Mimicry of phorbol ester responses by diacylglycerols. Differential effects on phosphatidylcholine biosynthesis, cell-cell communication and epidermal growth factor binding

The biosynthesis of phosphatidylcholine (PC) in HEL-37 cells was followed by measuring the incorporation of [32P]Pi into PC. Incorporation was stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and by the synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (diC8), but not by sn-1-oleoyl-2-acetylglycerol or sn-1,2-dihexanoylglycerol (diC6). DiC8 was rapidly metabolised by HEL-37 cells to the corresponding PC and phosphatidic acid derivatives. diC8, diC6 and oleoylacetylglycerol effectively displaced [3H]phorbol-12,13-dibutyrate bound to a soluble cell extract from HEL-37 cells, but only diC8 was able to displace the labelled phorbol ester from prelabelled cells. TPA, diC8, diC6 and oleoylacetylglycerol were all effective inhibitors of 125I-labelled epidermal growth factor binding to, and gap junctional communication between, HEL-37 cells. It is concluded that only cell-permeable diacylglycerols stimulate PC biosynthesis which may therefore require interaction with membranes other than the plasma membrane.     

Assembly of the endoplasmic reticulum phospholipid bilayer. Transporters for phosphatidylcholine and metabolites

Phosphatidylcholine is synthesized on the cytoplasmic surface of the endoplasmic reticulum and transported to the lumenal monolayer by a protein transporter, a phosphatidylcholine "flippase" (Bishop, W. R., and Bell, R. M. (1985) Cell 42, 51-60). Since the endoplasmic reticulum contains enzymes involved in phosphatidylcholine turnover that have different locations within the organelle, transport systems may exist for phosphatidylcholine metabolites. To test the hypothesis that rat liver microsomes contain a lysophosphatidylcholine transporter, sn-1-monobutyroylphosphatidylcholine was employed. Since this homolog is highly water-soluble, transport of lysophosphatidylcholine could be measured using standard transport methods. sn-1-Monobutyroylphosphatidylcholine entered the lumenal compartment of microsomal vesicles. Transport was saturable and dependent on time and on amount of microsomes and required an intact permeability barrier. sn-1-Monobutyroylphosphatidylcholine transport was inhibited by treatment of microsomes with trypsin, N-ethylmaleimide, and trinitrobenzene-sulfonic acid. These findings suggest that sn-1-monobutyroylphosphatidylcholine transport is protein-mediated. sn-1-Monobutyroylphosphatidylcholine transported into microsomes was degraded to glycerophosphorylcholine. Glycerophosphorylcholine was also transported across the microsomal membrane. Glycerophosphorylcholine transport was also saturable and dependent on time, amount of microsomes, and an intact permeability barrier but was not inhibited by treatment with trypsin or the two protein modification agents. Thus, separate and distinct transport systems exist for phosphatidylcholine metabolites. Molecular events of phosphatidylcholine turnover in the endoplasmic reticulum are discussed.     

1,2-Dioctanoyl-glycerol induces a discrete but transient translocation of protein kinase C as well as the inhibition of MCF-7 cell proliferation

Exposure of MCF-7 human breast cancer cells to phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) results in a dose-dependent inhibition of cell proliferation. One of the earliest biochemical events induced by TPA is the translocation of protein kinase C from the cytosolic to the particulate compartment. We have investigated the effects of permeant diacylglycerol 1,2-dioctanoyl-glycerol (diC8) on both protein kinase C activity and MCF-7 cell proliferation. DiC8 induces a discrete but significant translocation of protein kinase C within the first minutes of MCF-7 cell treatment (26 +/- 6%, mean +/- SD of 5 different experiments, upon 5 min incubation in the presence of 43 micrograms/ml diC8). However, this effect is only transient as the enzymatic activity returns to the control value after 60 min. DiC8 mimics the effect of TPA on MCF-7 cell proliferation. The dose-response curves for both protein kinase C translocation and cell growth inhibition show that diC8 exerts its effects on both parameters in the same range of concentrations, despite some discrepancies at the lowest doses. We also report that long-term treatment of the cells with diC8 does not lead to the protein kinase C disappearance observed during prolonged exposure to TPA. All together, our results reinforce the hypothesis of a negative modulatory role of protein kinase C in MCF-7 cell proliferation and suggest that the enzyme translocation but not its down-regulation could be a pre-requisite in the biological cell response.     

Contrasting effects of sn-1,2-dioctanoyl glycerol as compared to other protein kinase C activators in adrenal glomerulosa cells

Angiotensin II acts on adrenal glomerulosa cells to induce the phospholipase C-mediated generation of inositol trisphosphate and sn-1,2-diacylglycerol as the major products of inositol phospholipid breakdown. This last product is known to activate protein kinase C, but its role in the action of angiotensin II on steroidogenesis has not been defined. We report herein that, in bovine adrenal glomerulosa cells, protein kinase C activators, such as phorbol 12,13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate, mezerein and sn 1,2 oleoyl acetoylglycerol, each failed to increase steroidogenesis. These results contrast with our recent report on the enhancement of aldosterone output by sn-1,2-dioctanoylglycerol (DiC8) [J. Steroid Biochem. 35 (1990) 19-33]. In addition, the difference between DiC8 and the other protein kinase activators was also observed in the pattern of 86Rb efflux from preloaded glomerulosa cells; only DiC8 mimicked the effect of angiotensin II on ion fluxes. Furthermore, staurosporine, a potent inhibitor of protein kinase C, was capable of amplifying the aldosterone output induced by a maximally effective concentration of DiC8 or angiotensin II. These data suggest that the effect of the cell permeant DiC8 on aldosterone biosynthesis either is not mediated by protein kinase C activation, or is mediated by a phorbol ester-insensitive isoenzyme of protein kinase C.     

Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C

Bombesin caused a marked stimulation of 32Pi into phosphatidylinositol (PI), with no apparent lag, and into phosphatidylcholine (PC), after a lag of about 20 min. Stimulation was blocked by the bombesin receptor antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P, indicating that the effects on both PI and PC were mediated through the same receptor. The tumor-promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and dioctanoylglycerol (diC8) both directly activate protein kinase C and in this report were shown to stimulate 32Pi incorporation into PC but not into Pl. In addition, TPA stimulated the release of [3H]choline and [3H]phosphocholine and the accumulation of [3H]diacyglycerol from prelabelled cells. These results strongly suggest that TPA activates a phospholipase C specific for PC. Pretreatment of cells with phorbol-12, 13-dibutyrate (PDBu) for 24 h depleted cellular protein kinase C activity and inhibited the ability of TPA to induce these effects suggesting a direct involvement of protein kinase C. Similarly the bombesin stimulation of 32Pi into PC and of [3H]choline and [3H]phosphocholine release was inhibited by PDBu pretreatment. DiC8 and, to a lesser extent, TPA stimulated the translocation of CTP:phosphocholine cytidylytransferase from the cytosolic to the particulate fraction. DiC8 also stimulated this translocation in cells depleted of protein kinase C. It was concluded that both bombesin and TPA activated protein kinase C leading to activation of a phospholipase C specific for PC.     

Protein kinase C activation in mixed micelles. Mechanistic implications of phospholipid, diacylglycerol, and calcium interdependencies

The phospholipid, sn-1,2-diacylglycerol, and calcium dependencies of rat brain protein kinase C were investigated with a mixed micellar assay (Hannun, Y., Loomis, C., and Bell, R.M. (1985) J. Biol. Chem. 260, 10039-10043). Protein kinase C activity was independent of the number of Triton X-100, phosphatidylserine (PS), and sn-1,2-dioleoylglycerol (diC18:1) mixed micelles. Activation was strongly dependent on the mole per cent of PS and diC18:1. Activity of protein kinase C was dependent on PS, diC18:1, and calcium in mixed micelles prepared from detergents other than Triton X-100. This is consistent with the micelle providing an inert surface into which the lipid cofactors partition. Molecular sieve chromatography provided direct evidence for the homogeneity of Triton X-100, PS, and diC18:1 mixed micelles. Mixing studies and surface dilution studies indicated that PS and diC18:1 rapidly equilibrate among the mixed micelles. At saturating calcium, the diC18:1 dependence was strongly dependent on the mole per cent PS present. At 10 mol % PS, 0.25 mol % diC18:1 gave maximal activity whereas 6 mol % PS and 6 mol % diC18:1 did not give maximal activity. diC18:1 dependencies were hyperbolic at all PS levels tested. The data support the conclusion that a single molecule of diC18:1/micelle is sufficient to activate monomeric protein kinase C. The mole per cent PS required for maximal activation was reduced markedly as the mole per cent diC18:1 increased. Under all conditions tested, the PS dependence of protein kinase C activation lagged until greater than 3 mol % PS was present. Then activation occurred in a cooperative manner with Hill numbers near 4. These data indicate that 4 or more molecules of PS are required to activate monomeric protein kinase C. PS was the most effective of all the phospholipids tested in the mixed micelle assay. diC18:1 was found to modulate the amount of calcium required for maximal activity. As the level of Ca2+ increased, the mole per cent PS required reached a limiting value of 3 mol %. A number of sn-1,2-diacylglycerols containing short chain fatty acids activated protein kinase C in a saturable manner in mixed micelles. The data are discussed in relation to a model for protein kinase activation.     

Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine

A mixed micellar assay for protein kinase C was developed to investigate the specificity and stoichiometry of activation by phospholipids and diacylglycerols. Triton X-100 mixed micelles containing 8 mol % phosphatidylserine (PS) and 2.5 mol % sn-1,2-dioleoylglycerol (diC18:1) activated rat brain protein kinase C in the presence of Ca2+ to the same degree as sonicated PS/diC18:1 did in the standard assay. However, protein kinase C activity was more responsive to diC18:1 in the mixed micellar assay than the standard assay. At 8 mol % PS and 100 microM Ca2+, diC18:1 stimulated maximally at 1 mol %. At 2.5 mol % diC18:1 and 100 microM Ca2+, PS did not activate until 3 mol % and then did so cooperatively with maximal stimulation occurring at 6-8 mol %. Direct evidence for a Ca2+-, PS-, and diC18:1-dependent interaction of protein kinase C with mixed micelles was obtained by molecular sieve chromatography on Sephacryl S-200. These data permit inferences pertaining to the number of diC18:1 and PS molecules/micelle which are required for activation. For diC18:1, a single molecule may be sufficient but no more than 2 molecules are required. For PS, greater than 4 but less than 10 molecules are required. These data establish that a phospholipid bilayer is not required for protein kinase C activation and that activation of monomeric protein kinase C occurs.     

Cross-linking phosphatidylinositol-specific phospholipase C traps two activating phosphatidylcholine molecules on the enzyme

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), a bacterial model for the catalytic domain of mammalian PI-PLC enzymes, was cross-linked by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to probe for the aggregation and/or conformational changes of PI-PLC when bound to activating phosphatidylcholine (PC) interfaces. Dimers and higher order multimers (up to 31% of the total protein when cross-linked at pH 7) were observed when the enzyme was cross-linked in the presence of PC vesicles. Aggregates were also detected with PI-PLC bound to diheptanoyl-PC (diC(7)PC) micelles, although the fraction of cross-linked multimers (19% at pH 7) was lower than when the enzyme was cross-linked in the presence of vesicles. PI-PLC cross-linked in the presence of a diC(7)PC interface exhibited an enhanced specific activity for PI cleavage. The extent of this cross-linking-enhanced activation was reduced in PI-PLC mutants lacking either tryptophan in the rim (W47A and W242A) of this (betaalpha)(8)-barrel protein. The higher activity of the native protein cross-linked in the presence of diC(7)PC correlated with an increased affinity of the protein for two diC(7)PC molecules as detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In contrast to wild type protein, W47A and W242A had only a single diC(7)PC tightly associated when cross-linked in the presence of that activator molecule. These results indicate that (i) each rim tryptophan residue is involved in binding a PC molecule at interfaces, (ii) the affinity of the enzyme for an activating PC molecule is enhanced when the protein is bound to a surface, and (iii) this conformation of the enzyme with at least two PC bound that is stabilized by chemical cross-linking interacts more effectively with activating interfaces, leading to higher observed specific activities for the phosphotransferase reaction.     

Distribution of phosphatidylcholine molecular species between mixed micelles and phospholipid-cholesterol vesicles in human gallbladder bile: dependence on acyl chain length and unsaturation.

The partitioning of phosphatidylcholine (PC) molecular species between mixed micelles and vesicles was studied in each of seven human gallbladder biles. Biles were fractionated by Sephacryl S-300 SF gel filtration chromatography, and PC species in the micellar and vesicular fractions were quantitated by high performance liquid chromatography. Micelles were enriched in species containing unsaturated acyl groups (e.g., 16:1-18:2, 18:1-18:2, and 18:1-18:3); vesicles were enriched in more highly saturated species (e.g., 16:0-16:1, 16:0-18:1, and 18:0-18:1). Separate multivariate analyses for each bile demonstrated that the distribution of PC species between vesicles and micelles was related to the degree of sn-1 and sn-2 unsaturation, and sn-1, but not sn-2, chain length. In addition, the tendency to partition into the micellar phase was particularly marked when unsaturation was present at both the sn-1 and sn-2 positions. When this interaction was included in the multivariate analyses, the regression models accounted for virtually all of the variation in PC partitioning (for each of the seven patients r2 = 0.92-0.98, P less than 0.03). These results suggest that the partitioning of PC species between micelles and vesicles is strictly determined by sn-1 chain length and the degree of unsaturation at both the sn-1 and sn-2 positions. In light of recent reports that fatty acyl composition influences the cholesterol content of vesicles and micelles in model biles, these results raise the possibility that diet-induced alterations in the phospholipid species and the relative proportions of biliary lipid particles may influence the cholesterol-carrying capacity of bile.     

Specificity of the phosphatidylcholine exchange protein from bovine liver

The phosphatidylcholine exchange protein from bovine liver stimulates the specific transfer of phosphatidylcholine (PC) from rat liver microsomes to mitochondria or phospholipid vesicles (Wirtz, K.W.A., Kamp, H.H., and van Deenen, L.L.M. (1972), Biochim. Biophys. Acta 274, 606). In the present study, it has been established which components of the PC molecule are essential to the specific interaction with the protein. Radiochemically labeled analogues of PC have been synthesized with modifications in the polar and apolar moiety, and their transfer was measured between donor and acceptor vesicles. Relative to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (egg yolk PC), transfer is inhibited or abolished when (a) the distance between phosphorus and nitrogen is decreased or increased and (b) a methyl group on the quaternary nitrogen is removed or substituted by an ethyl or propyl group. Transfer is much less affected when (a) the ester bonds are replaced by ether or carbon-carbon bonds, (b) the PC molecule contains two saturated fatty acids, and (c) the D stereoisomer is used. It is concluded that the protein has a binding site which interacts specifically with the phosphorylcholine head group and which cannot accommodate substantial configurational changes. Interaction with the apolar moiety of PC is less specific. However, lyso-PC is not transferred, suggesting that two hydrocarbon chains are required to stabilize the exchange protein-phospholipid complex. Interaction of [14C]PC-labeled exchange protein with vesicles of different phospholipid compositon has been analyzed by measuring the release of [14C]PC into these vesicles. Vesicles of egg PC or dimethylphosphatidylethanolamine function as acceptors, in contrast to vesicles of sphingomyelin or phosphatidylethanolamine.     

IR spectroscopic determination of gel state miscibility in long-chain phosphatidylcholine mixtures.

We report on the gel-state microaggregation in binary mixtures of diacylphosphatidylcholines over temperatures ranging from -19 degrees C to near the gel-to-liquid crystal transition. Microaggregates with lateral dimensions in the range 1-100 chains were detected and measured with an isotope infrared method that relates the splitting or the shape of the methylene scissors band to aggregate size. Measurements were made on fully hydrated dispersions of diC18DPC/diC20HPC, diC18DPC/diC22HPC, and diC18DPC/diC24HPC at molar ratios of 4:1. Low levels of aggregation were determined with reference to the spectrum of the random mixture diC18DPC/diC18HPC. For diC18DPC/diC20HPC at -19 degrees C, which previous calorimetric measurements have indicated is a nearly ideal, we found about 4% of the minority component chains to be involved in aggregates. For diC18DPC/diC22HPC, the value increased to about 11%. DiC18DPC/diC24HPC was found to be highly fractionated, in agreement with the earlier studies. The unit subcell, which defines the type of acyl-chain packing, was determined for the components of the mixtures. The temperature behavior of the phases and the temperatures at which the minority component domains undergo dissolution were determined.     

Transbilayer diffusion of phospholipids: dependence on headgroup structure and acyl chain length

The kinetics and thermodynamics of the transmembrane movement (flip-flop) of fluorescent analogs of phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were investigated to determine the contributions of headgroup composition and acyl chain length to phospholipid flip-flop. The phospholipid derivatives containing n-octanoic, n-decanoic or n-dodecanoic acid in the sn-1 position and 9-(1-pyrenyl)nonanoic acid in the sn-2 position were incorporated at 3 mol% into sonicated single-bilayer vesicles of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC). The kinetics of diffusion of the pyrene-labeled phospholipids from the outer and inner monolayers of the host vesicles to a large pool of POPC acceptor vesicles were monitored by the time-dependent decrease of pyrene excimer fluorescence. The observed kinetics of transfer were biexponential, with a fast component due to the spontaneous transfer of pyrenyl phospholipids in the outer monolayer of labeled vesicles and a slower component due to diffusion of pyrenyl phospholipid from the inner monolayer of the same vesicles. Intervesicular transfer rates decreased approx. 8-fold for every two carbons added to the first acyl chain. Correspondingly, the free energy of activation for transfer increased approx. 1.3 kcal/mol. With the exception of PE, the intervesicular transfer rates for the different headgroups within a homologous series were nearly the same, with the PC derivative being the fastest. Transfer rates for the PE derivatives were 5-to 7-fold slower than the rates observed for PC. Phospholipid flip-flop, in contrast, was strongly dependent on headgroup composition with a smaller dependence on acyl chain length. At pH 7.4, flip-flop rates increased in the order PC less than PG less than PA less than PE, where the rates for PE were at least 10-times greater than those of the homologous PC derivative. Activation energies for flip-flop were large, and ranged from 38 kcal/mol for the longest acyl chain derivative of PC to 25 kcal/mol for the PE derivatives. Titration of the PA headgroup at pH 4.0 produced an approx. 500-fold increase in the flip-flop rate of PA, while the activation energy decreased 10 kcal/mol. Increasing acyl chain length reduced phospholipid flip-flop rates, with the greatest change observed for the PC analogs, which exhibited an approx. 2-fold decrease in flip-flop rate for every two methylene carbons added to the acyl chain at the sn-1 position.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipoprotein lipase-catalyzed hydrolysis of phospholipid monolayers: effect of fatty acyl composition on enzyme activity

The phospholipase A1 activity of lipoprotein lipase (LpL) was determined with monomolecular phospholipid films. Rates of phospholipid hydrolysis were dependent on apolipoprotein C-II (the activator protein for LpL) phospholipid fatty acyl composition, and lipid-packing density. In sphingomyelin: cholesterol (2:1, molar) monolayers containing 5 mol % disaturated phosphatidylcholines (PC) and at a surface pressure of 22 mNm-1, rates of LpL hydrolysis of diC14:0PC, diC16:0PC, and diC18:0PC were 74, 207, and 65 nmol h-1 mg LpL-1, respectively. At 22 mNm-1, phospholipids containing unsaturated fatty acyl chains were hydrolyzed at rates 5-10 times greater than saturated lipids. At higher lipid packing densities, the difference in hydrolysis rates between saturated and unsaturated lipids was less apparent. Comparison of molecular areas indicate no simple dependency between the rate of LpL catalysis and phospholipid fatty acyl chain length and saturation/unsaturation.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

Incorporation and remodeling of extracellular phosphatidylcholine with short acyl residues in Saccharomyces cerevisiae

The pem1/cho2 pem2/opi3 double mutant of Saccharomyces cerevisiae, which is auxotrophic for choline because of the deficiency in methylation activities of phosphatidylethanolamine, grew in the presence of 0.1 mM dioctanoyl-phosphatidylcholine (diC(8)PC). Analysis of the metabolism of methyl-(13)C-labeled diC(8)PC ((methyl-(13)C)(3)-diC(8)PC) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) revealed that it was rapidly converted to (methyl-(13)C)(3)-PCs containing C16 or C18 acyl chains. (Methyl-(13)C)(3)-8:0-lyso-PC, (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC, which are the probable intermediate molecular species of acyl chain remodeling, appeared immediately after 5 min of pulse-labeling and decreased during the subsequent chase period. These results indicate that diC(8)PC was taken up by the pem1 pem2 double mutant and that the acyl chains of diC(8)PC were exchanged with longer yeast fatty acids. The temporary appearance of (methyl-(13)C)(3)-8:0-lyso-PC suggests that the remodeling reaction may consist of deacylation and reacylation by phospholipase activities and acyltransferase activities, respectively. The detailed analyses of the structures of (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC by MS/MS and MS(3) strongly suggest that most (methyl-(13)C)(3)-8:0-16:0-PCs have a C16:0 acyl chain at sn-1 position, whereas (methyl-(13)C)(3)-8:0-16:1-PCs have a C16:1 acyl chain at either sn-1 or sn-2 position in a similar frequency, implying that the initial C16:0 acyl chain substitution prefers the sn-1 position; however, the C16:1 acyl chain substitution starts at both sn-1 and sn-2 positions. The current study provides a pivotal insight into the acyl chain remodeling of phospholipids in yeast.     

Sar1 translocation onto the ER-membrane for vesicle budding has different pathways for promotion and suppression of ER-to-Golgi transport mediated through H89-sensitive kinase and ER-resident G protein

ER-to-Golgi protein transport involves transport vesicles of which formation is initiated by assembly of Sar1. The assembly of Sar1 is suppressed by protein kinase inhibitor H89, suggesting that ER-to-Golgi transport is regulated progressively by H89 sensitive kinase. ER-resident G(i2) protein suppresses vesicle formation with inhibition of Sar1 assembly. This study examined whether these promotion and suppression of vesicle transport share the same signal pathway, by examining the effects of G(i/o) protein activator mastoparan 7 (Mp-7) and H89 on Sar1 and Sec23 recruitment onto microsomes. In a cell-free system for Sar1 translocation assay, GTPγS addition induced the translocation of Sar1 onto microsomes. Mp-7 and H89 decreased the Sar1 translocation. Double treatment of Mp-7 and H89 strongly decreased Sar1 translocation. In single and double treatments, however, G(i/o) protein inactivator pertussis toxin (IAP) partially restored the suppressive effect of Mp-7, but had not any effect on H89-induced effect. Then, the assembly of Sec23 onto the microsome was also increased by the addition of GTPγS. Sec23 translocation was decreased by Mp-7 and/or H89 treatment and recovered by IAP pretreatment except for H89 single treatment, similarly to Sar1 translocation in each treatment. Inhibitory effects of H89 and Mp-7on ER-to-Golgi vesicle transport by H89 or Mp-7 were also confirmed in a cell culture system by BFA-dispersion and BFA-reconstruction experiments. These findings indicate that promotion and suppression of ER-to-Golgi vesicle transport are modulated through separate signal pathways.     

Protein kinase C and membrane transport: divergent responses of Na+/K+/Cl- cotransport and sugar transport to exogenous diacylglycerol

Even though the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is known to bind to and activate protein kinase C (PKC), it is still not certain that all cellular responses to phorbol esters are necessarily mediated by PKC. In BALB/c 3T3 preadipose cells, TPA has previously been shown to rapidly inhibit Na+K+Cl- -cotransport activity, stimulate 2-deoxyglucose uptake and induce ornithine decarboxylase activity. The cell-permeable diacylglycerol sn-1,2-dioctanoylglycerol (DiC8) was used in order to distinguish between PKC-dependent and -independent responses of BALB/c 3T3 cells. DiC8 modulated 86Rb+ fluxes in BALB/c 3T3 cells in the same manner as TPA: furosemide-sensitive 86Rb+ influx and efflux was inhibited, while in cotransport-defective cells no effect was observed. In contrast, DiC8 did not stimulate 2-deoxyglucose uptake in either parental or cotransport-defective cell lines, even though TPA is a very effective inducer of this transport system in both cell types. Pretreatment of cells with DiC8 did not substantially alter the subsequent induction of 2-deoxyglucose uptake by TPA, although a slight but reproducible reduction in the magnitude of the response was observed in DiC8-pretreated cells. The PKC-dependent phosphorylation of an acidic 80-kDa protein was stimulated by both TPA and DiC8 in parental and cotransport-defective cell lines, suggesting that a gross defect in the primary effector system used by both TPA and diacylglycerols cannot explain any of our results. Ornithine decarboxylase was induced by DiC8 and the K1/2 was approximately the same as that for inhibition of Na+/K+/Cl- cotransport in these cells. Thus, our results suggest that PKC is clearly essential for some phorbol ester membrane transport responses (such as inhibition of Na+/K+/Cl- cotransport), but our results do not allow us to conclude that other responses (such as stimulation of 2-deoxyglucose uptake) necessarily require PKC activation.     

Slow translocon gating causes cytosolic exposure of transmembrane and lumenal domains during membrane protein integration

Integral membrane proteins are cotranslationally inserted into the endoplasmic reticulum via the protein translocation channel, or translocon, which mediates the transport of lumenal domains, retention of cytosolic domains and integration of transmembrane spans into the phospholipid bilayer. Upon translocon binding, transmembrane spans interact with a lateral gate, which regulates access to membrane phospholipids, and a lumenal gate, which controls the translocation of soluble domains. We analyzed the in vivo kinetics of integration of model membrane proteins in Saccharomyces cerevisiae using ubiquitin translocation assay reporters. Our findings indicate that the conformational changes in the translocon that permit opening of the lumenal and lateral channel gates occur less rapidly than elongation of the nascent polypeptide. Transmembrane spans and lumenal domains are therefore exposed to the cytosol during integration of a polytopic membrane protein, which may pose a challenge to the fidelity of membrane protein integration.     

Consequences of hydrophobic mismatch between lipids and melibiose permease on melibiose transport

The structural and functional consequences of a mismatch between the hydrophobic thickness d(P) of a transmembrane protein and that d(L) of the supporting lipid bilayer were investigated using melibiose permease (MelB) from Escherichia coli reconstituted in a set of bis saturated and monounsaturated phosphatidylcholine species differing in acyl-chain length. Influence of MelB on the midpoint gel-to-liquid-phase transition temperature, T(m), of the saturated lipids was investigated through fluorescence polarization experiments, with 1,6-diphenyl-1,3,5-hexatriene as the probe, for varying protein/lipid molar ratio. Diagrams in temperature versus MelB concentration showed positive or negative shifts in T(m) with the short-chain lipids DiC12:0-PC and DiC14:0-PC or the long-chain lipids DiC16:0-PC and DiC18:0-PC, respectively. Theoretical analysis of the data yielded a d(L) value of 3.0 +/- 0.1 nm for the protein, similar to the 3.02 nm estimated from hydropathy profiles. Influence of the acyl chain length on the carrier activity of MelB was investigated in the liquid phase, using the monounsaturated PCs. Binding of the sugar to the transporter showed no dependence on the acyl chain length. In contrast, counterflow and Deltapsi-driven experiments revealed strong dependence of melibiose transport on the lipid acyl chain length. Similar bell-shaped transport versus acyl chain length profiles were obtained, optimal activity being supported by diC16:1-PC. On account of a d(P) value of 2.65 nm for the lipid and of various local constraints which would all tend to elongate the acyl chains in contact with the protein, one can conclude that maximal activity was obtained when the hydrophobic thickness of the bilayer matched that of the protein.     

An ATP transporter is required for protein translocation into the yeast endoplasmic reticulum.

The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 x 10(-5) M. ATP transport into ER vesicles was specifically inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3'-O-(4-benzoyl)benzoyl-ATP (Bz2-ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2-ATP blocked protein translocation in vitro measured co- as well as post-translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS-treated membranes. These results demonstrate that translocation requires a DIDS and Bz2-ATP-sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.