The effects of insulin, cycloheximide and phalloidin on the content of actin and p35 in extracts prepared from the nuclear fraction of Krebs II ascites cells

The nuclear fraction isolated from Krebs II ascites cells following cell disruption by nitrogen cavitation was separated into four fractions by salt/detergent extraction: NP-40 soluble fraction, 130 mM KCl extract, DOC/Triton x 100 soluble fraction and salt/detergent treated nuclei. The protein composition of the individual fractions was studied by SDS-PAGE and the relative amounts of actin and a 35 kDa protein (p35) were measured from gel scans. There was a time-dependent shift of actin from the 130 mM KCl extract to the NP-40 soluble fraction upon storage of the nuclear fraction on ice, indicating a progressive depolymerization of microfilaments. Compared with actin there was a slower release of p35 into the NP-40 soluble fraction. The results suggest that p35 is not integrated in the microfilament network. Phalloidin, which stabilizes the microfilaments, enriched the amount of both proteins in the 130 mM KCl extracts, together with a series of other proteins in the range 50-205 kDa. The presence of phalloidin also resulted in a large increase in the actin content in both the DOC/Triton x 100 extract and the fraction containing salt/detergent treated nuclei. Incubation of cells with insulin and/or cycloheximide enriched the amount of actin in the 130 mM KCl fraction. The results show that short term incubation of cells with phalloidin, insulin or cycloheximide increases the actin content of the nuclear fraction and also affects the presence of several other proteins.     

Glucose-induced thermal stabilization of the native conformation of GLUT 1

The glucose transporter, GLUT 1, was purified from erythrocyte membranes and incorporated into vesicles of erythrocyte lipids. These protein-containing vesicles were studied with differential scanning calorimetry. It was found that the protein underwent an irreversible denaturation at 68.5 +/- 0.2 degreesC (at a scan rate of 0.25 degreesC/min) which was shifted to 72.6 +/- 0.2 degreesC in the presence of 500 mM D-glucose, while 500 mM L-glucose or 10 microM cytochalasin B did not produce a significant shift. The calorimetric enthalpy was found to be 150 kcal/mol, independent of the presence of D-glucose. On a weight basis this value is lower than that for soluble proteins, but it is comparable to values obtained with other integral membrane proteins. The van't Hoff enthalpy is similar to the calorimetric enthalpy, within the experimental error, indicating that the transition is not likely to be cooperative. The activation energy is estimated from both the scan rate dependence of the transition temperature and from the shape of the DSC curve. The presence of 500 mM D-glucose slightly decreases the activation energy. It is concluded that the shift to a higher denaturation transition temperature in the presence of D-glucose is not a result of increased kinetic stability of GLUT 1.     

Physical determinants of intermembrane protein transfer

Intermembrane protein transfer between erythrocytes and phospholipid vesicles was examined under a variety of conditions to investigate physical factors governing this process. Human erythrocytes were incubated with sonicated dimyristoylphosphatidylcholine vesicles containing trace [14C]dipalmitoylphosphatidylcholine. Protein-vesicle complexes were separated from cells and from membrane fragments by density gradient centrifugation. The yield of isolated protein vesicles was determined from the 14C-vesicle marker; protein compositions were analyzed by SDS-polyacrylamide gel electrophoresis. Enzymatic removal of portions of the cytoplasmic or exoplasmic domains of cell membrane proteins had little effect on the extent of protein transfer. Membrane additives such as cholate produced a 2-fold increase in protein-vesicle yield. The selectivity of protein transfer from erythrocytes was influenced by the lipid composition of recipient vesicles: inclusion of cholesterol increased band 3 content while the presence of anionic phospholipids reduced transfer. Proteins transferred from 32P-labeled cells differed in specific radioactivity from bulk cell proteins: glycophorin, highly phosphorylated in the cell membrane, showed no detectable labeling in the corresponding protein-vesicle band. These observations suggest that cell-to-vesicle protein transfer is insensitive to bulk steric and electrostatic properties of cell membranes, but enhanced by membrane defects. Recipient membrane composition influences the selectivity of transferred proteins and may reveal subtle differences in the membrane association of protein subpopulations.     

The myristoyl moiety of myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein is embedded in the membrane

Members of the myristoylated alanine-rich protein kinase C substrate (MARCKS) family are involved in several cellular processes such as secretion, motility, mitosis, and transformation. In addition to their ability to bind calmodulin and to cross-link actin filaments, reversible binding to the plasma membrane is most certainly an important component of the so far unknown functions of these proteins. We have therefore investigated the binding of murine MARCKS-related protein (MRP) to lipid vesicles. The partition coefficient, Kp, describing the affinity of myristoylated MRP for acidic lipid vesicles (20% phosphatidylserine, 80% phosphatidylcholine) is 5-8 x 10(3) M-1, which is only 2-4 times larger than the partition coefficient for the unmyristoylated protein. Interestingly, the affinity of MRP for acidic lipid membranes is 20-30-fold smaller than reported for murine MARCKS (Kim, J., Shishido, T., Jiang, X., Aderem, A. A., and McLaughlin, S. (1994) J. Biol. Chem. 269, 28214-28219). Since only a marginal binding could be observed with neutral phosphatidylcholine vesicles, we propose that electrostatic interactions are the major determinant of the binding of MRP to pure lipid membranes. Although the myristoyl moiety does not contribute drastically to the binding of MRP to vesicles, photolabeling experiments with a photoreactive phospholipid probe show that the fatty acid is embedded in the bilayer. The same membrane topology was found for bovine brain MARCKS. Since the relatively low affinity of MRP for vesicles is insufficient to account for a stable anchoring of the protein to cellular membranes, insertion of the myristoyl moiety into the bilayer might favor the interaction of MRP with additional factors required for the binding of the protein to intracellular membranes.     

Impaired development of Th2 cells in IL-13-deficient mice

Synaptic vesicles can be coated in vitro in a reaction that is ARF-, ATP-, and temperature-dependent and requires synaptic vesicle membrane proteins. The coat is largely made up of the heterotetrameric complex, adaptor protein 3, recently implicated in Golgi-to-vacuole traffic in yeast. Depletion of AP3 from brain cytosol inhibits small vesicle formation from PC12 endosomes in vitro. Budding from washed membranes can be reconstituted with purified AP3 and recombinant ARF1. We conclude that AP3 coating is involved in at least one pathway of small vesicle formation from endosomes.     

Subcellular compartmentalization of maize storage proteins in Xenopus oocytes injected with zein messenger RNAs

Maize storage proteins synthesized in oocytes were compartmentalized in membrane vesicles because they were resistant to hydrolysis by protease, unless detergent was present. The site of storage protein deposition within the oocyte was determined by subcellular fractionation. Optimal separation of oocyte membranes and organelles was obtained when EDTA and high concentrations of NaCl were included in the homogenization and gradient buffers. Under these conditions, fractions in sucrose gradients containing a heterogeneous mixture of smooth membranes (presumably endoplasmic reticulum, Golgi apparatus, and plasma membrane, density = 1.10-1.12 g/cm3), mitochondria (densities = 1.14 and 1.16 g/cm3), yolk platelets (density = 1.21 g/cm3), and a dense matrix material (density = 1.22 g/cm3) could be separated. Some zein proteins were recovered in the mixed membrane fraction, but the majority occurred in vesicles sedimenting with yolk platelets and granular material at a density of approximately 1.22 g/cm3. When metrizamide was included in the gradient to increase the density, little of the dense matrix material was isolated, and vesicles containing zein proteins were separated from other oocyte components. These vesicles were similar to protein bodies in maize endosperm because they were of identical density and contained the same group of polypeptides.     

Reconstitution of a functional acetylcholine receptor. Polypeptide chains, ultrastructure, and binding sites for acetylcholine and local anesthetics

The 'reconstitution cycle' is composed of the following sequence of operations. Highly purified receptor-rich membranes prepared from Torpedo marmorata electric organ are exposed to pH 11 to remove the 43,000-Mr protein and dispersed into solution by sodium cholate under conditions where more than 85% of the receptor protein is in its 9-S form. Elimination of the detergent by filtration on a Sephadex column (or dialysis) yields a 'reconstituted receptor' fraction, under conditions which conserve part of the endogenous lipids, or 'reconstituted vesicles' in the presence of an excess of exogenous lipids. The polypeptide composition of these fractions was analysed by sodium dodecylsulfate gel electrophoresis. Conditions are defined for quantitative measurements of the various polypeptide chains. The 40,000-Mr chain, which is labelled by the affinity reagent 4-(N-maleimido)phenyl [3H]trimethylammonium and therefore carries the acetylcholine receptor site, is the dominant polypeptide in the alkaline-treated membranes and the reconstituted acetylcholine receptor. Electron microscopy discloses that many of the alkaline-treated membranes no longer form closed vesicles and do not show the transverse asymmetry of the native membranes observed after tannic acid fixation. In the reconstituted receptor fractions, the receptor molecules reaggregate into discs and may be exposed on both faces of the discs. In the reconstituted vesicles, receptor rosettes are integrated to the lipid vesicles. With native membranes, the radioactive local anesthetic [3H]trimethisoquin binds to three classes of sites: non-specific, low-affinity and high-affinity. Carbamylcholine causes an increase in the number of high-affinity sites up to approximately 0.7 times the number of alpha-125I-bungarotoxin sites. This ratio, the three classes of binding sites, and their regulation by carbamylcholine are conserved through the reconstitution cycle.     

Association of N-ethylmaleimide sensitive fusion (NSF) protein and soluble NSF attachment proteins-alpha and -gamma with glucose transporter-4-containing vesicles in primary rat adipocytes

To investigate the role of N-ethylmaleimide sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAP)-containing fusion complexes in glucose transporter-4 (GLUT4) membrane trafficking, the subcellular distributions of NSF, alpha-SNAP, and gamma-SNAP in primary rat adipocytes were determined. A large fraction of the NSF and SNAPs were associated with intracellular membranes, distributed between the low-density microsomes (LDM) and high-density microsomes. Very little of the NSF and SNAPs were associated with the plasma membrane fraction. This distribution did not change after insulin stimulation. Approximately 75% of the NSF and SNAPs in the LDM fraction were coimmunoprecipitated with 85% of the GLUT4 and 60% of the vesicle associated membrane proteins (VAMPs; synaptobrevins) VAMP-2 and cellubrevin in anti-GLUT4 immunoadsorptions. In contrast to NSF and the SNAPs, the beta-coatomer protein (beta-COP) found in the LDM fraction was excluded from GLUT4 vesicles. When LDM fractions were solubilized with Thesit (octaethylene glycol dodecyl ether) or Triton X-100, approximately 40% of the alpha-SNAP was colocalized with NSF on glycerol gradients in large (approximately 20S), ATP-sensitive complexes. VAMP-2 and cellubrevin are concentrated in the LDM fractions and in GLUT4 vesicles; both were excluded from these complexes. These data suggest that the steady state association of NSF and the SNAPs with GLUT4 vesicles and cell membranes is independent of the formation of fusion complexes.     

Association of a cellular myosin II with anionic phospholipids and the neuronal plasma membrane

Myosin II has been observed in close proximity to the neuronal plasma membrane, suggesting the possibility that at least one isoform of neuronal myosin II may be capable of direct association. Here, we demonstrate that a significant fraction (> 30%, saturable around 90%) of brain myosin II, but not myosins from skeletal or cardiac muscle, can bind to lipid vesicles composed of the anionic phospholipid L-alpha-phosphatidyl-L-serine but not with vesicles made from the neutral phospholipid L-alpha-phosphatidylcholine. Binding to lipid vesicles made from L-alpha-phosphatidyl-L-serine is enhanced in the presence of millimolar amounts of free calcium. ATPase activity remains unimpaired after vesicle association. Myosin II was also shown to remain in tight association with purified plasma membranes, even after depletion of actin. The above observations suggest that mechanisms involving membrane-bound myosin II are required to facilitate metazoan cell motility.     

Chain length and temperature dependence of the reversible association of model acylated proteins with lipid bilayers

To study the binding of fatty-acylated proteins to lipid bilayers, we have specifically attached fatty acids to the N-terminus of chemically modified bovine pancreatic trypsin inhibitor. This was accomplished by reacting the protein with saturated fatty acid anhydrides ranging in length from 8 to 18 carbons. Following radiolabeling of the fatty-acylated proteins at Lys-15, binding of these proteins to palmitoyloleoyl phosphatidylcholine vesicles was examined as a function of temperature using ultracentrifugation to determine the fraction of bound protein. Binding of these fatty-acylated proteins exhibited a significant enthalpy change. We also examined the free-energy change of binding as a function of fatty acid chain length. Our results are complimentary to other binding studies of fatty-acylated peptides. Comparisons with other myristoylated proteins and peptides indicate that local protein structure, apart from electrostatic interactions, plays a significant role in determining the magnitude of the overall free-energy change of membrane binding of fatty-acylated proteins. Light-scattering experiments indicated that both myristoyl and palmitoyl groups can induce protein micelle formation in aqueous solution at high concentration, but that only palmitoyl groups do so at physiologically relevant concentrations. Our results support a model in which single lipid modifications are incapable of stably anchoring proteins to biological membranes but facilitate protein associations in conjunction with other modes of interaction.     

Characteristics of endoplasmic reticulum-derived transport vesicles

We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission. Inhibition of Ypt1p-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport. Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport. A 900-fold enrichment was developed using differential centrifugation and a series of velocity and equilibrium density gradients. Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack peripheral protein coats. Quantitative Western blot analysis indicates that protein markers of cytosol and cellular membranes are depleted throughout the purification, whereas the synaptobrevin-like Bet1, Sec22, and Bos1 proteins are highly enriched. Uncoated ER-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane. The ERV proteins may represent abundant cargo and additional targeting molecules.     

Hepatocellular carcinoma: power Doppler US with a contrast agent--preliminary results

Tetracaine-induced biphasic structure-functional alterations were investigated in acetylcholinesterase (AChE)-associated membrane vesicles from the electric organ of Torpedo californica. Enzyme assays showed that tetracaine exhibits a biphasic effect on the activity of membrane-bound AChE: increasing it at low concentrations (< 12 mM) and decreasing it at high concentrations (> 12 mM). Fluorescence-polarization experiments demonstrated that tetracaine affects the fluidity of lipid hydrocarbon chains of these membranes in a biphasic manner: increasing it at < 20 mM and decreasing it at > 20 mM. This small molecule also alters the fluidity of the negatively charged lipid head group: increasing it at < 13 mM and remaining essentially at the same level at > 13 mM. The positively charged lipid head group is unaffected. Contrasting effects on AChE activity with changes in membrane fluidity showed that [tetracaine] for AChE activity is comparable to that for the fluidity of the negatively charged lipid head group (12 mM versus 13 mM), but lower than that for a biphasic effect on the fluidity of lipid hydrocarbon chains (12 mM versus 20 mM). Differential scanning microcalorimetry showed that, due to membrane protein-lipid interaction, the lipid-phase transition temperature (tml) is higher for AChE-associated membrane vesicles than for isolated lipids from these membranes. An overall disordering of the membranes by tetracaine, as inferred from the lowering of tml, was also demonstrated. These findings suggested that binding of tetracaine to the lipid polar head group and membrane protein-lipid interaction may contribute to a higher [tetracaine] in inducing a comparable biphasic effect on membrane fluidity. At high [tetracaine], charge interactions between the tetracaine cation and the negatively charged lipid head group may result in a new lipid phase in the membranes, which could reverse the increase in membrane fluidity, resulting in the observed biphasic effect. Although both tetracaine and alcohol are amphiphilic species, they exhibit distinctive structure-functional effects on the membranes, as shown by comparing the results obtained on tetracaine with those previously reported for alcohol. The present observations may have significant physiological implications and may be of importance in understanding the biochemical effects of tetracaine in correlation with its physiological impact.     

Intracellular events in the "selective" transport of lipoprotein-derived cholesteryl esters

The current study utilizes human, apoE-free high density lipoprotein reconstituted with a highly specific fluorescent-cholesteryl ester probe to define the initial steps and regulatory sites associated with the "selective" uptake and intracellular itinerary of lipoprotein-derived cholesteryl esters. Bt2cAMP-stimulated ovarian granulosa cells were used as the experimental model, and both morphological and biochemical fluorescence data were obtained. The data show that cholesteryl ester provided through the selective pathway is a process which begins with a temperature-independent transfer of cholesteryl ester to the cell's plasma membrane. Thereafter transfer of the lipid proceeds rapidly and accumulates prominently in a perinuclear region (presumed to be the Golgi/membrane sorting compartment) and in lipid storage droplets of the cells. The data suggest that lipid transfer proteins (or other small soluble proteins) are not required for the intracellular transport of the cholesteryl esters, nor is an intact Golgi complex or an intact cell cytoskeleton (although the transfer is less efficient in the presence of certain microtubule-disrupting agents). The intracellular transfer of the cholesteryl esters is also somewhat dependent on an energy source in that a glucose-deficient culture medium or a combination of metabolic inhibitors reduces the efficiency of the transfer. A protein-mediated event may be required for cholesteryl ester internalization from the plasma membrane, in that N-ethylmaleimide dramatically blocks the internalization phase of the selective uptake process. Taken together these data suggest that the selective pathway is a factor-dependent, energy-requiring cholesteryl ester transport system, in which lipoprotein-donated cholesteryl esters probably flow through vesicles or intracellular membrane sheets and their connections, rather than through the cell cytosol.     

A conformational model for the action of general anesthetics at the membrane level. II. Experimental observations on the effects of anesthetics on lipid fluidity and lipid protein interactions

We have investigated the effect of general anesthetics (the normal alcohol series up to pentanol, halothane, pentrane, ether, chloroform, and ketamine) on lipid fluidity of phospholipid vesicles and mitochondrial and erythrocyte membranes by using spin labels and fluorescent probes. The spin labels used (5- and 16-doxyl stearic acids) show that all anesthetics tested have a slight fluidizing effect on lipid vesicles but induce a very strong increase in mobility of spin labels in mitochondria and lower in erythrocyte ghosts. These results are interpreted as a labilization of lipid protein interactions at all depths in the bilayer. The fluorescent molecules ANS and NPN, which probe the glycerol region and the core of the bilayer respectively, show a decrease of fluorescence induced by alcohols, halothane, ether, chloroform in both lipid vesicles and membranes. The decrease of fluorescence is due to decreased quantum yield as shown by double reciprocal plots of probe fluorescence against membrane concentration. The fluorescence decrease is interpreted mainly as an increase in fluidity of the lipid bilayer and not as an increase of polarity of the probe environment. The effect of ketamine is that of fluidization in the bilayer core (NPN) but of increased rigidity in the glycerol region (ANS) perhaps due to the amphipathic character of this anesthetic, that is supposed to bind in the polar region of the bilayer. Pentrane also induces fluidization in the bilayer core (NPN) but has a peculiar effect near the surface (ANS): in lipid vesicles it induces a fluorescence decrease, whereas an increase is seen in mitochondrial membranes. These complex effects are considered as the result of some specific change in the lipid protein interactions in the region probed by ANS. The effects of anesthetics on maximal NPN fluorescence (Fo) have been usually found to be stronger in mitochondrial membranes than in lipid vesicles, thus confirming the results of the spin label studies, showing a labilization of lipid protein interactions induced by anesthetics. The effects on Fo of ANS, however, appear to be stronger in lipid vesicles than in membranes. These findings indicate that the presence of the proteins counteracts the perturbation induced by anesthetics at the level of the membrane surface, in contrast with the disruption of lipid protein interactions observed in the membrane hydrophobic areas.     

Na+,K+-ATPase was found to be the membrane component responsible for the hydrophobic behavior of the brain membrane tubulin

We have previously described that the tubulin isolated from brain membranes as a hydrophobic compound by partitioning into Triton X-114 is a peripheral membrane protein [corrected]. The hydrophobic behavior of this tubulin is due to its interaction with membrane protein(s) and the interaction occurs principally with the acetylated tubulin isotype. In the present work we identified the membrane protein that interacts with tubulin as the Na+,K+-ATPase alpha subunit by amino acid sequencing. Using purified brain Na+,K+-ATPase we were able to isolate part of the total hydrophilic tubulin as a hydrophobic compound which contains a high proportion of the acetylated tubulin isotype.     

The additional predictive value contributed by quantitative chromatin pattern description as compared to DNA ploidy level measurement in 257 superficial bladder transitional cell carcinomas

The purpose of the present study was to demonstrate the post-translational modifications of sperm plasma membrane proteins by fatty acid acylation during sperm maturation in the epididymis. Rat epididymal spermatozoa were incubated at 37 degrees C with various concentrations (100 microCi and 1 mCi) of [9-10(n)3H]palmitic acid in a medium containing Tyrode's solution supplemented with sodium bicarbonate, sodium pyruvate and sodium lactate. The incorporation of [3H]palmitate in vitro was determined in epididymal spermatozoa and an attempt was made to identify the lipid-linked proteins of purified plasma membranes of maturing epididymal spermatozoa by autoradiography. The studies demonstrated that [3H]palmitate was covalently linked to a subset of membrane cytoskeleton proteins of maturing rat spermatozoa. The pattern of incorporation of lipid was a maturation-associated phenomenon as caput spermatozoa incorporated more radioactivity than did caudal spermatozoa. The labelled proteins appeared to be membrane-bound since 82% of radioactivity was associated with membrane fractions. Autoradiograms of SDS-PAGE gels of labelled caput sperm extract showed three prominent palmitate-incorporating protein bands of about 70, 56 and 36 kDa and few minor bands. Most of these proteins were present in the membrane fraction of caput spermatozoa. Labelled gels of both the sperm extracts and of purified membranes showed resistance to hydroxylamine treatment, suggesting that there are amide bonds between lipid and proteins. The higher incorporation of labelled palmitate by immature spermatozoa of the caput epididymis compared with mature spermatozoa from the cauda epididymis and the addition of palmitate to plasma membrane proteins of caput epididymal spermatozoa suggest that fatty acylation is a post-translational modification of sperm membrane proteins.     

Amplified expression, purification and functional reconstitution of the dipeptide and tripeptide transport protein of Lactococcus lactis

Transport of hydrophilic dipeptides and tripeptides into Lactococcus lactis is mediated by a proton-motive-force-driven peptide-transport protein (DtpT) that shares similarity to eukaryotic peptide transporters, e.g. from yeasts, plants, and the kidney and small intestine of rabbit, man and rat. The expression level of DtpT protein in L. lactis was increased (20-40-fold) to approximately 10% of total integral membrane protein by means of a low-copy-number vector and selecting the appropriate growth conditions. Membrane vesicles bearing the DtpT-His6 protein (containing a C-terminal factor-Xa cleavage site and a six-histidine-tag) showed a Pro-Ala uptake activity that was half that of membranes containing the wild-type protein. The activity in the DtpT-His6 membrane vesicles increased at least 50% upon removal of the His6 tag from the protein. More than 95% DtpT was solubilized from L. lactis membranes in the presence of 1% (mass/vol.) n-dodecyl-beta-D-maltoside, and approximately 2 mg DtpT-His6 was purified by Ni2+-chelate affinity chromatography from 100 mg membrane protein. Purified DtpT-His6 was reconstituted unidirectionally into detergent-saturated formed liposomes, which were prepared from Escherichia coli phospholipid and egg phosphatidylcholine; the detergent was removed by adsorption to polystyrene beads. The highest uptake activities were obtained when DtpT was incorporated into liposomes that were treated with a low amount of n-dodecyl-beta-D-maltoside (onset of liposome solubilization). The uptake activity could be improved by addition of NaCl (200 mM) and lipids (2 mg/ml) during the solubilization, purification and reconstitution steps.     

A comparative study of fixation techniques in the open Bankart operation using either a cannulated screw or suture-anchors

Neuroendocrine PC12 cells contain small microvesicles that closely resemble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous size and their unique protein composition. Since synaptic vesicles arise by endocytosis from the plasma membrane, nerve terminals and PC12 cells must contain the molecular machinery to sort synaptic vesicles from other membrane proteins and pinch off vesicles of the correct diameter from a precursor compartment. A cell-free reconstitution system was developed that generates vesicles from PC12 membrane precursors in the presence of ATP and brain cytosol and is temperature dependent. At 15 degrees C, surface-labeled synaptic vesicle proteins accumulate in a donor compartment, while labeled synaptic vesicles cannot be detected. The block of synaptic vesicle formation at 15 degrees C enables the use of the monoclonal antibody, KT3, a specific marker for the epitope-tagged synaptic vesicle protein, VAMP-TAg, to label precursors in the synaptic vesicle biogenesis pathway. From membranes labeled in vivo at 15 degrees C, vesicles generated in vitro at 37 degreesC had the sedimentation characteristics of neuroendocrine synaptic vesicles on glycerol velocity gradients, and excluded the transferrin receptor. Therefore, vesiculation and sorting can be studied in this cell-free system.     

Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a Fourier transform infrared spectroscopy study of two-dimensional membrane crystals

Aquaporins are integral membrane proteins found in diverse animal and plant tissues that mediate the permeability of plasma membranes to water molecules. Projection maps of two-dimensional crystals of aquaporin-1 (AQP1) reconstituted in lipid membranes suggested the presence of six to eight transmembrane helices in the protein. However, data from other sequence and spectroscopic analyses indicate that this protein may adopt a porin-like beta-barrel fold. In this paper, we use Fourier transform infrared spectroscopy to characterize the secondary structure of highly purified native and proteolyzed AQP1 reconstituted in membrane crystalline arrays and compare it to bacteriorhodopsin. For this analysis the fractional secondary structure contents have been determined by using several different algorithms. In addition, a neural network-based evaluation of the Fourier transform infrared spectra in terms of numbers of secondary structure segments and their interconnections [sij] has been performed. The following conclusions were reached: 1) AQP1 is a highly helical protein (42-48% alpha-helix) with little or no beta-sheet content. 2) The alpha-helices have a transmembrane orientation, but are more tilted (21 degrees or 27 degrees, depending on the considered refractive index) than the bacteriorhodopsin helices. 3) The helices in AQP1 undergo limited hydrogen/deuterium exchange and thus are not readily accessible to solvent. Our data support the AQP1 structural model derived from sequence prediction and epitope insertion experiments: AQP1 is a protein with at least six closely associated alpha-helices that span the lipid membrane.