Characterization of phosphatidylserine transport to the locus of phosphatidylserine decarboxylase 2 in permeabilized yeast

In yeast, nascent phosphatidylserine (PtdSer) can be transported to the mitochondria and Golgi/vacuole for decarboxylation to synthesize phosphatidylethanolamine (PtdEtn). In strains with a psd1Delta allele for the mitochondrial PtdSer decarboxylase, the conversion of nascent PtdSer to PtdEtn can serve as an indicator of lipid transport to the locus of PtdSer decarboxylase 2 (Psd2p) in the Golgi/vacuole. We have followed the metabolism of [(3)H]serine into PtdSer and PtdEtn to study lipid transport in permeabilized psd1Delta yeast. The permeabilized cells synthesize (3)H-PtdSer and, after a 20-min lag, decarboxylate it to form [(3)H]PtdEtn. Formation of [(3)H]PtdEtn is linear between 20 and 100 min of incubation and does not require ongoing PtdSer synthesis. PtdSer transport can be resolved into a two-component system using washed, permeabilized psd1Delta cells as donors and membranes isolated by ultracentrifugation as acceptors. With this system, the transport-dependent decarboxylation of nascent PtdSer is dependent upon the concentration of acceptor membranes, requires Mn(2+) but not nucleotides, and is inhibited by EDTA. High speed membranes isolated from a previously identified PtdSer transport mutant, pstB2, contain normal Psd2p activity but fail to reconstitute PtdSer transport and decarboxylation. Reconstitution with permutations of wild type and pstB2Delta donors and acceptors identifies the site of the mutant defect as the acceptor side of the transport reaction.     

Adriamycin disrupts phosphatidylserine import into the mitochondria of permeabilized CHO-K1 cells

The action of adriamycin (an inhibitor of precursor protein import into mitochondria) upon phosphatidylserine (PtdSer) import into mitochondria was examined in permeabilized CHO-K1 cells. The decarboxylation of nascent PtdSer to phosphatidylethanolamine was used as an indicator reaction for the lipid translocation process. Adriamycin was without effect upon new PtdSer synthesis but blocked the time- and translocation-dependent decarboxylation of this lipid at the mitochondrial inner membrane of permeabilized cells. The effect of adriamycin was concentration-dependent with an IC50 of 150 microM and was not due to direct inhibition of PtdSer decarboxylase. To determine at which level of PtdSer transport adriamycin was working, the adriamycin-treated permeabilized cells were incubated with 1-acyl-2-[N-(6-[(7-nitrobenz-2-oxa-1,3-diazo-4-yl)] aminocaproyl)]phosphatidyl[1'-14C] serine (NBD-Ptd[1'-14C]Ser), and its decarboxylation was determined. Since the NBD-Ptd[1'-14C]Ser freely partitions into all cell membranes, it can partition into the outer mitochondrial membrane in an ATP-independent fashion. The NBD-Ptd[1'-14C]Ser was readily decarboxylated in an ATP-independent manner in permeabilized cells. Adriamycin inhibited the decarboxylation of NBD-Ptd[1'-14C]Ser, thereby indicating that it can act upon lipid transport processes between the outer and inner mitochondrial membrane.     

Organellar DNA synthesis in permeabilized soybean cells

Summary Cultured cells of Glycine max (L.) Merr. v. Corsoy were permeabilized by treatment with L--lysophosphatidylcholine (LPC). The permeabilized cells were capable of uptake and incorporation of deoxynucleoside triphosphates into DNA. Incorporation of exogenous nucleotides into DNA was linear for at least 90 minutes and the initial rate of incorporation approached 50% of the theoretical in vivo rate of DNA synthesis. However, DNA synthesis in the permeabilized cells was unaffected by the potent DNA polymerase inhibitor, aphidicolin. Analysis of newly synthesized DNA by molecular hybridization revealed that only organellar DNA was synthesized by the permeabilized cells. The LPC treated cells were also permeable to a protein as large as DNase I. The permeabilized cells were capable of RNA and protein synthesis as indicated by incorporation of radiolabeled UTP and leucine, respectively, into acid-precipitable material.     

DNA synthesis in permeabilized mouse L cells.

Mouse L cells are rendered permeable to nucleoside triphosphates by a cold shock with a near isotonic buffer. These cells retain their morphologic integrity and use exogenously supplied nucleotides and deoxynucleotides to synthesize RNA and DNA. The newly synthesized DNA is nuclear and is the product of semiconservative replication. Incorporation of deoxynucleotides into DNA by thymidine kinase-deficient cells were used to conform rigorously that the exogenously supplied deoxynucleotides were incorporated into DNA without intermediate processing through nucleosides. DNA synthesis requires the presence of Na+, ATP, all 4 deoxynucleotides, and Mg2+. The reaction is inhibited by N-ethylmaleimide, p-hydroxymercuribenzoate and actinomycin D. Hydroxy-urea and arabinosylcytosine do not inhibit the reaction whereas cytosine arabinoside triphosphate shows competitive inhibition with the deoxynucleotides. These findings indicate that the permeable cell system can be used for in situ evaluations of the replicative DNA polymerase using the endogenous DNA template.     

Nanopore-facilitated, voltage-driven phosphatidylserine translocation in lipid bilayers--in cells and in silico

Nanosecond, megavolt-per-meter pulses--higher power but lower total energy than the electroporative pulses used to introduce normally excluded material into biological cells--produce large intracellular electric fields without destructively charging the plasma membrane. Nanoelectropulse perturbation of mammalian cells causes translocation of phosphatidylserine (PS) to the outer face of the cell, intracellular calcium release, and in some cell types a subsequent progression to apoptosis. Experimental observations and molecular dynamics (MD) simulations of membranes in pulsed electric fields presented here support the hypothesis that nanoelectropulse-induced PS externalization is driven by the electric potential that appears across the lipid bilayer during a pulse and is facilitated by the poration of the membrane that occurs even during pulses as brief as 3 ns. MD simulations of phospholipid bilayers in supraphysiological electric fields show a tight association between PS externalization and membrane pore formation on a nanosecond time scale that is consistent with experimental evidence for electropermeabilization and anode-directed PS translocation after nanosecond electric pulse exposure, suggesting a molecular mechanism for nanoelectroporation and nanosecond PS externalization: electrophoretic migration of the negatively charged PS head group along the surface of nanometer-diameter electropores initiated by field-driven alignment of water dipoles at the membrane interface.     

DNA synthesis in permeabilized WI38 and MRC5 cells

DNA synthesis was examined in cultures of growing WI38 and MRC5 cells made permeable to deoxyribonucleotides. Cells from late passage cultures showed a reduced rate of deoxythymidine triphosphate (dTTP) uptake as compared to cells from early- to mid-passage cultures. This reduction became evident earlier in WI38 cultures (passage 33) than in MRC5 cultures (passage 41). Although this reduced rate of incorporation appeared to be primarily due to a reduced percentage of replicating (S phase) cells in later passage cultures, some effect on the rate of DNA synthesis in replicating cells was also evident.     

Disruption of phosphatidylserine translocation to the mitochondria in baby hamster kidney cells

Phosphatidylserine synthase is found predominantly in the microsomal fraction, and phosphatidylserine decarboxylase is found predominantly in the mitochondrial fraction of baby hamster kidney (BHK-21) cells. This segregation of enzymes of phosphatidylserine metabolism allows serine metabolism to phosphatidylserine and phosphatidylethanolamine to be used as an indicator of the intracellular movement of phosphatidylserine. After BHK-21 cells were pulse-labeled with [3H]serine, phosphatidylserine was efficiently labeled, and subsequently 40-50% of this radiolabeled lipid turned over to form phosphatidylethanolamine during a 7.5-h chase. Treatment of cells with NaN3 plus NaF or cycloheximide at the end of the pulse labeling period markedly inhibited the rate and extent of phosphatidylserine turnover during the chase period. The inhibition of phosphatidylserine turnover could not be attributed to inhibition of either phosphatidylserine decarboxylase or phosphatidylserine exchange protein activity. Subcellular fractionation of the BHK-21 cells demonstrated that cells poisoned with NaN3 plus NaF accumulated phosphatidylserine in the microsomal fraction relative to unpoisoned cells. The results indicate that metabolic energy is required for the transport of phosphatidylserine to the mitochondria.     

Inhibition of protein translocation in permeabilized cells of Schizosaccharomyces pombe by puromycin.

To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-alpha-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.     

Inhibition of DNA synthesis in permeabilized L cells by novobiocin

Novobiocin was equipotent in inhibiting DNA and RNA synthesis in cultured mouse L cells. It also suppressed in vitro DNA and RNA synthesis in permeabilized L cells and nuclei; 50 percent inhibition of DNA and RNA synthesis was obtained by 1 mM and 20 mM novobiocin, respectively. ATP antagonized the effect of novobiocin. Nalidixic acid had a weak inhibitory effect on in vitro DNA synthesis; 10 mM nalidixic acid showed 60 percent inhibition. ATP did not antagonize nalidixic acid. The inhibitory effect of novobiocin exceeded that of aphidicolin. These findings suggest a participation of a gyrase- and/or type II topoisomerase-like enzyme in the DNA replication machinery in L cells.     

Enhancement of transport-dependent decarboxylation of phosphatidylserine by S100B protein in permeabilized Chinese hamster ovary cells

Phosphatidylethanolamine synthesis through the phosphatidylserine (PtdSer) decarboxylation pathway requires PtdSer transport from the endoplasmic reticulum or mitochondrial-associated membrane to the mitochondrial inner membrane in mammalian cells. The transport-dependent PtdSer decarboxylation in permeabilized Chinese hamster ovary (CHO) cells was enhanced by cytosolic factors from bovine brain. A cytosolic protein factor exhibiting this enhancing activity was purified, and its amino acid sequence was partially determined. The sequence was identical to part of the amino acid sequence of an EF-hand type calcium-binding protein, S100B. A His(6)-tagged recombinant CHO S100B protein was able to remarkably enhance the transport-dependent PtdSer decarboxylation in permeabilized CHO cells. Under the standard assay conditions for PtdSer decarboxylase, the recombinant S100B protein did not stimulate PtdSer decarboxylase activity and exhibited no PtdSer decarboxylase activity. These results implicated the S100B protein in the transport of PtdSer to the mitochondrial inner membrane.     

Plasma membrane translocation of phosphatidylserine in human spermatozoa

The purpose of the study was to examine the phosphatidylserine translocation in human spermatozoa membrane during capacitation. Material consisted of human semen from normozoospermic men. Spermatozoa were stained with fluorescein-labelled annexin V. The presence and distribution of annexin V binding sites were analysed using the fluorescence microscope. Within first 60 min afterejaculation, 5-39% viable annexin V-positive spermatozoa were detected. The annexin V binding sites were found mainly in the midpiece. After 4 to 8 h of incubation of spermatozoa in capacitation medium (BMI), the number of cells positively stained with annexin V increased. After capacitation, the localisations of phosphatidylserine was changed and the annexin V binding sites were found also in the acrosomal region but never in the equatorial area. The process of the phosphatidylserine translocation observed during our experiments may reflect changes of the plasma membrane occurring during capacitation or, less likely, apoptosis of spermatozoa.     

DNA synthesis in permeabilized karyoplasts from cytochalasin B-enucleated mouse L cells

DNA synthesis was examined in karyoplasts permeabilized by hypotonic buffer or lysolecithin. DNA synthesis was not affected by the omission of a single deoxyribonucleated triphosphate but was greatly reduced in the absence of three of the deoxyribonucleotide triphosphates. The rate of DNA synthesis was linear for 40 min and continued for up to 2–3 h. The DNA synthesized in the permeabilized karyoplasts was a continuation of semi-conservative replication occurring in the intact cell.     

Optimized synthesis of phosphatidylserine

The synthesis of phosphatidyl serine containing saturated fatty acids was thoroughly studied and optimized in order to establish a protocol amenable to large-scale synthesis. The key step was a one-pot multicomponent reaction involving an O-benzyl phosphorodiamidite, protected serine and diacylglycerol, followed by in situ oxidation of the resulting phosphite. In order to replace expensive and poorly stable tetrazole, a screening of substitutes was carried out and imidazolium chloride was selected as the best suited one.     

Effect of alpha-1-antichymotrypsin on DNA synthesis in permeabilized human cells

The effect of Alpha-1-antichymotrypsin(ACT) on DNA synthesis was studied using lysolecithin-permeabilized cultured human stomach carcinoma cells. ACT added in medium inhibited DNA synthesis and the degree of inhibition is incubation time dependent. It is proportional to ACT concentration and the concentration of ACT required for 50% inhibition was 0.8 mg/ml.     

Protein synthesis in transformed 3T3 cells permeabilized by exogenous ATP

Exogenous ATP has been shown to cause a rapid and reversible increase in permeability in transformed 3T3 cells (3T6 and SV3T3) but not in untransformed 3T3 cells. The cells remain viable, but lose intracellular acid-soluble pools. Treatment of transformed cells with ATP greatly reduces incorporation of ¹⁴C-leucine into protein, which is restored by the incubation of the cells with Dulbecco's modified Eagle's medium or by the external additions of certain ions and energy sources. tRNA is not required for the restoration of protein synthesis. In the permeabilized cells the energy for protein synthesis can be provided by glycolysis, oxidative phosphorylation, or direct addition of ATP. These studies demonstrate the usefulness of this method for studying the control of metabolism and macromolecular synthesis in monolayer cultures of transformed mammalian cells.     

Inhibition of phosphatidylserine synthesis in Jurkat T cells by hydrogen peroxide.

Incubation of Jurkat cells in the presence of H2O2 either directly added to the culture medium or generated with glucose oxidase, menadione or the couple xanthine/xanthine oxidase induced a marked decrease of phosphatidylserine synthesis in the absence of changes in the synthesis of phosphatidylcholine and phosphatidylethanolamine. Concentration dependent response curves indicated that H2O2 induced inhibition of phosphatidylserine synthesis with an IC(50)=5 microM while both induction of tyrosine phosphorylation of proteins and Ca(2+) signals were obtained with an EC(50)=300 microM. The tyrosine kinase and Ca(2+) independent mechanism was confirmed by comparing the H2O2-induced and the CD3-induced inhibition of phosphatidylserine synthesis using several Jurkat clones differing in the expression of cell surface receptors such as CD3/TCR and CD45 and protein tyrosine kinase such as p72syk, ZAP-70 and p56lck. While CD3-induced inhibition of phosphatidylserine synthesis necessitates protein tyrosine phosphorylation and Ca(2+) signals, H2O2 provoked its effect in all the clones studied independently of the presence or absence of the proteins previously shown to be key elements in T cell signal transduction. Conversely, the antioxidant molecule, butylated hydroxanisole, generates an increased PtdSer synthesis, suggesting that the synthesis of this phospholipid is regulated by the redox status of the cells.