The response of giant phospholipid vesicles to pore-forming peptide melittin

The interaction between the pore-forming peptide melittin (MLT) and giant phospholipid vesicles was explored experimentally. Micromanipulation and direct optical observation of a vesicle (loaded with sucrose solution and suspended in isomolar glucose solution) enabled the monitoring of a single vesicle response to MLT. Time dependences of the vesicle size, shape and the composition of the inner solution were examined at each applied concentration of MLT (in the range from 1 to 60 μg/ml). The response varied with MLT concentration from slight perturbation of the membrane to disintegration of the vesicle. A model for MLT-vesicle interaction is proposed that explains the observed phenomena in the entire span of MLT concentrations and is consistent with deduced underlying mechanisms of MLT action: trans-membrane positioning and dimerization of MLT, the lipid flow from the outer to the inner membrane leaflet induced by MLT translocation, formation of pores and the consequent transport of small molecules through the membrane. The results of the theoretical analysis stress the role of dimers in the MLT-membrane interaction and demonstrate that the MLT-induced membrane permeability for sugar molecules in this experimental set-up depends on both MLT concentration and time.     

The response of giant phospholipid vesicles to pore-forming peptide melittin

The interaction between the pore-forming peptide melittin (MLT) and giant phospholipid vesicles was explored experimentally. Micromanipulation and direct optical observation of a vesicle (loaded with sucrose solution and suspended in isomolar glucose solution) enabled the monitoring of a single vesicle response to MLT. Time dependences of the vesicle size, shape and the composition of the inner solution were examined at each applied concentration of MLT (in the range from 1 to 60 microg/ml). The response varied with MLT concentration from slight perturbation of the membrane to disintegration of the vesicle. A model for MLT-vesicle interaction is proposed that explains the observed phenomena in the entire span of MLT concentrations and is consistent with deduced underlying mechanisms of MLT action: trans-membrane positioning and dimerization of MLT, the lipid flow from the outer to the inner membrane leaflet induced by MLT translocation, formation of pores and the consequent transport of small molecules through the membrane. The results of the theoretical analysis stress the role of dimers in the MLT-membrane interaction and demonstrate that the MLT-induced membrane permeability for sugar molecules in this experimental set-up depends on both MLT concentration and time.     

Are gap junction membrane plaques implicated in intercellular vesicle transfer?

Gap junction channels are concentrated in specialised plaques of plasma membrane where cells are in close apposition. In this communication evidence is provided showing that these specialised regions of membrane also provide a site for vesicular transfer between cells. Vesicle distribution in eye lenses was found to generally reflect the reported distribution of gap junction membrane plaques. In certain areas of the lens gap junction membrane plaques and vesicles could be seen to form combined, complex structures. Ultrastructure of the vesicle and gap junction membrane plaque complexes was consistent with the vesicles moving through membrane plaques from one lens fibre cell to the next. To investigate whether transport of substances was consistent with intercellular vesicle transfer, transport of various markers was investigated. Time course experiments showing the rate of uptake of various markers into the lens did not show dramatic differences for molecules smaller or larger then gap junction pores formed by connexons. While considered as a primary intercellular transport mechanism in the lens, connexon pores were not the sole agent mediating the observed transport. Other reported mechanisms of intercellular transport in the lens can only account for the movement of relatively small molecules. Vesicular transport may therefore be a major form of transport into the outer lens layers for larger molecules. Implicit in these observations is a new hypothesis for intercellular vesicle movement via gap junction membrane plaques. Intercellular vesicle movement could possibly provide a path for large molecules associated with intact vesicles to be transported into the eye lens tissue.     

Oligomerization and pore formation by equinatoxin II inhibit endocytosis and lead to plasma membrane reorganization

Pore-forming toxins have evolved to induce membrane injury by formation of pores in the target cell that alter ion homeostasis and lead to cell death. Many pore-forming toxins use cholesterol, sphingolipids, or other raft components as receptors. However, the role of plasma membrane organization for toxin action is not well understood. In this study, we have investigated cellular dynamics during the attack of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina, by combining time lapse three-dimensional live cell imaging, fluorescence recovery after photobleaching, FRET, and fluorescence cross-correlation spectroscopy. Our results show that membrane binding by equinatoxin II is accompanied by extensive plasma membrane reorganization into microscopic domains that resemble coalesced lipid rafts. Pore formation by the toxin induces Ca(2+) entry into the cytosol, which is accompanied by hydrolysis of phosphatidylinositol 4,5-bisphosphate, plasma membrane blebbing, actin cytoskeleton reorganization, and inhibition of endocytosis. We propose that plasma membrane reorganization into stabilized raft domains is part of the killing strategy of equinatoxin II.     

Permeability properties of Escherichia coli outer membrane containing, pore-forming proteins: comparison between lambda receptor protein and porin for saccharide permeation.

Outer membrane permeability conferred by lambda receptor protein and porins to maltose-maltodextrins and other oligosaccharides was studied in vitro with reconstituted vesicle membranes and in vivo with mutant strains lacking either one of these proteins. The vesicle membranes reconstituted from phospholipids, lipopolysaccharide, and purified lambda receptor allowed rapid diffusion of maltose and maltose-maltodextrins of up to six glucose residues, but the membranes acted essentially as a molecular sieve for sucrose, raffinose, stachyose, and inulins of molecular weights 800, 920, and 1,380. The vesicle membranes containing porins allowed rapid diffusion of maltose but not of maltose-maltodextrins larger than maltose. The apparent transport Km values for maltose-maltodextrins of up to six glucose residues from the strain carrying lamB+ ompB (lambda receptor+, porin-) were similar (about 5 X 10(-6) M), whereas the transport Km values for maltose- and maltotriose of the strain carrying lamB ompB+ (lambda receptor-, porin+) alleles appeared to be 300 and about 20,000 X 10(-6) M. These results suggest that lambda receptor protein forms permeability pores that facilitate the diffusion of maltose-maltodextrins and function as a molecular sieve for other saccharides.     

Cytotoxicity of equinatoxin II from the sea anemoneActinia equina involves ion channel formation and an increase in intracellular calcium activity

Summary Equinatoxin Il is a 20-kDa basic protein isolated from the sea anemoneActinia equina. The aim of our work was to investigate the primary molecular basis for the cytotoxic effects of equinatoxin II in two model systems: single bovine lactotrophs and planar lipid bilayers. Previous work has shown that equinatoxin II produces rapid changes in cell morphology, which are dependent on external calcium. It has also been reported that addition of equinatoxin II increases membrane electrical conductance, which suggests that the cytotoxic action of equinatoxin II involves an increase in the permeability of membranes to Ca²⁺. Extensive changes in cytosolic Ca²⁺ activity are thought to invoke irreversible changes in cell physiology and morphology. In this paper, we show that morphological changes brought about by equinatoxin II in bovine lactotrophs are associated with a rapid rise in cytosolic Ca²⁺ activity, monitored with a fura-2 video imaging apparatus. Moreover, incorporation of equinatoxin II into planar lipid bilayers produces Ca²⁺ permeable ion channels. This suggests that the mode of equinatoxin II cytotoxicity involves the formation of cation (Ca²⁺) permeable channels in cell membranes.     

Cascades of transient pores in giant vesicles: line tension and transport

Under ordinary circumstances, the membrane tension of a giant unilamellar vesicle is essentially nil. Using visible light, we stretch the vesicles, increasing the membrane tension until the membrane responds by the sudden opening of a large pore (several micrometers in size). Only a single pore is observed at a time in a given vesicle. However, a cascade of transient pores appear, up to 30-40 in succession, in the same vesicle. These pores are transient: they reseal within a few seconds as the inner liquid leaks out. The membrane tension, which is the driving force for pore opening, is relaxed with the opening of a pore and the leakage of the inner liquid; the line tension of the pore's edge is then able to drive the closure of a pore. We use fluorescent membrane probes and real-time videomicroscopy to study the dynamics of the pores. These can be visualized only if the vesicles are prepared in a viscous solution to slow down the leakout of the internal liquid. From measurements of the closure velocity of the pores, we are able to infer the line tension,. We have studied the effect of the shape of inclusion molecules on. Cholesterol, which can be modeled as an inverted cone-shaped molecule, increases the line tension when incorporated into the bilayers. Conversely, addition of cone-shaped detergents reduces. The effect of some detergents can be dramatic, reducing by two orders of magnitude, and increasing pore lifetimes up to several minutes. We give some examples of transport through transient pores and present a rough measurement of the leakout velocity of the inner liquid through a pore. We discuss how our results can be extended to less viscous aqueous solutions which are more relevant for biological systems and biotechnological applications.     

Immunolocalization of glucose transporter GLUT1 in the rat placental barrier: possible role of GLUT1 and the gap junction in the transport of glucose across the placental barrier

GLUT1 is an isoform of facilitated-diffusion glucose transporters and has been shown to be abundant in cells of blood-tissue barriers. Using antibodies against GLUT1, we investigated the immunohistochemical localization of GLUT1 in the rat placenta. Rat placenta is of the hemotrichorial type. Three cell layers (from the maternal blood side inward) cytotrophoblast and syncytiotrophoblasts I and II, lie between the maternal and fetal bloodstreams. GLUT1 was abundant along the invaginating plasma membrane facing the cytotrophoblast and the syncytiotrophoblast I. Also, the infolded basal plasma membrane of the syncytiotrophoblast II was rich in GLUT1. Apposing plasma membranes of syncytiotrophoblasts I and II, however, had only a small amount of GLUT1. Numerous gap junctions were seen between syncytiotrophoblasts I and II. Taking into account the localization of GLUT1 and the gap junctions, we suggest a possible major transport route of glucose across the placental barrier, as follows: glucose in the maternal blood passes freely through pores of the cytotrophoblast. Glucose is then transported into the cytoplasm of the syncytiotrophoblast I via GLUT1. Glucose enters the syncytiotrophoblast II throught the gap junctions. Finally glucose leaves the syncytiotrophoblast II via GLUT1 and enters the fetal blood through pores of the endothelial cells.     

The static head method for determining the charge stoichiometry of coupled transport systems Applications to the sodium-coupled d-glucose transporters of the renal proximal tubule

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled d-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.     

The static head method for determining the charge stoichiometry of coupled transport systems. Applications to the sodium-coupled D-glucose transporters of the renal proximal tubule

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled D-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.     

Structural Features of N-Glycans of Seaweed Glycoproteins: Predominant Occurrence of High-Mannose Type N-Glycans in Marine Plants

An ice nucleation-active strain of Erwinia uredovora shed vesicles when cultured at low temperature (l0?C). We isolated an ice nucleation-active vesicle fraction from the culture medium by ultrafiltration, ultracentrifugation, and gel filtration. Western blot analysis showed that this cell-free vesicle fraction contained an ice nucleation-active protein (InaU). The process of the InaU transport to a shed vesicle was examined by immunohistochemical analysis using electron microscopy. The examination showed the following successive processes: InaU molecules first assemble around the inner membrane, then the assembly enters a vesicle justforming on the surface of the outer membrane, and finally the vesicle, 100–400 nm in diameter, leaves the surface to be shed with InaU molecules occluded.     

Solution structure of the eukaryotic pore-forming cytolysin equinatoxin II: implications for pore formation

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. The structure of the actinoporin equinatoxin II in aqueous solution, determined from NMR data, consists of two short helices packed against opposite faces of a beta-sandwich structure formed by two five-stranded beta-sheets. The protein core has extensive hydrophobic interfaces formed by residues projecting from the internal faces of the two beta-sheets. 15N relaxation data show uniform backbone dynamics, implying that equinatoxin II in solution is relatively rigid, except at the N terminus; its inferred rotational correlation time is consistent with values for monomeric proteins of similar mass. Backbone amide exchange rate data also support the view of a stable structure, even though equinatoxin II lacks disulfide bonds. As monitored by NMR, it unfolds at around 70 degrees C at pH 5.5. At 25 degrees C the structure is stable over the pH range 2.5-7.3 but below pH 2.5 it undergoes a slow transition to an incompletely unfolded structure resembling a molten globule. Equinatoxin II has two significant patches of positive electrostatic potential formed by surface-exposed Lys and Arg residues, which may assist its interaction with charged regions of the lipid head groups. Tyr and Trp residues on the surface may also contribute by interacting with the carbonyl groups of the acyl chains of target membranes. Data from mutational studies and truncated analogues identify two regions of the protein involved in membrane interactions, the N-terminal helix and the Trp-rich region. Once the protein is anchored, the N-terminal helix may penetrate the membrane, with up to four helices lining the pore, although other mechanisms of pore formation cannot be ruled out.     

Differential interaction of equinatoxin II with model membranes in response to lipid composition

Equinatoxin II is a 179-amino-acid pore-forming protein isolated from the venom of the sea anemone Actinia equina. Large unilamellar vesicles and lipid monolayers of different lipid compositions have been used to study its interaction with membranes. The critical pressure for insertion is the same in monolayers made of phosphatidylcholine or sphingomyelin (approximately 26 mN m(-1)) and explains why the permeabilization of large unilamellar vesicles by equinatoxin II with these lipid compositions is null or moderate. In phosphatidylcholine-sphingomyelin (1:1) monolayers, the critical pressure is higher (approximately 33 mN m(-1)), thus permitting the insertion of equinatoxin II in large unilamellar vesicles, a process that is accompanied by major conformational changes. In the presence of vesicles made of phosphatidylcholine, a fraction of the protein molecules remains associated with the membranes. This interaction is fully reversible, does not involve major conformational changes, and is governed by the high affinity for membrane interfaces of the protein region comprising amino acids 101-120. We conclude that although the presence of sphingomyelin within the membrane creates conditions for irreversible insertion and pore formation, this lipid is not essential for the initial partitioning event, and its role as a specific receptor for the toxin is not so clear-cut.     

Modulation of glucose uptake in animal cells. Studies using plasma membrane vesicles isolated from nontransformed and simian virus 40-transformed mouse fibroblast cultures.

Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation.     

Interplay between membrane dynamics, diffusion and swelling pressure governs individual vesicular exocytotic events during release of adrenaline by chromaffin cells

Release of adrenaline by chromaffin cells occurs through a process involving docking and then fusion of a secretory vesicle to the cytoplasmic membrane of the cell. Fusion proceeds in two main stages. The first one leads to the creation of a stable fusion pore passing through the two membranes and which gives a constant release flux of neurotransmitter (pore-release stage). After a few milliseconds, this initial stage which is not investigated here proceeds through a sudden enlargement of the initial pore (full-fusion stage) up to the complete incorporation of the vesicle membrane into that of the cell and total exposure of the initial matrix vesicle core to the extracellular fluid. The precise time-resolved dynamics of the release and of the vesicle membrane during the full-fusion phase can be extracted with a precision never achieved so far by de-convolution of experimental chronoamperometric currents monitored during individual exocytotic secretion events. The peculiar dynamics of the vesicle membrane proves that exocytotic events are powered by the swelling of the matrix polyelectrolyte core of the vesicle, although they are kinetically regulated by diffusion in the matrix and by the dynamics of the vesicle and cell membranes. Two simple theoretical models based on the dynamics of pores are developed to account for these dynamics and are shown to predict behaviors which are essentially identical to the experimental ones. This offers a new view of the kinetic grounds which control the full-fusion stage, and therefore provides a new interpretation of the sudden transition between the pore-release and the full-fusion stages. This transition occurs when the increasing membrane surface tension energy due to the refrained internal swelling pressure overcomes the edge energy of the pore, so that the initial fusion pore becomes unstable and is disrupted. This new view predicts that secretory vesicles which contain matrixes energetically similar to those of the adrenal cells investigated here can be separated into two classes according to their radius and catecholamine content. Small vesicles (less than ca. 25 nm radius, and containing less than ca. 20000 molecules) should always release through pores. Larger vesicles should always end into fusing except if another mechanism closes the pore before ca. 10000 molecules of catecholamines have been released.     

Membrane associated nonmuscle myosin II functions as a motor for actin-based vesicle transport in clam oocyte extracts

Nonmuscle myosin II (Myo2) has been shown to associate with membranes of the trans-Golgi network and to be involved in Golgi to ER retrograde protein transport. Here, we provide evidence that Myo2 not only associates with membranes but functions to transport vesicles on actin filaments (AFs). We used extracts from unactivated clam oocytes for these studies. AFs assembled spontaneously in these extracts and myosin-dependent vesicle transport was observed upon activation. In addition, actin bundles formed and moved relative to each other at an average speed of 0.30 microm/s. Motion analysis revealed that vesicles moved on the spontaneously assembled AFs at speeds greater than 1 microm/s. The motor on these vesicles was identified as a member of the nonmuscle Myo2 family based on sequence determination by Edman chemistry. Vesicles in these extracts were purified by sucrose gradient centrifugation and movement was reconstituted in vitro using skeletal muscle actin coated coverslips. When peripheral membrane proteins of vesicles including Myo2 were removed by salt stripping or when extracts were treated with an antibody specific to clam oocyte nonmuscle Myo2, vesicle movement was inhibited. Blebbistatin, a Myo2 specific inhibitor, also blocked vesicle movement. Myo2 light chain kinase activity was found to be essential for vesicle movement and sliding of actin bundles. Together, our data provide direct evidence that nonmuscle Myo2 is involved in actin-dependent vesicle transport in clam oocytes.     

The role of sterols in the lipid vesicle response induced by the pore-forming agent nystatin

The influences of ergosterol and cholesterol on the activity of the nystatin were investigated experimentally in a POPC model membrane as well as theoretically. The behavior of giant unilamellar vesicles (GUVs) under osmotic stress due to the formation of transmembrane pores was observed on single vesicles at different nystatin concentrations using phase-contrast microscopy. A significant shift of the typical vesicle behavior, i.e., morphological alterations, membrane bursts, slow vesicle ruptures and explosions, towards lower nystatin concentrations was detected in the ergosterol-containing vesicles and a slight shift towards higher nystatin concentrations was detected in the cholesterol-containing membranes. In addition, the nystatin activity was shown to be significantly affected by the ergosterol membrane’s molar fraction in a non-proportional manner. The observed tension-pore behavior was interpreted using a theoretical model based on the osmotic phenomena induced by the occurrence of size-selective nystatin pores. The number of nystatin pores for different vesicle behavior was theoretically determined and the role of the different mechanical characteristics of the membrane, i.e., the membrane's expansivity and bending moduli, the line tension and the lysis tension, in the tension-pore formation process was quantified. The sterol-induced changes could not be explained adequately on the basis of the different mechanical characteristics, and were therefore interpreted mainly by the direct influences of the membrane sterols on the membrane binding, the partition and the pore-formation process of nystatin.     

Fluid phase and receptor-mediated endocytosis in Paramecium primaurelia by fluorescence confocal laser scanning microscopy

In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.     

Insulin-stimulated glucose uptake involves the transition of glucose transporters to a caveolae-rich fraction within the plasma membrane: implications for type II diabetes.

BACKGROUND: Adipose and muscle tissues express an insulin-sensitive glucose transporter (GLUT4). This transporter has been shown to translocate from intracellular stores to the plasma membrane following insulin stimulation. The molecular mechanisms signalling this event and the details of the translocation pathway remain unknown. In type II diabetes, the cellular transport of glucose in response to insulin is impaired, partly explaining why blood-glucose levels in patients are not lowered by insulin as in normal individuals. MATERIALS AND METHODS: Isolated rat epididymal adipocytes were stimulated with insulin and subjected to subcellular fractionation and to measurement of glucose uptake. A caveolae-rich fraction was isolated from the plasma membranes after detergent solubilization and ultracentrifugal floatation in a sucrose gradient. Presence of GLUT4 and caveolin was determined by immunoblotting after SDS-PAGE. RESULTS: In freshly isolated adipocytes, insulin induced a rapid translocation of GLUT4 to the plasma membrane fraction, which was followed by a slower transition of the transporter into a detergent resistant caveolae-rich region of the plasma membrane. The insulin-stimulated appearance of transporters in the caveolae-rich fraction occurred in parallel with enhanced glucose uptake by cells. Treatment with isoproterenol plus adenosine deaminase rapidly inhibited insulin-stimulated glucose transport by 40%, and at the same time GLUT4 disappeared from the caveolae-rich fraction and from plasma membranes as a whole. CONCLUSIONS: Insulin stimulates glucose uptake in adipocytes by rapidly translocating GLUT4 from intracellular stores to the plasma membrane. This is followed by a slower transition of GLUT4 to the caveolae-rich regions of the plasma membrane, where glucose transport appears to take place. These results have implications for an understanding of the defect in glucose transport involved in type II diabetes.