scholarly journals Directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a P4-ATPase

2016 ◽  
Vol 113 (31) ◽  
pp. E4460-E4466 ◽  
Author(s):  
Bartholomew P. Roland ◽  
Todd R. Graham
Keyword(s):  
Directed Evolution ◽  
Cell Polarity ◽  
Molecular Mechanisms ◽  
Transmembrane Segment ◽  
Membrane Asymmetry ◽  
Mutagenesis Screen ◽  
Type Iv ◽  
Neurologic Development ◽  
P Type ◽  
Evolution Approach

Phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophospholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known. We used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeast Saccharomyces cerevisiae has identified several gain-of-function mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. We found that a highly conserved asparagine (N220) in the first transmembrane segment is a key enforcer of glycerophospholipid selection, and specific substitutions at this site allow transport of sphingomyelin.

scholarly journals The PQ-loop protein Any1 segregates Drs2 and Neo1 functions required for viability and plasma membrane phospholipid asymmetry

2019 ◽  
Vol 60 (5) ◽  
pp. 1032-1042 ◽  
Author(s):  
Mehmet Takar ◽  
Yannan Huang ◽  
Todd R. Graham
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Critical Role ◽  
Protein Protein Interactions ◽  
Membrane Asymmetry ◽  
Growth Defects ◽  
Type Iv ◽  
Organizational Feature ◽  
Membrane Type ◽  
P Type

Membrane asymmetry is a key organizational feature of the plasma membrane. Type IV P-type ATPases (P4-ATPases) are phospholipid flippases that establish membrane asymmetry by translocating phospholipids, such as phosphatidylserine (PS) and phospatidylethanolamine, from the exofacial leaflet to the cytosolic leaflet. Saccharomyces cerevisiae expresses five P4-ATPases: Drs2, Neo1, Dnf1, Dnf2, and Dnf3. The inactivation of Neo1 is lethal, suggesting Neo1 mediates an essential function not exerted by the other P4-ATPases. However, the disruption of ANY1, which encodes a PQ-loop membrane protein, allows the growth of neo1Δ and reveals functional redundancy between Golgi-localized Neo1 and Drs2. Here we show Drs2 PS flippase activity is required to support neo1Δ any1Δ viability. Additionally, a Dnf1 variant with enhanced PS flipping ability can replace Drs2 and Neo1 function in any1Δ cells. any1Δ also suppresses drs2Δ growth defects but not the loss of membrane asymmetry. Any1 overexpression perturbs the growth of cells but does not disrupt membrane asymmetry. Any1 coimmunoprecipitates with Neo1, an association prevented by the Any1-inactivating mutation D84G. These results indicate a critical role for PS flippase activity in Golgi membranes to sustain viability and suggests Any1 regulates Golgi membrane remodeling through protein-protein interactions rather than a previously proposed scramblase activity.

scholarly journals Cross Talk between Sphingolipids and Glycerophospholipids in the Establishment of Plasma Membrane Asymmetry

2004 ◽  
Vol 15 (11) ◽  
pp. 4949-4959 ◽  
Author(s):  
Akio Kihara ◽  
Yasuyuki Igarashi
Keyword(s):  
Plasma Membrane ◽  
Cross Talk ◽  
Abc Transporters ◽  
Transcriptional Factor ◽  
Drug Efflux ◽  
Lipid Asymmetry ◽  
Membrane Asymmetry ◽  
P Type ◽  
Yeast Mutants ◽  
Long Chain

Glycerophospholipids and sphingolipids are distributed asymmetrically between the two leaflets of the lipid bilayer. Recent studies revealed that certain P-type ATPases and ATP-binding cassette (ABC) transporters are involved in the inward movement (flip) and outward movement (flop) of glycerophospholipids, respectively. In this study of phytosphingosine (PHS)-resistant yeast mutants, we isolated mutants for PDR5, an ABC transporter involved in drug efflux as well as in the flop of phosphatidylethanolamine. The pdr5 mutants exhibited an increase in the efflux of sphingoid long-chain bases (LCBs). Genetic analysis revealed that the PHS-resistant phenotypes exhibited by the pdr5 mutants were dependent on Rsb1p, a putative LCB-specific transporter/translocase. We found that the expression of Rsb1p was increased in the pdr5 mutants. We also demonstrated that expression of RSB1 is under the control of the transcriptional factor Pdr1p. Expression of Rsb1p also was enhanced in mutants for the genes involved in the flip of glycerophospholipids, including ROS3, DNF1, and DNF2. These results suggest that altered glycerophospholipid asymmetry induces the expression of Rsb1p. Conversely, overexpression of Rsb1p resulted in increased flip and decreased flop of fluorescence-labeled glycerophospholipids. Thus, there seems to be cross talk between sphingolipids and glycerophospholipids in maintaining the functional lipid asymmetry of the plasma membrane.

scholarly journals Celsr1 and CAMSAP3 differently regulate intercellular and intracellular cilia orientation in oviduct multiciliated cells

2020 ◽  
Author(s):  
Fumiko Matsukawa Usami ◽  
Masaki Arata ◽  
Dongbo Shi ◽  
Sanae Oka ◽  
Yoko Higuchi ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Planar Cell Polarity ◽  
Molecular Mechanisms ◽  
The Other ◽  
Basal Bodies ◽  
Generating Functional ◽  
Tissue Polarity ◽  
Other Hand ◽  
Multiciliated Cells ◽  
Polarity Regulation

SummaryThe molecular mechanisms by which cilia orientation is coordinated within and between multiciliated cells (MCCs) is not fully understood. By observing the orientation of basal bodies (BB) in MCCs of mouse oviducts, here, we show that Celsr1, a planar cell polarity (PCP) factor involved in tissue polarity regulation, is dispensable for determining BB orientation in individual cells, whereas CAMSAP3, a microtubule minus-end regulator, is critical for this process but not for PCP. MCCs exhibit a characteristic BB orientation and microtubule gradient along the tissue axis, and these intracellular polarities were maintained in the cells lacking Celsr1, although the intercellular coordination of the polarities was partly disrupted. On the other hand, CAMSAP3 regulated the assembly of microtubules interconnecting BBs by localizing at the BBs, and its mutation led to disruption of intracellular coordination of BB orientation, but not affecting PCP factor localization. Thus, both Celsr1 and CAMSAP3 are responsible for BB orientation but in distinct ways; and therefore, their cooperation should be critical for generating functional multiciliated tissues.

scholarly journals Axenic Biofilm Formation and Aggregation bySynechocystissp. Strain PCC 6803 Are Induced by Changes in Nutrient Concentration and Require Cell Surface Structures

2019 ◽  
Vol 85 (7) ◽  
Author(s):  
Rey Allen ◽  
Bruce E. Rittmann ◽  
Roy Curtiss
Keyword(s):  
Biofilm Formation ◽  
Molecular Mechanisms ◽  
Type Iv Pili ◽  
Biofuel Production ◽  
Oxygenic Photosynthesis ◽  
Phototrophic Biofilm ◽  
Bg11 Medium ◽  
Phototrophic Biofilms ◽  
Type Iv ◽  
Cell Surface Structures

ABSTRACTPhototrophic biofilms are key to nutrient cycling in natural environments and bioremediation technologies, but few studies describe biofilm formation by pure (axenic) cultures of a phototrophic microbe. The cyanobacteriumSynechocystissp. strain PCC 6803 (hereSynechocystis) is a model microorganism for the study of oxygenic photosynthesis and biofuel production. We report here that wild-type (WT)Synechocystiscaused extensive biofilm formation in a 2,000-liter outdoor nonaxenic photobioreactor under conditions attributed to nutrient limitation. We developed a biofilm assay and found that axenicSynechocystisforms biofilms of cells and extracellular material but only when cells are induced by an environmental signal, such as a reduction in the concentration of growth medium BG11. Mutants lacking cell surface structures, namely type IV pili and the S-layer, do not form biofilms. To further characterize the molecular mechanisms of cell-cell binding bySynechocystis, we also developed a rapid (8-h) axenic aggregation assay. Mutants lacking type IV pili were unable to aggregate, but mutants lacking a homolog to Wza, a protein required for type 1 exopolysaccharide export inEscherichia coli, had a superbinding phenotype. In WT cultures, 1.2× BG11 medium induced aggregation to the same degree as 0.8× BG11 medium. Overall, our data support that Wza-dependent exopolysaccharide is essential to maintain stable, uniform suspensions of WTSynechocystiscells in unmodified growth medium and that this mechanism is counteracted in a pilus-dependent manner under altered BG11 concentrations.IMPORTANCEMicrobes can exist as suspensions of individual cells in liquids and also commonly form multicellular communities attached to surfaces. Surface-attached communities, called biofilms, can confer antibiotic resistance to pathogenic bacteria during infections and establish food webs for global nutrient cycling in the environment. Phototrophic biofilm formation is one of the earliest phenotypes visible in the fossil record, dating back over 3 billion years. Despite the importance and ubiquity of phototrophic biofilms, most of what we know about the molecular mechanisms, genetic regulation, and environmental signals of biofilm formation comes from studies of heterotrophic bacteria. We aim to help bridge this knowledge gap by developing new assays forSynechocystis, a phototrophic cyanobacterium used to study oxygenic photosynthesis and biofuel production. With the aid of these new assays, we contribute to the development ofSynechocystisas a model organism for the study of axenic phototrophic biofilm formation.

scholarly journals The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase

Science ◽  
2020 ◽  
Vol 369 (6511) ◽  
pp. eabc5809 ◽  
Author(s):  
Michael J. McKenna ◽  
Sue Im Sim ◽  
Alban Ordureau ◽  
Lianjie Wei ◽  
J. Wade Harper ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Targeting ◽  
Transmembrane Helix ◽  
Protein Composition ◽  
Binding Pocket ◽  
Transmembrane Segment ◽  
Substrate Binding Pocket ◽  
Cryo Electron Microscopy ◽  
Endogenous Substrate ◽  
P Type

Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A–adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail–anchored proteins. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo–electron microscopy structures of Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis.

scholarly journals FppA, a Novel Pseudomonas aeruginosa Prepilin Peptidase Involved in Assembly of Type IVb Pili

2006 ◽  
Vol 188 (13) ◽  
pp. 4851-4860 ◽  
Author(s):  
Sophie de Bentzmann ◽  
Marianne Aurouze ◽  
Geneviève Ball ◽  
Alain Filloux
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Type Iv Pili ◽  
Reading Frame ◽  
Type Iv ◽  
Bacterial Aggregation ◽  
Epithelial Cell Surface ◽  
Prepilin Peptidase ◽  
Pilus Assembly

ABSTRACT Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Whereas molecular mechanisms of type IVa pilus assembly have been well documented for Pseudomonas aeruginosa and involve the PilD prepilin peptidase, no type IVb pili have been described in this microorganism. One subclass of type IVb prepilins has been identified as the Flp prepilin subfamily. Long and bundled Flp pili involved in tight adherence have been identified in Actinobacillus actinomycetemcomitans, for which assembly was due to a dedicated machinery encoded by the tad-rcp locus. A similar flp-tad-rcp locus containing flp, tad, and rcp gene homologues was identified in the P. aeruginosa genome. The function of these genes has been investigated, which revealed their involvement in the formation of extracellular Flp appendages. We also identified a gene (designated by open reading frame PA4295) outside the flp-tad-rcp locus, that we named fppA, encoding a novel prepilin peptidase. This is the second enzyme of this kind found in P. aeruginosa; however, it appears to be truncated and is similar to the C-terminal domain of the previously characterized PilD peptidase. In this study, we show that FppA is responsible for the maturation of the Flp prepilin and belongs to the aspartic acid protease family. We also demonstrate that FppA is required for the assembly of cell surface appendages that we called Flp pili. Finally, we observed an Flp-dependent bacterial aggregation process on the epithelial cell surface and an increased biofilm phenotype linked to Flp pilus assembly.

scholarly journals aPKC: the Kinase that Phosphorylates Cell Polarity

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 903 ◽  
Author(s):  
Yang Hong
Keyword(s):  
Subcellular Localization ◽  
Cell Polarity ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Cell Polarization ◽  
Kinase C ◽  
Dividing Cells ◽  
Cell Embryo ◽  
Powerful Mechanism ◽  
Anterior Posterior

Establishing and maintaining cell polarity are dynamic processes that necessitate complicated but highly regulated protein interactions. Phosphorylation is a powerful mechanism for cells to control the function and subcellular localization of a target protein, and multiple kinases have played critical roles in cell polarity. Among them, atypical protein kinase C (aPKC) is likely the most studied kinase in cell polarity and has the largest number of downstream substrates characterized so far. More than half of the polarity proteins that are essential for regulating cell polarity have been identified as aPKC substrates. This review covers mainly studies of aPKC in regulating anterior-posterior polarity in the worm one-cell embryo and apical-basal polarity in epithelial cells and asymmetrically dividing cells (for example, Drosophila neuroblasts). We will go through aPKC target proteins in cell polarity and discuss various mechanisms by which aPKC phosphorylation controls their subcellular localizations and biological functions. We will also review the recent progress in determining the detailed molecular mechanisms in spatial and temporal control of aPKC subcellular localization and kinase activity during cell polarization.

scholarly journals Pathogenic siderophore ABC importer YbtPQ adopts a surprising fold of exporter

2020 ◽  
Vol 6 (6) ◽  
pp. eaay7997 ◽  
Author(s):  
Zhiming Wang ◽  
Wenxin Hu ◽  
Hongjin Zheng
Keyword(s):  
Bound States ◽  
Molecular Mechanisms ◽  
Transmembrane Helix ◽  
Human Pathogens ◽  
Binding Domains ◽  
Type Iv ◽  
Cryo Electron Microscopy ◽  
Open Conformation ◽  
Multiple Structures ◽  
First Time

To fight for essential metal ions, human pathogens secrete virulence-associated siderophores and retake the metal-chelated siderophores through a subfamily of adenosine triphosphate (ATP)–binding cassette (ABC) importer, whose molecular mechanisms are completely unknown. We have determined multiple structures of the yersiniabactin importer YbtPQ from uropathogenic Escherichia coli (UPEC) at inward-open conformation in both apo and substrate-bound states by cryo–electron microscopy. YbtPQ does not adopt any known fold of ABC importers but surprisingly adopts the fold of type IV ABC exporters. To our knowledge, it is the first time an exporter fold of ABC importer has been reported. We have also observed two unique features in YbtPQ: unwinding of a transmembrane helix in YbtP upon substrate release and tightly associated nucleotide-binding domains without bound nucleotides. Together, our study suggests that siderophore ABC importers have a distinct transport mechanism and should be classified as a separate subfamily of ABC importers.

scholarly journals Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili

2017 ◽  
Vol 114 (25) ◽  
pp. E4978-E4986 ◽  
Author(s):  
Vivianne J. Goosens ◽  
Andreas Busch ◽  
Michaella Georgiadou ◽  
Marta Castagnini ◽  
Katrina T. Forest ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Type Iv Pili ◽  
Macromolecular Complex ◽  
Heterologous Host ◽  
Synthetic Genes ◽  
Macromolecular Assemblies ◽  
Type Iv ◽  
Filament Assembly ◽  
Multistep Process ◽  
Synthetic Biology Approach

Type IV pili (Tfp), which are key virulence factors in many bacterial pathogens, define a large group of multipurpose filamentous nanomachines widespread in Bacteria and Archaea. Tfp biogenesis is a complex multistep process, which relies on macromolecular assemblies composed of 15 conserved proteins in model gram-negative species. To improve our limited understanding of the molecular mechanisms of filament assembly, we have used a synthetic biology approach to reconstitute, in a nonnative heterologous host, a minimal machinery capable of building Tfp. Here we show that eight synthetic genes are sufficient to promote filament assembly and that the corresponding proteins form a macromolecular complex at the cytoplasmic membrane, which we have purified and characterized biochemically. Our results contribute to a better mechanistic understanding of the assembly of remarkable dynamic filaments nearly ubiquitous in prokaryotes.

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