Cell-free transfer and sorting of membrane lipids in spinach

Summary The donor and acceptor specificity of cell-free transfer of radiolabeled membrane constituents, chiefly lipids, was examined using purified fractions of endoplasmic reticulum, Golgi apparatus, nuclei, plasma membrane, tonoplast, mitochondria, and chloroplasts prepared from green leaves of spinach. Donor membranes were radiolabeled with [¹⁴C]acetate. Acceptor membranes were unlabeled and immobilized on nitrocellulose filters. The assay was designed to measure membrane transfer resulting from ATP-and temperature-dependent formation of transfer vesicles by the donor fraction in solution and subsequent attachment and/or fusion of the transfer vesicles with the immobilized acceptor. When applied to the analysis of spinach fractions, significant ATP-dependent transfer in the presence of cytosol was observed only with endoplasmic reticulum as donor and Golgi apparatus as acceptor. Transfer in the reverse direction, from Golgi apparatus to endoplasmic reticulum, was only 0.2 to 0.3 that from endoplasmic reticulum to Golgi apparatus. ATP-dependent transfers also were indicated between nuclei and Golgi apparatus from regression analysis of transfer kinetics. Specific transfer between Golgi apparatus and plasma membrane and, to a lesser extent, from plasma membrane to Golgi apparatus was observed at 25°C compared to 4°C but was not ATP plus cytosol-dependent. All other combinations of organelles and membranes exhibited no ATP plus cytosol-dependent transfer and only small increments of specific transfer comparing transfer at 37°C to transfer at 4°C. Thus, the only combinations of membranes capable of significant cell-free transfer in vitro were those observed by electron microscopy of cells and tissues to be involved in vesicular transport in vivo (endoplasmic reticulum, Golgi apparatus, plasma membrane, nuclear envelope). Of these, only with endoplasmic reticulum (or nuclear envelope) and Golgi apparatus, where transfer in situ is via 50 to 70 nm transition vesicles, was temperature-and ATP-dependent transfer of acetatelabeled membrane reproduced in vitro. Lipids transferred included phospholipids, mono-and diacylglycerols, and sterols but not triacylglycerols or steryl esters, raising the possibility of lipid sorting or processing to exclude transfer of triacylglycerols and steryl esters at the endoplasmic reticulum to Golgi apparatus step.     

Monensin blocks the transfer of very long chain fatty acid containing lipids to the plasma membrane of leek seedlings. Evidence for lipid sorting based on fatty acyl chain length.

Delivery of newly synthesized fatty acids and lipids to the plasma membrane in leek seedlings via the endoplasmic reticulum (ER)-Golgi apparatus pathway is primarily by bulk transport (without sorting). However, pulse-chase experiments revealed kinetics of transport of lipids with VLCFA (very long chain fatty acids having more than 18 carbon atoms) in favor of a preferential transfer of these molecules to the plasma membrane. Use of monensin showed the accumulation of lipids in the Golgi apparatus and a related decrease of the amount of lipids transported to the plasma membrane. Lipid and fatty acid analyses revealed that transport of VLCFA-containing phospholipids was most strongly inhibited by the monensin block. These results taken together with an inability of the plasma membrane to synthesize VLCFA support a role for the Golgi apparatus in VLCFA delivery to the plasma membrane and leads to the hypothesis of a sorting function as well, based on fatty acyl chain length.     

Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts

The trafficking and function of cell surface proteins in eukaryotic cells may require association with detergent-resistant sphingolipid- and sterol-rich membrane domains. The aim of this work was to obtain evidence for lipid domain phenomena in plant membranes. A protocol to prepare Triton X-100 detergent-resistant membranes (DRMs) was developed using Arabidopsis (Arabidopsis thaliana) callus membranes. A comparative proteomics approach using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins. They included eight glycosylphosphatidylinositol-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins, and proteins of the stomatin/prohibitin/hypersensitive response family, suggesting that the DRMs originated from PM domains. We also identified a plant homolog of flotillin, a major mammalian DRM protein, suggesting a conserved role for this protein in lipid domain phenomena in eukaryotic cells. Lipid analysis by gas chromatography-mass spectrometry showed that the DRMs had a 4-fold higher sterol-to-protein content than the average for Arabidopsis membranes. The DRMs were also 5-fold increased in sphingolipid-to-protein ratio. Our results indicate that the preparation of DRMs can yield a very specific set of membrane proteins and suggest that the PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition. Our results also suggest a conserved role of lipid modification in targeting proteins to both the intracellular and extracellular leaflet of these domains. The proteins associated with these domains provide important new experimental avenues into understanding plant cell polarity and cell surface processes.     

Protein-mediated lipid transfer. The effects of lipid-phase transition and of charged lipids.

The protein-mediated phospholipid exchange between small unilamellar vesicles was investigated by fluorescence polarization measurements with diphenylhexatriene as optical probe. Thermotropic phase-transition measurements were taken after mixing two vesicle preparations of distinct and different phase-transition temperatures or having different states of charge. From the heights of each phase-transition step, we were able to follow the lipid-exchange process in the presence, as well as in the absence (natural exchange), of so-called transfer protein isolated from beef liver. A strong enhancement of the lipid transfer was observed at the corresponding lipid-phase-transition temperature, which is explained by the presence of fluctuating fluid and ordered domains co-existing at the lipid-phase-transition temperature. A unidirectional lipid transfer of the neutral component was observed between negatively charged phosphatidic acid and neutral phosphatidylcholine vesicles. Fluorescence polarization measurements showed the disappearance of the phosphatidylcholine phase transition, whereas the phosphatidic acid phase transition broadened and its phase transition temperature became lower.     

Evidence of direct interaction between actin and membrane lipids

Actin is a protein component of the cystoskeleton and is involved in cell motility. It is believed generally that actin filaments are attached to the cell membrane through an interaction with membranous actin-binding proteins. By using an in vitro system composed of liposomes and actin, we have shown that actin may also interact directly with the phospholipids of the membrane. Actin deposited at the surface of the liposome is organized in two regular patterns: a paracrystalline sheet of parallel filaments in register, or a netlike organization. These interactions of actin with membrane lipids occur only in the presence of millimolar concentrations of Mg2+. These results suggest that the interaction of the cytoskeleton with the membrane involves, at least in part, a direct association of actin with phospholipids.     

Control of membrane lipids in Mycoplasma gallisepticum: effect on lipid order.

Adaptation of Mycoplasma gallisepticum, a sterol-requiring Mycoplasma sp., to growth in a serum-free medium supplemented with cholesterol in decreasing concentrations and with various saturated or unsaturated fatty acids enabled us to control both the cholesterol levels and the membrane fatty acid composition. An estimate of the membrane physical state from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene indicated that the membrane lipids of native M. gallisepticum were highly ordered. Elongation of the saturated fatty acid chains from 14 to 18 carbon atoms caused only a small increase in the membrane lipid ordering, whereas the introduction of a cis double bond reduced it significantly. Lipid-phase transitions were observed in low-cholesterol-adapted organisms, whose membrane lipids were still highly ordered at the growth temperature.     

Modification of membrane lipids. Phenethyl alcohol-induced alteration of lipid composition in Tetrahymena membranes.

Tetrahymena pyriformis NT-I cells in the early-logarithmic phase were incubated with phenethyl alcohol (2-phenylethanol) and effects on the lipid composition were examined in various membranes. 1. There was a marked modification in phospholipid head, as well as fatty acyl group composition in pellicles, mitochondria and microsomes of the phenethyl alcohol-treated cells. Compared with membranes of the control cells, the membranes from phenethyl alcohol-treated cells were found to contain a higher level of phosphatidylcholine content with the compensating decrease in phosphatidylethanolamine, while 2-aminoethylphosphonolipid showed only a slight decrease in these membranes. The acyl group profile of membrane phospholipids in the presence of phenethyl alcohol was also modified so that a profound elevation of the content of polyunsaturated fatty acids, linoleic and gamma-linolenic acids. The major monounsaturate, palmitoleate decreased. Such lipid alteration is a reversible process, and therefore upon removal of phenethyl alcohol the modified lipid composition returned to normal. 2. By freeze-fracture electron microscopy in combination with temperature quenching, the outer alveolar membrane of the phenethyl alcohol-treated cell was observed to reveal less aggregation of intercalated-membrane particles, as compared with the control membrane. The quantitative analysis of the thermotropic lateral movement of membrane particles provided evidence that the membrane in the phenethyl alcohol-treated cell became more fluid. Such fluidizing effects may result from an increase in the acyl group unsaturation and also in the phosphatidylcholine content. 3. With regard to the mechanism responsible for the marked decrease in palmitoleate in membrane phospholipids, there was found a depressed conversion of the palmitate to palmitoleate in the phenethyl alcohol-treated cells. It was further suggested that the drug may have an inhibitory effect on the synthesis of palmitoyl-CoA desaturase involving the (16 : 0 leads to 16 : 1) conversion. Also, it was demonstrated that the increase in a precursor-product fashion of phosphatidylcholine with the corresponding decrease in phosphatidylethanolamine was not due to transformation of phosphatidylethanolamine to phosphatidylcholine through stepwise methylation.     

Radiation-induced lipid peroxidation and the fluidity of erythrocyte membrane lipids

The effect of radiation-induced peroxidation on the fluidity of the phospholipids of the erythrocyte membrane was studied using both erythrocyte ghosts and liposomes formed from the polar lipids of erythrocytes. In liposomes, the oxidation of the phospholipids increased with radiation dose, but there was no change in the fluidity of the lipids as measured by spin-label motion. Under the same conditions of irradiation, no oxidation of phospholipid was detected in erythrocyte ghosts, although changes occurred in the motion of spin labels intercalated with the membrane. These changes were attributed to radiation-induced alterations in the membrane proteins. It is concluded that alterations in motion of spin labels, observed with intact membranes after irradiation, are most likely the result of changes in the structure of membrane proteins rather than the lipids.     

Spiroplasma membrane lipids.

Membranes of six spiroplasma strains belonging to different Spiroplasma species and subgroups were isolated by a combination of osmotic lysis and sonication in the presence of EDTA to block endogenous phospholipase activity. Analysis of membrane lipids showed that in addition to free and esterified cholesterol the spiroplasmas incorporated exogenous phospholipids from the growth medium. Sphingomyelin was preferentially incorporated from phosphatidylcholine-sphingomyelin vesicles or from the serum used to supplement the growth medium. Palmitate was incorporated better than oleate into membrane lipids synthesized by the organisms during growth. The major phospholipid synthesized by the spiroplasmas was phosphatidylglycerol. The positional distribution of the fatty acids in phosphatidylglycerol of Spiroplasma floricola resembled that found in Mycoplasma species, in which the saturated fatty acids prefer position 2 in the glycerol backbone and not position 1 as found in Acholeplasma species and elsewhere in nature. Electron paramagnetic resonance analysis of spin-labeled fatty acids incorporated into S. floricola membranes exhibited homogeneous single-component spectra without immobilized regions. The S. floricola membranes were more rigid than those of Acholeplasma laidlawii and less rigid than those of Mycoplasma gallisepticum.     

Cell-free processing of nascent proteins destined to be linked to the plasma membrane by a phosphatidylinositol-glycan anchor.

Certain proteins are anchored to the outer plasma membrane by a phosphatidylinositol-glycan (PI-G) linker. Nascent forms of PI-G anchored proteins contain both NH2- and COOH-terminal signal peptides. The function and structural requirements of the COOH-terminal signal peptide as discussed and some studies on the cell-free processing of a nascent protein to its mature PI-G tailed form are presented.     

Role of lipids in theNeurospora crassa membrane. I. Influence of fatty acid composition on membrane lipid phase transitions

Summary The relationship between lipid composition and phase transition was investigated by differential scanning calorimetry for intact and membrane phospholipid extracts of wild-type (w/t) and thecel ^– (Tw 40) mutant ofNeurospora crassa. Thecel ^– (Tw 40) mutant (grown on minimal, sucrose medium supplemented with Tween 40 at 34 °C) had approximately twice the saturated fatty acid content ofw/t organisms grown at 22 °C. The gel-liquid crystal phase transitions of ergosterol-free extracts derived fromw/t andcel ^– (Tw 40) occur at –31 and –11 °C, respectively. The heats of transition (H) of these extracts were 1 and 13 cal/g, respectively. The addition of ergosterol (the predominant sterol inNeurospora) to the phospholipid extracts decreased the observed heats of transition, but did not alter the transition temperature. IntactNeurospora, whetherw/t orcel ^– (Tw 40) did not manifest similar gel-liquid crystal phase transitions in the differential scanning calorimeter. However, an endothermic peak at approximately 30 °C was observed in intact cells and extracted phospholipids of bothw/t andcel ^– (Tw 40) organisms. This peak was insensitive to the addition of ergosterol, had a low heat content (H1 cal/g), and was reversible.     

Chloroplast biogenesis. Cell-free transfer of envelope monogalactosylglycerides to thylakoids.

An ATP- and temperature-dependent transfer of monogalactosylglycerides from the chloroplast envelope to the chloroplast thylakoids was reconstituted in a cell-free system prepared from isolated chloroplasts of garden pea (Pisum sativum) or spinach (Spinacia oleracea). Isolated envelope membranes, in which the label was present exclusively in monogalactosylglycerides, were prepared radiolabeled in vitro with [14C]galactose from UDP-[14C]galactose to label galactolipids as the donor. ATP-dependent transfer of radioactivity from donor to unlabeled acceptor thylakoids, immobilized on nitrocellulose strips, was observed. In some experiments linear transfer for longer than 30 min of incubation was facilitated by the addition of stroma proteins but in other experiments stroma was without effect or inhibitory suggesting no absolute requirements for a soluble protein carrier. Transfer was donor specific. No membrane fraction tested (plasma membrane, tonoplast, endoplasmic reticulum, nuclei, Golgi apparatus, mitochondria or thylakoids) (isolated from tissue radiolabeled in vivo with [14C]acetate) other than chloroplast envelopes demonstrated any significant ability to transfer labeled membrane lipids to immobilized thylakoids. Acceptor specificity, while not absolute, showed a 3-10-fold greater ATP-dependent transfer of labeled galactolipids from chloroplast envelopes to immobilized thylakoids than to other leaf membranes. The results provide independent confirmation of the potential for transfer of galactolipids between chloroplast envelopes and thylakoids suggested previously from ultrastructural studies and of the known location of thylakoid galactolipid biosynthetic activities in the chloroplast envelope.     

Cell-free transfer of sterols from dictyosome-like structures to plasma membrane vesicles of guinea pig testes

Summary Transfer of radiolabeled lipids from dictyosome-like structures (DLS) from testis tubules of the guinea pig as donor to unlabeled plasma membrane from testis tubules immobilized on nitrocellulose as acceptor was studied in a completely cell-free system. As a general label for lipids of the donor DLS, isolated testis tubules were incubated with [¹⁴C]acetate. Time- and temperature-dependent transfer of [¹⁴C]acetate labeled constituents was observed in the cellfree system. However, despite the fact that phospholipids and other constituents were highly labeled in the donor fraction, primarily radioactive sterols were transferred to the plasma membrane acceptor vesicles. Transfer at 37°C represented 0.4 to 0.7% of the total radiolabeled cholesterol at 37°C but little or no transfer occurred at 4°C. The sterols transferred exhibited Chromatographic mobilities corresponding to those of cholesterol and lanosterol. Similar results were obtained with [¹⁴C]mevalonic acid. In subsequent experiments, cholesterol transfer from DLS to plasma membrane was demonstrated by incubation of DLS with [³H]squalene which was converted into sterol or with [¹⁴C]cholesterol. Transfer of sterols required ATP, but not cytosol, and was both time- and temperature-dependent. DLS were more effective than either endoplasmic reticulum or plasma membrane as the donor fraction. The results from the cell-free analysis suggest a possible functional role of the DLS in sterol biogenesis and transfer to the plasma membrane during spermatid development.Abbreviations DLS dictyosome-like structure(s) - PBS phosphatebuffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin     

Cell-free processing and segregation of insulin precursors

The biosynthesis, segregation, and processing of preproinsulin (116 amino acids) was investigated to determine the mechanism(s) by which it is translocated across the endoplasmic reticulum membrane. Islet mRNA was translated in the wheat germ cell-free system, and at various times during preproinsulin synthesis, puromycin was added, followed by addition of microsomal membranes. Neither processing of preproinsulin nor translocation of proinsulin into microsomal membranes occurred in the presence of puromycin. Synchronization of preproinsulin translation by addition of 7-methylguanosine 5'-phosphate enabled the timing of preproinsulin synthesis and proinsulin (91 amino acids) segregation into microsomal membranes to be determined. Membrane binding occurs when about 60 amino acids have been polymerized, i.e. prior to the completion of the polypeptide chain. The binding of signal recognition particle to the nascent signal is demonstrated to be an absolute requirement for translocation and processing of preproinsulin. The results indicate that segregation and processing of preproinsulin are co-translational events; no evidence for a post-translational mechanism was found. Furthermore, this work, together with similar studies, suggests that presecretory polypeptides must be synthesized as part of a precursor with a minimum size of 60-80 amino acids in order to effect membrane binding and translocation of the polypeptide chain within the intracisternal space of the endoplasmic reticulum.     

Role of lipids in the Neurospora crassa membrane. I. Influence of fatty acid composition on membrane lipid phase transitions.

The relationship between lipid composition and phase transition was investigated by differential scanning calorimetry for intact and membrane phospholipid extracts of wild-type (w/t) and the cel-(Tw 40) mutant of Neurospora crassa. The cel-(Tw 40) mutant (grown on minimal, sucrose medium supplemented with Tween 40 at approximately 34 degrees C) had approximately twice the saturated fatty acid content of w/t organisms grown at approximately 22 degrees C. The gel-liquid crystal phase transitions of ergosterol-free extracts derived from w/t and cel-(Tw 40) occur at -31 and -11 degrees C, respectively. The heats of transition (delta H) of these extracts were 1 and 13 cal/g, respectively. The addition of ergosterol (the predominant sterol in Neurospora) to the phospholipid extracts decreased the observed heats of transition, but did not alter the transition temperature. Intact Neurospora, whether w/t or cal-(Tw 40) did not manifest similar gel-liquid crystal phase transitions in the differential scanning calorimeter. However, an endothermic peak at approximately 30 degrees C was observed in intact cells and extracted phospholipids of both w/t and cel-(Tw 40) organisms. This peak was insensitive to the addition of ergosterol, had a low heat content (delta H congruent to 1 cal/g), and was reversible.