substrates across lipid bilayers. P4 ATPases differ in their cellular membrane location and their substrates. The structures of the endosome- and Golgi-localized class-1 phosphatidylserine flippases--such as the yeast Drs2 and human ATP8A1--have recently been reported, revealing a substrate binding site on the lumenal side and several transport states. However, a substrate binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases in other classes are still unknown. Here we report a systematic structural and functional study on two plasma-membrane localized, class-3 P4 ATPases that have broader substrate specificity, the S. cerevisiae Dnf1-Lem3 and Dnf2-Lem3 complexes. We have captured substrate lipids on both the exoplasmic and cytosolic sides, and we found that these two enzymes have very similar structures, consistent with their high sequence identity and redundant function. Unexpectedly, Lem3 contributes to substrate binding near the cytosolic surface. We found that the conformational transitions through the substrate transport cycle of these two class-3 enzymes match those of the class-1 enzymes, suggesting a conserved lipid-flipping mechanism among all classes of the P4 ATPases. Our study also revealed a helix-turn-helix insertion in the cytosolic P domain that is unique to the class-3 enzymes and plays a crucial role in their function. Therefore, the P4 ATPases may have retained an overall transport mechanism while evolving distinct features for cellular membrane localization, regulatory mechanisms, and transporting different lipid substrates.

biochemistry↗

bioRxiv2020

Exofacial membrane composition and lipid metabolism regulates plasma membrane P4-ATPase substrate specificity

The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. The regulation of membrane asymmetry is a fundamental characteristic of membrane biology, and is crucial for signal transduction, vesicle transport, and cell division. The type-IV family of P-ATPases, or P4-ATPases, establish membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is still unclear how these enzymes sort through the spectrum of lipids within the membrane to identify their desired substrate(s) and how the membrane environment modulates this activity. Therefore, we tested how the yeast plasma membrane P4-ATPase, Dnf2, responds to changes in membrane composition induced by perturbation of endogenous lipid biosynthetic pathways or exogenous application of lipid. The primary substrates of Dnf2 are two chemically divergent lipids, glucosylceramide (GlcCer) and phosphatidylcholine ((PC) or their lyso-lipid derivatives), and we find that these substrates compete with each other for transport. Acutely inhibiting sphingolipid synthesis using myriocin attenuates transport of exogenously applied GlcCer without perturbing PC transport. Deletion of genes controlling later steps of glycosphingolipid production also perturb GlcCer transport to a greater extent than PC transport. Surprisingly, application of lipids that are poor transport substrates differentially affect PC and GlcCer transport by Dnf2, thus altering substrate preference. Our data indicate that Dnf2 exhibits exquisite sensitivity to the membrane composition; thus, providing feedback onto the function of the P4-ATPases.

cell biology↗