Absence of anionic phospholipids in Kluyveromyces lactis cells is fatal without F1-catalysed ATP hydrolysis

2012 ◽  
Vol 58 (6) ◽  
pp. 694-702 ◽  
Author(s):  
Viktoria Palovicova ◽  
Annamaria Bardelcikova ◽  
Margita Obernauerova
Keyword(s):  
Membrane Potential ◽  
Atp Hydrolysis ◽  
Kluyveromyces Lactis ◽  
Phenotypic Characterization ◽  
Endogenous Respiration ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Hydrolytic Reaction ◽  
Biogenesis Of Mitochondria ◽  
Petite Negative Yeast

We have shown in previous research that the loss of phosphatidylglycerol and cardiolipin caused by disruption of the PGS1 gene is lethal for the petite-negative yeast Kluyveromyces lactis . This present study demonstrates the role and mechanism of atp2.1 in the suppression of pgs1 lethality in K. lactis cells. Phenotypic characterization has shown that a strain lacking the phosphatidylglycerolphosphate synthase (atp2.1pgs1Δ) possessed a markedly impaired respiratory chain, very low endogenous respiration, and uncoupled mitochondria. As a result the mutant strain was unable to generate a sufficient mitochondrial membrane potential via respiration. The atp2.1 suppressor mutation enabled an increase in the affinity of F1-ATPase for ATP in the hydrolytic reaction, resulting in the maintenance of sufficient membrane potential for the biogenesis of mitochondria and survival of cells lacking anionic phospholipid biosynthesis.

scholarly journals Size-Tunable Paclitaxel Nanoparticles Stabilized by Anionic Phospholipids for Transdermal Delivery Applications

2020 ◽  
Vol 15 (3) ◽  
pp. 1934578X1990068
Author(s):  
Noriyuki Uchida ◽  
Masayoshi Yanagi ◽  
Hiroki Hamada
Keyword(s):  
Stratum Corneum ◽  
Transdermal Delivery ◽  
Skin Tissue ◽  
Composite Nanoparticles ◽  
Rat Skin ◽  
Cooling Process ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Subsequent Heating

Composite nanoparticles composed of an anionic phospholipid of 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG) and paclitaxel (PTX) were successfully prepared by mixing them in water followed by a subsequent heating/cooling process. The size of DPPG-PTX nanoparticle could be easily tuned by ultrasonic fragmentation. Upon addition of small-sized fluorescently labeled paclitaxel (FLPTX) nanoparticles with DPPG (DPPG-FLPTX) to rat skin tissue, part of the FLPTX molecules permeated to the stratum corneum.

scholarly journals Expression of the Saccharomyces cerevisiae Gene YME1 in the Petite-Negative Yeast Schizosaccharomyces pombe Converts It to Petite-Positive

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 147-154 ◽  
Author(s):  
Douglas J Kominsky ◽  
Peter E Thorsness
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Schizosaccharomyces Pombe ◽  
Atp Synthase ◽  
Kluyveromyces Lactis ◽  
Blot Hybridization ◽  
Inner Mitochondrial Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitochondrial Atp Synthase ◽  
Petite Negative Yeast

Abstract Organisms that can grow without mitochondrial DNA are referred to as “petite-positive” and those that are inviable in the absence of mitochondrial DNA are termed “petite-negative.” The petite-positive yeast Saccharomyces cerevisiae can be converted to a petite-negative yeast by inactivation of Yme1p, an ATP- and metal-dependent protease associated with the inner mitochondrial membrane. Suppression of this yme1 phenotype can occur by virtue of dominant mutations in the α- and γ-subunits of mitochondrial ATP synthase. These mutations are similar or identical to those occurring in the same subunits of the same enzyme that converts the petite-negative yeast Kluyveromyces lactis to petite-positive. Expression of YME1 in the petite-negative yeast Schizosaccharomyces pombe converts this yeast to petite-positive. No sequence closely related to YME1 was found by DNA-blot hybridization to S. pombe or K. lactis genomic DNA, and no antigenically related proteins were found in mitochondrial extracts of S. pombe probed with antisera directed against Yme1p. Mutations that block the formation of the F1 component of mitochondrial ATP synthase are also petite-negative. Thus, the F1 complex has an essential activity in cells lacking mitochondrial DNA and Yme1p can mediate that activity, even in heterologous systems.

scholarly journals Extension of the Scope of Anionic Phospholipid-Based Nanoformulation to Kaempferol and Indometacin

2021 ◽  
Vol 16 (3) ◽  
pp. 1934578X2110026
Author(s):  
Noriyuki Uchida ◽  
Masayoshi Yanagi ◽  
Kei Shimoda ◽  
Hiroki Hamada
Keyword(s):  
Phosphatidic Acid ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Dispersion Agent

In this work, resveratrol was dispersed with anionic phospholipids of 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, and 1,2-distearoyl-sn-glycero-3-phosphoglycerol. Moreover, small-sized nanoparticles of kaempferol and indometacin were successfully prepared by using DPPG as a dispersion agent.

scholarly journals Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids

2016 ◽  
Vol 148 (3) ◽  
pp. 227-237 ◽  
Author(s):  
Sun-Joo Lee ◽  
Feifei Ren ◽  
Eva-Maria Zangerl-Plessl ◽  
Sarah Heyman ◽  
Anna Stary-Weinzinger ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Membrane Lipids ◽  
Inward Rectifier ◽  
Primary Site ◽  
Structural Basis ◽  
Channel Activity ◽  
Anionic Phospholipid ◽  
X Ray ◽  
Anionic Phospholipids ◽  
Kir Channel

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL−) with a distinct second site is required for high PIP2 sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP2 sensitivity, even in the absence of PL−. Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP2 (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL− binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP2 site and explaining the positive allostery between PL− binding and PIP2 sensitivity.

scholarly journals Molecular determinants of KA1 domain-mediated autoinhibition and phospholipid activation of MARK1 kinase

2017 ◽  
Vol 474 (3) ◽  
pp. 385-398 ◽  
Author(s):  
Ryan P. Emptage ◽  
Mark A. Lemmon ◽  
Kathryn M. Ferguson
Keyword(s):  
Membrane Surface ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Disease States ◽  
C Terminus ◽  
Regulatory Module ◽  
Correct Location ◽  
Mechanistic Basis

Protein kinases are frequently regulated by intramolecular autoinhibitory interactions between protein modules that are reversed when these modules bind other ‘activating’ protein or membrane-bound targets. One group of kinases, the MAP/microtubule affinity-regulating kinases (MARKs) contain a poorly understood regulatory module, the KA1 (kinase associated-1) domain, at their C-terminus. KA1 domains from MARK1 and several related kinases from yeast to humans have been shown to bind membranes containing anionic phospholipids, and peptide ligands have also been reported. Deleting or mutating the C-terminal KA1 domain has been reported to activate the kinase in which it is found — also suggesting an intramolecular autoinhibitory role. Here, we show that the KA1 domain of human MARK1 interacts with, and inhibits, the MARK1 kinase domain. Using site-directed mutagenesis, we identify residues in the KA1 domain required for this autoinhibitory activity, and find that residues involved in autoinhibition and in anionic phospholipid binding are the same. We also demonstrate that a ‘mini’ MARK1 becomes activated upon association with vesicles containing anionic phospholipids, but only if the protein is targeted to these vesicles by a second signal. These studies provide a mechanistic basis for understanding how MARK1 and its relatives may require more than one signal at the membrane surface to control their activation at the correct location and time. MARK family kinases have been implicated in a plethora of disease states including Alzheimer's, cancer, and autism, so advancing our understanding of their regulatory mechanisms may ultimately have therapeutic value.

scholarly journals Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity

2009 ◽  
Vol 284 (24) ◽  
pp. 16099-16107 ◽  
Author(s):  
Mingming Pu ◽  
Xiaomin Fang ◽  
Alfred G. Redfield ◽  
Anne Gershenson ◽  
Mary F. Roberts
Keyword(s):  
Enzyme Activity ◽  
Phospholipase C ◽  
Lateral Diffusion ◽  
Lipid Vesicles ◽  
Membrane Lipid ◽  
Correlation Spectroscopy ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Substrate Analogue ◽  
Nmr Methods

The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (τc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased τc. PI-PLC activity and phospholipid τc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC τc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins.

Ligand-regulated secretion of recombinant annexin V from cultured thyroid epithelial cells

2002 ◽  
Vol 282 (6) ◽  
pp. C1313-C1321 ◽  
Author(s):  
Xiuqiong Wang ◽  
Marcia A. Kaetzel ◽  
Sung E. Yoo ◽  
Paul S. Kim ◽  
John R. Dedman
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
External Surface ◽  
Annexin V ◽  
Therapeutic Agents ◽  
Anionic Phospholipid ◽  
Thyroid Cells ◽  
Tissue Ischemia ◽  
Anionic Phospholipids ◽  
Activated Platelets

The exposure of anionic phospholipids on the external surface of injured endothelial cells and activated platelets is a primary biological signal to initiate blood coagulation. Disease conditions that promote the formation of ectopic thrombi result in tissue ischemia. Annexins, Ca2+-dependent anionic phospholipid binding proteins, are potential therapeutic agents for the inhibition of coagulation. We have designed a transgene that targets secretion of annexin V from cultured thyroid cells under the control of doxycycline. Our results indicate that annexin V in the endoplasmic reticulum (ER)/Golgi lumen does not affect the synthesis, processing, and secretion of thyroglobulin. ER luminal Ca2+was moderately increased and can be released by inositol 1,4,5-trisphosphate. Our study demonstrates that targeting and secretion of annexin V through the secretory pathway of mammalian cells does not adversely affect cellular function. Regulated synthesis and release of annexin V may exert anticoagulatory and anti-inflammatory effects systemically and may prove useful in further developing therapeutic strategies for conditions including antiphospholipid syndrome.

scholarly journals Oyxsterols induce membrane procoagulant activity in monocytic THP-1 cells

1996 ◽  
Vol 314 (3) ◽  
pp. 1027-1033 ◽  
Author(s):  
Karine AUPEIX ◽  
Florence TOTI ◽  
Nathalie SATTA ◽  
Pierre BISCHOFF ◽  
Jean-Marie FREYSSINET
Keyword(s):  
Tissue Factor ◽  
Annexin V ◽  
Antigen Expression ◽  
Resting Cells ◽  
Dependent Manner ◽  
Anionic Phospholipid ◽  
Monocytic Cells ◽  
Anionic Phospholipids ◽  
Phosphatidylserine Exposure ◽  
And Control

Oxidized cholesterol compounds or oxysterols are thought to be potent membrane-destabilizing agents. Anionic phospholipids, chiefly phosphatidylserine, have a procoagulant potential due to their ability to favour the membrane assembly of the characteristic clotting enzyme complexes including the tissue factor-dependent initiating complex. However, in resting cells, phosphatidylserine is sequestered in the inner leaflet of the plasma membrane. When THP-1 monocytic cells were cultured in the presence of 7β-hydroxycholesterol (7β-OH) or 25-hydroxycholesterol (25-OH), prothrombinase, which reflects anionic phospholipid exposure and tissue factor (TF) procoagulant activities, increased in a time- and dose-dependent manner. 7β-OH appeared 1.5- to 2-fold more potent than 25-OH. Interestingly, no effect of cholesterol itself could be detected on procoagulant activities. Nevertheless, no difference in TF activity could be detected between oxysterol-treated and control cells after disruption. TF antigen expression was the same in oxysterol-treated and control cells as shown by flow cytometry. In contrast, the use of labelled annexin V, a protein probe of anionic phospholipids, revealed an elevated number of cells with exposed phosphatidylserine. Because the latter also constitutes a signal for phagocyte recognition of apoptotic cells and fragments, and a proportion of cells displayed altered morphology with condensed chromatin and membrane blebs, analysis of DNA was performed and indicated apoptosis in oxysterol-treated cells. Hence, oxysterol-induced phosphatidylserine exposure and enhanced TF activity may result from apoptosis. These results suggest relationships between oxysterol and the amplification of coagulation reactions by monocytic cells resulting from induced phosphatidylserine exposure.

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