Branchial elimination of superhydrophobic organic compounds by rainbow trout (Oncorhynchus mykiss).

The branchial elimination of pentachloroethane and four congeneric polychlorinated biphenyls by rainbow trout was measured using a fish respirometer-metabolism chamber and an adsorption resin column. Branchial elimination was characterized by calculating a set of apparent in vivo blood:water partition coefficients (P(BW)). Linear regression was performed on the logarithms of P(BW) estimates and the log K(OW) value for each compound to give the fitted equation: log P(BW)=0.76 x log K(OW)-1.0 (r(2)=0.98). The linear nature of this relationship provides support for existing models of chemical flux at fish gills and suggests that a near equilibrium condition was established between chemical in venous blood entering the gills, including dissolved and bound forms, and dissolved chemical in expired branchial water. In vivo P(BW) estimates were combined with P(BW) values determined in vitro for a set of lower log K(OW) compounds (Bertelson et al., Environ. Toxicol. Chem. 17 (1998) 1447-1455) to give the fitted relationship: log P(BW)=0.73 x log K(OW)-0.88 (r(2)=0.98). The slope of this equation is consistent with the suggestion that chemical binding to non-lipid organic material contributes substantially to blood:water chemical partitioning. An equation based on the composition of trout blood (water content and the total amount of organic material) was then derived to predict blood:water partitioning for compounds with log K(OW) values ranging from 0 to 8: log P(BW)=log[(10(0.73 log K(ow)) x 0.16)+0.84].

Dietary uptake kinetics of 2,2',5,5'-tetrachlorobiphenyl in rainbow trout.

The disposition of [UL-(14)C]2,2',5,5'-tetrachlorobiphenyl (TCB) in rainbow trout (Oncorhynchus mykiss) was studied in acute dietary exposures using TCB-contaminated fathead minnows (Pimephales promelas). Trout were sampled at several postfeeding time points and TCB-derived radioactivity was measured in gut contents and selected tissues. Gastric evacuation was exponential with time and was 95% complete within 36 h of feeding. The ratio of activity in upper intestinal tissue to that in blood declined between 6 and 48 h, as did the lumenal contents/tissue ratio. Stomach content lipid declined between 0 and 24 h, while the lipid content of chyme remained relatively constant. These observations are consistent with liquid phase emptying of lipid and TCB to the upper intestine followed by rapid coassimilation. Tissue/blood activity ratios for the stomach, lower intestine, muscle, liver, and kidney were constant and probably represented near equilibrium conditions. The fat/blood activity ratio increased through 96 h, indicating that TCB was redistributing to fat. The lower intestinal tissue/feces activity ratio increased between 6 and 24 h and then declined rapidly. Fecal lipid content also increased between 6 and 24 h, but the amount of this increase was insufficient to explain observed changes in the distribution of TCB-derived activity. A small amount of 3-hydroxy TCB was detected in feces. Generally, however, metabolism had little or no impact on the uptake, distribution or elimination of TCB. Measured assimilation efficiencies exceeded 90% and are the highest ever reported in fish feeding studies with TCB.

NBD-labeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane.

The internalization and distribution of fluorescent analogs of phosphatidylcholine (M-C6-NBD-PC) and phosphatidylethanolamine (M-C6-NBD-PE) were studied in Saccharomyces cerevisiae. At normal growth temperatures, M-C6-NBD-PC was internalized predominantly to the vacuole and degraded. M-C6-NBD-PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded. At 2 degrees C, both were internalized to the nuclear envelope/ER and mitochondria by an energy-dependent, N-ethylmaleimide-sensitive process, and transport of M-C6-NBD-PC to and degradation in the vacuole was blocked. Internalization of neither phospholipid was reduced in the endocytosis-defective mutant, end4-1. However, following pre-incubation at 37 degrees C, internalization of both phospholipids was inhibited at 2 degrees C and 37 degrees C in sec mutants defective in vesicular traffic. The sec18/NSF mutation was unique among the sec mutations in further blocking M-C6-NBD-PC translocation to the vacuole suggesting a dependence on membrane fusion. Based on these and previous observations, we propose that M-C6-NBD-PC and M-C6-NBD-PE are transported across the plasma membrane to the cytosolic leaflet by a protein-mediated, energy-dependent mechanism. From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria. Subsequently, M-C6-NBD-PC, but not M-C6-NBD-PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases.

Energy-dependent flip of fluorescence-labeled phospholipids is regulated by nutrient starvation and transcription factors, PDR1 and PDR3.

The yeast Saccharomyces cerevisiae readily accumulates short-chain, fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled phosphatidylcholine and phosphatidylethanolamine at the nuclear envelope/endoplasmic reticulum and mitochondria. The net intracellular accumulation reflects the sum of their inwardly and outwardly directed transbilayer translocation across the plasma membrane (flip and flop, respectively). The rate of flop is negligible in energy-depleted cells as well as at low temperature (2 degrees C). Although flip is reduced at 2 degrees C, it can still be measured by flow cytometry, allowing the rate of flip, independent of flop, to be characterized at this temperature. Flip requires the energy of the plasma membrane proton electrochemical gradient and is down-regulated as cells pass through the diauxic shift and enter stationary phase. Furthermore, drug-resistant, gain-of-function mutations in the transcription factors, PDR1 and PDR3, result in a dramatic down-regulation of flip in addition to their already established up-regulation of flop. These results imply that down-regulation of the NBD-phospholipid flip pathway is a physiological response to environmental stress.

Loss of Drs2p does not abolish transfer of fluorescence-labeled phospholipids across the plasma membrane of Saccharomyces cerevisiae.

The yeast DRS2 gene, which is required for growth at 23 degreesC or below, encodes a member of a P-type ATPase subgroup reported to transport aminophospholipids between the leaflets of the plasma membrane. Here, we evaluated the potential role of Drs2p in phospholipid transport. When examined by fluorescence microscopy, a drs2 null mutant showed no defect in the uptake or distribution of fluorescent-labeled 1-palmitoyl-2[6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD))aminocaproyl]phosphatidylserine) or 1-myristoyl-2[6-NBD-aminocaproyl]phosphatidylethanolamine. Quantification of the amount of cell-associated NBD fluorescence using flow cytometry indicated a significant decrease in the absence of Drs2p, but this decrease was not restricted to the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) and was dependent on culture conditions. Furthermore, the absence of Drs2p had no effect on the amount of endogenous PE exposed to the outer leaflet of the plasma membrane as detected by labeling with trinitrobenzene sulfonic acid. The steady state pool of Drs2p, which was shown to reside predominantly in the plasma membrane, increased upon shift to low temperature or exposure to various divalent cations (Mn2+, Co2+, Ni2+, and Zn2+ but not Ca2+ or Mg2+), conditions that also inhibited the growth of a drs2 null mutant. The data presented here call into question the identification of Drs2p as the exclusive or major aminophospholipid translocase in yeast plasma membranes (Tang, X., Halleck, M. S., Schlegel, R. A., and Williamson, P. (1996) Science 272, 1495-1497).

Trace element trophic transfer in aquatic organisms: a critique of the kinetic model approach.

The bioaccumulation of trace elements in aquatic organisms can be described with a kinetic model that includes linear expressions for uptake and elimination from dissolved and dietary sources. Within this model, trace element trophic transfer is described by four parameters: the weight-specific ingestion rate (IR); the assimilation efficiency (AE); the physiological loss rate constant (ke); and the weight-specific growth rate (g). These four parameters define the trace element trophic transfer potential (TTP = IR.AE/[ke + g]) which is equal to the ratio of the steady-state trace element concentration in a consumer due to trophic accumulation to that in its prey. Recent work devoted to the quantification of AE and ke for a variety of trace elements in aquatic invertebrates has provided the data needed for comparative studies of trace element trophic transfer among different species and trophic levels and, in at least one group of aquatic consumers (marine bivalves), sensitivity analyses and field tests of kinetic bioaccumulation models. Analysis of the trophic transfer potentials of trace elements for which data are available in zooplankton, bivalves, and fish, suggests that slight variations in assimilation efficiency or elimination rate constant may determine whether or not some trace elements (Cd, Se, and Zn) are biomagnified. A linear, single-compartment model may not be appropriate for fish which, unlike many aquatic invertebrates, have a large mass of tissue in which the concentrations of most trace elements are subject to feedback regulation.

Dithionite quenching rate measurement of the inside-outside membrane bilayer distribution of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled phospholipids.

Measurement of the extent of dithionite quenching of the fluorescence of 7-nitrobenz-2-oxa-1,3-diazol-4-yl- (NBD-) labeled lipids inserted into cellular and organellar membranes has been used to quantify their topological distribution and translocation. This assay provides a straightforward method for determining the fraction of NBD-lipid exposed to the outer leaflet of membranes that are impermeant to dithionite. However, it appears that many, if not all, cellular membranes are relatively permeable to dithionite. The present work describes a method in which the initial rate of dithionite quenching, rather than the extent of quenching, was used to determine the fraction of different NBD-labeled phospholipids exposed to the outer leaflet. This method permits the estimation of the translocation process even in experimental conditions where the membrane is semipermeable to dithionite. This technique was used to measure the translocation of several NBD-labeled phospholipids across two biological membranes: brush border membranes vesicles (BBMV) from rabbit intestine and secretory vesicles (SV) from sec 6-4 mutant yeast cells. BBMV were shown to passively equilibrate N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)monopalmitoylphosphatidylethanolamine (N-NBD-PPE) and 1-palmitoyl-2-[6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)aminocaproyl]phosphatidylcholine (P-C6-NBD-PC) to approximately 50% in the inner leaflet by a protein-mediated process. In addition, P-C6-NBD-PC was shown to passively equilibrate across SV to approximately 20% in the inner leaflet. The addition of Mg2+ increased the amount on the inner leaflet to approximately 30% by an unknown mechanism, but no evidence for ATP-dependent inward translocation across the SV was found. In the case of BBMV, several different NBD-phospholipids were translocated from the outer to inner leaflet in a matter of minutes and reached an equilibrium distribution of approximately 50% inside and outside. This movement was inhibitable by N-bromosuccinimide. The inward translocation rate and distribution of headgroup labeled N-NBD-lysophosphatidylethanolamine, having one titratable negative charge, was increased in the presence of an inward basic pH gradient. The same NBD-phospholipids were also translocated across SV to roughly 50% in both leaflets with the exception of NBD-phosphatidic acid, which was passively distributed with 80% in the inner leaflet.

ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p.

The Saccharomyces cerevisiae genome encodes 15 full-size ATP binding cassette transporters (ABC), of which PDR5, SNQ2, and YOR1 are known to be regulated by the transcription factors Pdr1p and Pdr3p (pleiotropic drug resistance). We have identified two new ABC transporter-encoding genes, PDR10 and PDR15, which were up-regulated by the PDR1-3 mutation. These genes, as well as four other ABC transporter-encoding genes, were deleted in order to study the properties of Yor1p. The PDR1-3 gain-of-function mutant was then used to overproduce Yor1p up to 10% of the total plasma membrane proteins. Overexpressed Yor1p was photolabeled by [gamma-32P]2', 3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP (K0.5 = 45 microM) and inhibited by ATP (KD = 0.3 mM) in plasma membranes. Solubilization and partial purification on sucrose gradient allowed to detect significant Yor1p ATP hydrolysis activity (approximately 100 nmol of Pi.min-1.mg-1). This activity was phospholipid-dependent and sensitive to low concentrations of vanadate (I50 = 0.3 microM) and oligomycin (I50 = 8.5 microg/ml). In vivo, we observed a correlation between the amount of Yor1p in the plasma membrane and the level of resistance to oligomycin. We also demonstrated that Yor1p drives an energy-dependent, proton uncoupler-insensitive, cellular extrusion of rhodamine B. Furthermore, cells lacking both Yor1p and Pdr5p (but not Snq2p) showed increased accumulation of the fluorescent derivative of 1-myristoyl-2-[6-(NBD)aminocaproyl]phosphatidylethanolamine. Despite their different topologies, both Yor1p and Pdr5p mediated the ATP-dependent translocation of similar drugs and phospholipids across the yeast cell membrane. Both ABC transporters exhibit ATP hydrolysis in vitro, but Pdr5p ATPase activity is about 15 times higher than that of Yor1p, which may indicate mechanistic or regulatory differences between the two enzymes.

Plasma membrane translocation of fluorescent-labeled phosphatidylethanolamine is controlled by transcription regulators, PDR1 and PDR3.

The transcription regulators, PDR1 and PDR3, have been shown to activate the transcription of numerous genes involved in a wide range of functions, including resistance to physical and chemical stress, membrane transport, and organelle function in Saccharomyces cerevisiae. We report here that PDR1 and PDR3 also regulate the transcription of one or more undetermined genes that translocate endogenous and fluorescent-labeled (M-C6-NBD-PE) phosphatidylethanolamine across the plasma membrane. A combination of fluorescence microscopy, fluorometry, and quantitative analysis demonstrated that M-C6-NBD-PE can be translocated both inward and outward across the plasma membrane of yeast cells. Mutants, defective in the accumulation of M-C6-NBD-PE, were isolated by selectively photokilling normal cells that accumulated the fluorescent phospholipid. This led to the isolation of numerous trafficking in phosphatidylethanolamine (tpe) mutants that were defective in intracellular accumulation of M-C6-NBD-PE. Complementation cloning and linkage analysis led to the identification of the dominant mutation TPE1-1 as a new allele of PDR1 and the semidominant mutation tpe2-1 as a new allele of PDR3. The amount of endogenous phosphatidylethanolamine exposed to the outer leaflet of the plasma membrane was measured by covalent labeling with the impermeant amino reagent, trinitrobenzenesulfonic acid. The amount of outer leaflet phosphatidylethanolamine in both mutant strains increased four- to fivefold relative to the parent Tpe+ strain, indicating that the net inward flux of endogenous phosphatidylethanolamine as well as M-C6-NBD-PE was decreased. Targeted deletions of PDR1 in the new allele, PDR1-11, and PDR3 in the new allele, pdr3-11, resulted in normal M-C6-NBD-PE accumulation, confirming that PDR1-11 and pdr3-11 were gain-of-function mutations in PDR1 and PDR3, respectively. Both mutant alleles resulted in resistance to the drugs cycloheximide, oligomycin, and 4-nitroquinoline N-oxide (4-NQO). However, a previously identified drug-resistant allele, pdr3-2, accumulated normal amounts of M-C6-NBD-PE, indicating allele specificity for the loss of M-C6-NBD-PE accumulation. These data demonstrated that PDR1 and PDR3 regulate the net rate of M-C6-NBD-PE translocation (flip-flop) and the steady-state distribution of endogenous phosphatidylethanolamine across the plasma membrane.

Transbilayer movement of fluorescent phospholipids in Bacillus megaterium membrane vesicles.

We investigated the transbilayer movement or flip-flop of phospholipids in vesicles derived from the cytoplasmic membrane of Bacillus megaterium. Since common assay techniques were found to be inapplicable to the Bacillus system, we exploited and elaborated a newly described method in which fluorescent phospholipids (1-myristoyl-2-C6-NBD phospholipids) are used as tracers to monitor flip-flop. These lipids were introduced into Bacillus vesicles from synthetic donor vesicles containing a fluorescence quencher. Transport was measured by monitoring the increase in fluorescence as the tracers departed the quenched environment of the donor vesicle and entered first the outer membrane leaflet and subsequently the inner leaflet of Bacillus vesicles. Independent experiments involving cobalt quenching of NBD fluorescence provided results consistent with the existence of pools of fluorescent phospholipid in the outer and inner leaflets of Bacillus vesicles at the completion of transport. Using the assay we show that phospholipid flip-flop in Bacillus vesicles occurs rapidly (half-time approximately 30 s at 37 degrees C) with no preference for a particular phospholipid headgroup and that it is sensitive to proteolysis. We also establish that flip-flop does not occur in synthetic phospholipid vesicles or vesicles made from Bacillus phospholipids. We conclude that Bacillus vesicles possess the ability to promote rapid transbilayer movement of phospholipids, and that the transport is probably protein (flippase)-mediated.

Allele-specific suppression of a defective trans-Golgi network (TGN) localization signal in Kex2p identifies three genes involved in localization of TGN transmembrane proteins.

Kex2 protease (Kex2p) and Ste13 dipeptidyl aminopeptidase (Ste13p) are required in Saccharomyces cerevisiae for maturation of the alpha-mating factor in a late Golgi compartment, most likely the yeast trans-Golgi network (TGN). Previous studies identified a TGN localization signal (TLS) in the C-terminal cytosolic tail of Kex2p consisting of Tyr-713 and contextual sequences. Further analysis of the Kex2p TLS revealed similarity to the Ste13p TLS. Mutation of the Kex2p TLS results in transport of Kex2p to the vacuole by default. When expression of a GAL1 promoter-driven KEX2 gene is shut off in MAT(alpha) cells, the TGN becomes depleted of Kex2p, resulting in a gradual decline in mating competence which is greatly accelerated by TLS mutations. To identify the genes involved in localization of Kex2p, we isolated second-site suppressors of the rapid loss of mating competence observed upon shutting off expression of a TLS mutant form of Kex2p (Y713A). Seven of 58 suppressors were allele specific, suppressing point mutations at Tyr-713 but not deletions of the TLS or entire C-terminal cytosolic tail. By linkage analysis, the allele-specific suppressors defined three genetic loci, SOI1, S0I2, and S0I3. Pulse-chase analysis demonstrated that these suppressors increased net TGN retention of both Y713A Kex2p and a Ste13p-Pho8p fusion protein containing a point mutation in the Ste13p TLS. SOI1 suppressor alleles reduced the efficiency of localization of wild-type Kex2p to the TGN, implying an impaired ability to discriminate between the normal TLS and a mutant TLS. soi1 mutants also exhibited a recessive defect in vacuolar protein sorting. Suppressor alleles of S0I2 were dominant. These results suggest that the SOI1 and S0I2 genes encode regulators or components of the TLS recognition machinery.

Dermal absorption of three waterborne chloroethanes in rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus).

In vivo estimates of xenobiotic chemical flux across the dermal surface of intact fish were obtained by measuring chemical loss from venous blood to expired water. An experimental system was developed to separate the dermal route of exposure from all other routes. The system was then used to measure dermal absorption of tetrachloroethane (TCE), pentachloroethane (PCE), and hexachloroethane (HCE) in channel catfish (Ictalurus punctatus) and rainbow trout (Oncorhynchus mykiss), two fish with very different skin anatomies. The kinetics of accumulation varied among chemicals, but for each compound were similar among species. TCE accumulated rapidly, reaching steady state in blood within 48 hr. Steady state was not reached in 48 hr with PCE or HCE, although blood levels of PCE were probably close to steady-state values. Dermal flux estimates (based on branchial efflux) for TCE, PCE, and HCE were two to four times greater in catfish than in trout. Arterial blood concentrations of each compound were three to six times greater in catfish. These observations are indicative of greater flux across catfish skin, augmented by higher blood:water chemical partitioning. Trout skin is covered with scales and has no taste buds, while catfish skin does not possess scales and has numerous taste bud papillae. Both scales and taste bud papillae originate in the dermis and extend to the skin surface through the epidermis. In catfish these taste buds may offer channels through which chemicals diffuse across the epidermis to the more vascularized dermis. A comparison of dermal and branchial uptake was made by estimating zero-time dermal and branchial fluxes for all three chloroethanes. The mean dermal fluxes for TCE, PCE, and HCE ranged from 1.4 to 2.8, 1.8 to 3.6, and 1.4 to 3.2% of the total flux (branchial plus dermal) in rainbow trout and channel catfish, respectively. This research demonstrates that dermal absorption of waterborne chemicals occurs in large adult fish and results in distribution kinetics similar to those observed in inhalation exposures. Compared to branchial uptake, the dermal route of exposure appears to be relatively unimportant in large fish. It may, however, be very important in smaller fish and for juveniles of larger species.

A physiologically based toxicokinetic model for dermal absorption of organic chemicals by fish.

A physiologically based toxicokinetic model was developed to describe dermal absorption of waterborne organic chemicals by fish. The skin was modeled as a discrete compartment into which compounds diffuse as a function of chemical permeability and the concentration gradient. The model includes a countercurrent description of chemical flux at fish gills and was used to simulate dermal-only exposures, during which the gills act as a route of elimination. The model was evaluated by exposing adult rainbow trout and channel catfish to hexachloroethane (HCE), pentachloroethane (PCE), and 1,1,2,2-tetrachloroethane (TCE). Skin permeability coefficients were obtained by fitting model simulations to measured arterial blood data. Permeability coefficients increased with the number of chlorine substituent groups, but not in the manner expected from a directly proportional relationship between dermal permeability and skin:water chemical partitioning. An evaluation of rate limitations on dermal flux in both trout and catfish suggested that chemical absorption was limited more by diffusion across the skin than by blood flow to the skin. Modeling results from a hypothetical combined dermal and branchial exposure indicate that dermal uptake could contribute from 1.6% (TCE) to 3.5% (HCE) of initial uptake in trout. Dermal uptake rates in catfish are even higher than those in trout and could contribute from 7.1% (TCE) to 8.3% (PCE) of initial uptake in a combined exposure.

Time-resolved fluorescence anisotropy of fluorescent-labeled lysophospholipid and taurodeoxycholate aggregates.

Previous work from this laboratory demonstrated that the environment-sensitive lysolipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- monomyristoylphosphatidylethanolamine (N-NBD-MPE), at concentrations below its critical micelle concentration (CMCN-NBD-MPE = 4 microM), reached maximum fluorescence yield upon the addition of taurodeoxycholate (TDC) at concentrations well below its CMC (CMCTDC = 2.5 mM). These data indicated the formation of micellar aggregates of the two amphiphiles at concentrations below both of their CMCs. In the present study, fluorescence lifetime and differential polarization measurements were made to determine the size of these aggregates. In the absence of TDC and at 0.5 mM TDC a single lifetime (tau) and rotational correlation time (phi) were measured for N-NBD-MPE at the submicellar concentration of 2 microM, indicating a lack of interaction between the two molecules at this concentration. Above 0.5 mM TDC, two discrete lifetimes were resolved. Based on these lifetimes, two distinct rotational correlation times were established through polarization measurements. The shorter phi(0.19-0.73 ns) was ascribed to local probe motions, whereas the longer phi was in a time range expected for global rotation of aggregates the size of simple bile salt micelles (3-6.5 ns). From the longer phi, molecular volume and hydrodynamic radii were calculated, ranging from approximately 15 A at 1 mM to approximately 18 A at 5 mM TDC. These data support the conclusion that monomeric lysolipids in solution seed the aggregation of numerous TDC molecules (aggregation number = 16 at 1 mM TDC) to form a TDC micelle with a lysolipid core at concentrations below which they both self-aggregate.

Protein-mediated transfer of fluorescent-labeled phospholipids across brush border of rabbit intestine.

Monoacyl and diacyl phospholipids labeled with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) were used to measure the rate of phospholipid transfer between artificial large unilammelar vesicles and rabbit intestine brush-border membrane vesicles (BBMV). This assay demonstrated that transfer into and out of BBMV occurred in two kinetic phases. The fast rate of transfer resulted from interbilayer transfer between the outer membrane leaflets. The slower rate resulted from transmembrane transfer across the BBMV. Treatment of the BBMV with the protease pronase E and with the histidyl reagent diethylpyrocarbonate inhibited the rate of transmembrane transfer. Phospholipids containing one or two acyl chains and a wide range of head-group structures were transported across the membranes with rates that increased as a direct function of their water solubility. These data demonstrated the existence of a protein transporter in the intestinal brush border that functions to absorb slightly water-soluble lysophospholipids and short-chain diacyl phospholipids without redistributing the highly water-insoluble long-chain diacyl phospholipids between the outer and inner leaflets of the brush-border membrane.

Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane.

Digital, video-enhanced fluorescence microscopy and spectrofluorometry were used to follow the internalization into the yeast Saccharomyces cerevisiae of phosphatidylcholine molecules labeled on one acyl chain with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). Two pathways were found: (1) transport by endocytosis to the vacuole and (2) transport by a non-endocytic pathway to the nuclear envelope and mitochondria. The endocytic pathway was inhibited at low temperature (< 2 degrees C) and by ATP depletion. Mutations in secretory (SEC) genes that are necessary for membrane traffic through the secretory pathway (including SEC1, SEC2, SEC4, SEC6, SEC7, SEC12, SEC14, SEC17, SEC18, and SEC21) almost completely blocked endocytic uptake. In contrast, mutations in the SEC63, SEC65, or SEC11 genes, required for translocation of nascent secretory polypeptides into the ER or signal peptide processing in the ER, only slightly reduced endocytic uptake. Phospholipid endocytosis was also independent of the gene encoding the clathrin heavy chain, CHC1. The correlation of biochemical analysis with fluorescence microscopy indicated that the fluorescent phosphatidylcholine was degraded in the vacuole and that degradation was, at least in part, dependent on the vacuolar proteolytic cascade. The non-endocytic route functioned with a lower cellular energy charge (ATP levels 80% reduced) and was largely independent of the SEC genes. Non-endocytic transport of NBD-phosphatidylcholine to the nuclear envelope and mitochondria was inhibited by pretreatment of cells with the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzenesulfonic acid, suggesting the existence of protein-mediated transmembrane transfer (flip-flop) of phosphatidylcholine across the yeast plasma membrane. These data establish a link between lipid movement during secretion and endocytosis in yeast and suggest that phospholipids may also gain access to intracellular organelles through non-endocytic, protein-mediated events.

Kinetic analysis of phospholipid exchange between phosphatidylcholine/taurocholate mixed micelles: effect of the acyl chain moiety of the micellar phosphatidylcholine.

A fluorescent assay based on concentration-dependent self-quenching of the fluorescent phospholipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine was used to measure the rate of phospholipid exchange between taurocholate/phosphatidylcholine mixed micelles. Two NBD-labeled phosphatidylethanolamine probes (dilauryl and dimyristoyl) were tested in taurocholate/phosphatidylcholine mixed micelles prepared from phosphatidylcholine molecules varying in saturated chain length from 12 to 18. All combinations of probes and micellar phosphatidylcholines gave kinetic results that were best described by a transfer model in which phospholipids exchange predominantly through the water phase at low micellar concentrations and through transient micelle fusions at higher concentrations. Increasing the chain length of the micellar-saturated diacylphosphatidylcholine from 12 to 18 carbons resulted in a decrease in the overall rate of exchange by a factor of 127 for NBD-labeled dilaurylphosphatidylethanolamine and a factor of 2490 for NBD-labeled dipalmitoylphosphatidylethanolamine. The reduction in the overall rate resulted from decreases in both mechanisms of transfer. These results argue that the hydrophobicity of the lipophilic core of bile salt/phospholipid mixed micelles is the predominant determinant of the rate of formation of transfer-competent, transient micelle fusions and a major determinant of the rate of micelle to water phospholipid dissociation.

Interaction of lysophospholipid/taurodeoxycholate submicellar aggregates with phospholipid bilayers.

The equilibrium partitioning and the rate of transfer of monoacylphosphatidylethanolamines (lysoPEs) between phospholipid bilayers and lysoPE/taurodeoxycholate submicellar aggregates (SMAs) were examined with a series of environment-sensitive fluorescent-labeled N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-1-monoacylphosphatidyletha nolamine (N-NBD-lysoPE) probes of differing acyl chain length. Our previous work has demonstrated the formation of SMAs between bile salts and lysophospholipids [Shoemaker & Nichols (1990) Biochemistry 29, 5837-5842]. The experiments in the current work demonstrate that SMAs can coexist with phospholipid vesicles and can function as shuttle carriers for the transfer of lysophospholipids between membranes. The formation of submicellar aggregates of N-NBD-lysoPE and taurodeoxycholate (TDC) in equilibrium with 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles was determined from the increase in fluorescence generated upon addition of TDC to POPC vesicles containing 3 mol% N-NBD-lysoPE and 3 mol% N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE) as a nonextractable fluorescence energy-transfer quencher. The fraction of lysolipid extracted increased as a function of decreasing acyl chain length of the N-NBD-lysoPE molecule. The half-time for equilibration was independent of acyl chain length and averaged 44 ms at 10 degrees C. The delivery of N-NBD-lysoPE from preformed N-NBD-lysoPE/TDC SMAs into POPC vesicles containing the energy-transfer quencher N-Rh-PE was measured by the rate of fluorescence decline. The initial rate of insertion increased with decreasing acyl chain length of the N-NBD-lysoPE molecule and as a function of vesicle concentration.(ABSTRACT TRUNCATED AT 250 WORDS)