Sedimentation rate of erythrocytes as an indicator for phase transitions in the membrane]

Temperature dependent sedimentation measurements with human erythrocytes showed that the sedimentation velocity at 21 degrees C in Krebs-Ringer-solution, pH 7.4, has a peak shaped minimum [1]. In further investigations it could be shown that a phase transition of membrane lipids is the main reason of this peak. Membrane proteins influence the sedimentation behavior of the erythrocytes only by changing the interaction with membrane lipids.     

Erythrocyte sedimentation rate. I. Volume fraction dependence in saline solution

We have measured volume fraction dependence of the sedimentation curve of swine erythrocytes in a physiological saline solution at 10 degrees C, 20 degrees C, 30 degrees C and 40 degrees C. The sedimentation curves were found to consist of initial constant velocity region and final plateau region at the lower temperatures of 10 degrees C and 20 degrees C, while modified S-shaped curves were observed at the higher temperatures of 30 degrees C and 40 degrees C. The volume fraction dependence of the initial slope v of the sedimentation curve was fitted well to the following exponential type equation at all the temperatures: v = vs,exp (1 - H)exp[-(BH + CH2)] where vs,exp is the velocity in infinite dilution corresponding to the Stokes velocity and H is the volume fraction of erythrocytes. The volume fraction dependence of the relative velocity v/vs,exp was in close agreement with a semi-empirical equation derived for slurrys in the field of chemical engineering at the lower temperatures, while a small deviation between the observed and calculated curves was found at the higher temperatures. The volume fraction dependence of v at 20 degrees C was also analyzed on a theory recently developed by Oka. The explicit functional form of the medium up-flow factor phi (H) and the deformability factor f in the theory were determined using the experimental data.     

Temperature dependence of the apparent affinity and the maximum velocity of the membrane-bound monosaccharide transport system in the yeast Rhodotorula gracilis

Analysis of the temperature dependence of the monosaccharide transport system in the yeast Rhodotorula gracilis (ATCC 26194, CBS 6681), as tested with D-xylose, revealed that the apparent affinity of the transport system, measured as the reciprocal of the half-saturation constant KT, increased when transport velocity was stimulated by temperature (15--30 degrees C) and decreased when the rate of uptake was reduced at temperatures aboce 30 degrees C. Breaks in Arrhenius plots were accompanied by corresponding breaks in van't Hoff plots. Whereas untreated cells exhibited in the van't Hoff plot a discontinuity at 28--30 degrees C this was not observed in heat-treated cells (at either 37 or 45 degrees C). In heat-treated cells the maximum transport velocity was always lower and the apparent affinity higher than in untreated cells at the same temperature; the optimum temperature for both transport velocity and apparent affinity was shifted to higher values. The data are interpreted in terms of a reversible phase transition of membrane lipids effecting an irreversible alteration of membrane structure. The temperature-induced reversible alkalinization of unbuffered yeast suspensions supports this interpretation.     

Binding of two spin-labelled derivatives of chlorpromazine to human erythrocytes.

The binding to human intact erythrocytes of two different spin-labelled derivatives of chlorpromazine has been studied. The influence of the positively charged side chain of the drug has been the focus of our attention. The positively charged amphiphilic compound (spin derivative I) is water-soluble up to 80 microM at pH values below 5.9. The apolar analogue (spin derivative II) aggregates in aqueous buffer from the lowest concentration tested. Both spin derivatives undergo a slow reduction inside the erythrocyte. The reduced nitroxides are readily reoxidized by adding a low, non-quenching, concentration of potassium ferricyanide to the intact erythrocytes. The fractions of spin label I and II bound to the erythrocyte membrane or to the erythrocyte-extracted lipids remain constant as a function of the temperature (3-42 degrees C) and as a function of the concentration of the spin label up to 150 microM. E.s.r. spectra of both spin labels show a two-component lineshape when they are bound to intact erythrocytes. Below 35 degrees C for the positively charged spin probe, and below 32 degrees C for the apolar spin probe, the simulation of the lineshape shows that more than 50% of the spectrum originates from a slow-motion component. This slow-motion component is also found in erythrocyte-extracted lipids probed by the positively charged spin label below 25 degrees C. In contrast, no slow-motion component is detected in the range 4-40 degrees C for the apolar spin label in erythrocyte-extracted lipids. In this environment the apolar probe experiences a single fast anisotropic motion with an exponential dependence on 1/temperature. Detailed lineshape simulations take into account the exchange frequency between binding sites where the probe experiences a fast motion and binding sites where it experiences a slow motion. The exchange frequency is strongly temperature-dependent. Characterization of the different motions experienced inside the different locations has been achieved and compared for whole erythrocytes and for the extracted lipids. The biochemical nature of the binding sites (membrane protein/acidic phospholipid) giving rise to the slow-motion component is discussed as a function of the polarity of the spin-labelled drug and as a function of the temperature controlling the fluidity of the lipid bulk and influencing the distribution of the drug inside the membrane.     

Evidence for a correlation between swimming velocity and membrane fluidity of Tetrahymena cells

The influence of the physical state of the membrane on the swimming behaviour of Tetrahymena pyriformis was studied in cells with lipid-modified membranes. When the growth temperature of Tetrahymena cells was increased from 15 degrees C to 34 degrees C or decreased from 39 degrees C to 15 degrees C, their swimming velocity changed gradually in a similar to the adaptive change in membrane lipid composition. Therefore, such adaptive changes in swimming velocity were not observed during short exposures to a different environment. Tetrahymena cells adapted to 34 degrees C swam at 570 microns/s. On incubation at 15 degrees C these cells swam at 100 microns/s. When the temperature was increased to 34 degrees C after a 90-min incubation at 15 degrees C, the initial velocity was immediately recovered. On replacement of tetrahymanol with ergosterol, the swimming velocity of 34 degrees C-grown cells decreased to 210 microns/s, and the cells ceased to move when the temperature was decreased to 15 degrees C. To investigate the influence of the physical state of the membrane on the swimming velocity, total phospholipids were prepared from Tetrahymena cells grown under these different conditions. The fluidities of liposomes of these phospholipid were measured using stearate spin probe. The membrane fluidity of the cells cooled to 15 degrees C increased gradually during incubation at 15 degrees C. On the other hand, the fluidity of the heated cell decreased during incubation at 34 degrees C. Replacement of tetrahymanol with ergosterol decreased the membrane fluidity markedly. Consequently, a good correlation was observed between swimming velocity and membrane fluidity; as the membrane fluidity increased, the swimming velocity increased linearly up to 600 microns/s. These results provide evidence for the regulation of the swimming behaviour by physical properties of the membrane.     

Lectin-mediated attachment and ingestion of yeast cells and erythrocytes by hamster fibroblasts

Yeast cell attachment to Concanavalin A (ConA)-coated fibroblasts depends on the degree of saturation of ConA-binding sites on the fibroblast. Under comparable conditions, fresh mouse erythrocytes fail to establish stable contacts with ConA-coated fibroblasts. The interaction of ConA-coated erythrocytes with fibroblasts and of non-coated erythrocytes with wheat germ agglutinin (WGA)-coated fibroblasts is remarkably less efficient than that of yeast cells interacting with ConA-coated fibroblasts. Ingestion of attached cells was not observed in any of the above lectin-mediated cell-cell interactions. Yeast cells coated with ConA show a high extent of attachment to fibroblasts (three-fold that of yeast cell attachment to ConA-coated fibroblasts). The attachment is highly temperature sensitive, being 3 times more at 37 °C than at 14 °C. A significant fraction of attached yeast cells (˜46%) is ingested by the fibroblasts during the 60 min incubation at 37 °C. The ingestion exhibits a strong temperature dependence, being nil at 14 °C and amounting to 150 and 600 ingested yeast cells per 100 fibroblasts at 24 °C and 37 °C, respectively. Transmission and scanning electron microscopy of ConA-mediated yeast cell-fibroblast interaction indicates a tighter interaction when the yeast cells are coated with ConA than when the fibroblasts are coated with ConA. Thus spreading of the plasma membrane around the attached yeast cell as well as transduction of attachment to ingestion could be triggered only under conditions of a very extensive multibridge interaction between the two apposing surfaces. Such an interaction is not achieved when the mobility of ConA-receptors within the fibroblast membrane plane is restricted as a result of crosslinking with ConA.     

Factors influencing survival and growth of mammalian cells exposed to hypothermia. I. Effects of temperature and membrane lipid perturbers

The Arrhenius plot of the rate of V79 Chinese hamster cell inactivation due to hypothermia has a "break" around 7-10 degrees C with optimum storage temperature for unprotected cells being about 10 degrees C. Addition of the membrane lipid perturber, butylated hydroxytoluene, improves survival of cells when compared to controls at temperatures below this break but not above. Arrhenius plots of growth rates of the cells show breaks at 30 and 40 degrees C. Measurements of membrane fluidity by electron spin resonance or membrane polarization anisotropy by fluorescence spectrophotometry techniques as a function of temperature in these cells also reveal "breaks" centered around 8 and 30 degrees C. Hence, the changes in the rate of cell inactivation and growth as a function of temperature may be related to membrane lipid phase changes.     

Direct measurement of the internal viscosity of sickle erythrocytes as a function of cell density

The rotational dynamics of TEMPAMINE can be used to study directly the intracellular environment. The extracellular signal from TEMPAMINE is broadened away by the use of potassium ferricyanide which does not enter the cell. The EPR signal which results when 1 mM TEMPAMINE, 120 mM ferricyanide, and erythrocytes are mixed together arises from TEMPAMINE only in the intracellular aqueous space. The relative viscosity measured by the motion of TEMPAMINE in various control environments is: water at 37 degrees C = 1; human plasma at 37 degrees C = 1.1; internal aqueous environment of washed erythrocytes or whole blood at 37 degrees C = 4.92 +/- 0.32. Erythrocytes can be fractionated by density. In sickle-cell anemia (SS), the percentage of cells we find with density greater than 1.128 g/ml is 15-40%, in normals (AA) and sickle trait (AS) 1%. By direct spin-label measurements with TEMPAMINE we show, for the first time, that the relative internal viscosity (eta mu) of these dense erythrocytes is markedly elevated and density-dependent. Our results show that (1) eta mu increases with increasing cell density; (2) eta mu obtained from sickle cells is higher than eta mu obtained from normal cells at a given density, and this effect is greater at 37 degrees C than at 20 degrees C; (3) eta mu is proportional to MCHC, but eta mu in erythrocytes is higher than eta mu obtained from in vitro preparations of hemoglobin S at equivalent concentrations. We conclude that the relative internal viscosity of erythrocytes is affected by three factors: the state of cell hydration, the amount of hemoglobin polymer present, and the potential interactions of the cell membrane with intracellular hemoglobin.     

Thermal transitions of red blood cell deformability. Correlation with membrane rheological properties

Red blood cell deformability has been studied by the initial filtration flow rate as a function of temperature. The well-known transition at 49-50 degrees C (probably due to spectrin denaturation) is shown. Another transition is demonstrated around 18 degrees C (the cell becomes stiffer below this temperature range). The erythrocyte membranes prepared by a mild dialysis technique have the same deformability as intact erythrocytes at room temperature; they also show the same low-temperature transition. No such transition has been found for hemoglobin solutions of viscosity 30 g X dl-1. It is interesting to compare these results with those obtained by other methods which measure the properties of natural or artificial lipid membranes and which also demonstrate a thermal transition at 15-20 degrees C. Therefore, the deformability of intact normal erythrocytes seems to depend mainly on the rheological properties of the membrane.     

Temperature-dependent changes in the swimming behaviour of Tetrahymena pyriformis-NT1 and their interrelationships with electrophysiology and the state of membrane lipids

The swimming velocity and the amplitude of the helical swimming path of T. pyriformis-NT1 cells grown at 20 degrees C (Tg 20 degrees C) and 38 degrees C (Tg 38 degrees C) were monitored between 0 and 40 degrees C in the presence and absence of electric fields. Within physiological limits the swimming velocity increased and the amplitude decreased as temperature was raised. The temperature profiles of these properties were not linear, and showed discontinuities at different temperatures for the different cultures. The break points in Arrhenius plots of the resting potential, regenerative spike magnitude, repolarization time, swimming velocity and swimming amplitude are tabulated and compared. The initial breakpoints upon cooling were clustered about the breakpoints in fluorescence polarization of D.P.H. in extracted phospholipids, and around the transition temperatures estimated from the literature for the pellicular membrane of these cells. The average of the initial breakpoints on cooling was 22.9 degrees C for Tg 38 degrees C cells and 13.7 degrees C for Tg 20 degrees C cells, a shift of 9.2 degrees C. Unlike Paramecium there is no depolarizing receptor potential in Tetrahymena upon warming. It is suggested that this may be the basis of a behavioural difference between Tetrahymena and Paramecium--namely that in Tetrahymena maximum swimming velocity occurs above growth temperature whereas in Paramecium the two points coincide. Swimming velocity and resting potential were correlated with membrane fluidity within physiological limits, but for other parameters the relationship with fluidity was more complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of nucleoside transport in human erythrocytes. Alterations during blood preservation

The transmembrane equilibration of radiolabeled uridine was measured by rapid kinetic techniques in human erythrocytes from freshly drawn blood and in the same cells during conventional storage of the blood as well as in cells from outdated blood. Our results confirm earlier reports that the maximum velocity of uridine equilibrium exchange (Vee) at 25 degrees C is about 30% lower in outdated than fresh red cells, whereas the opposite is the case for the Michaelis-Menten constant for equilibrium exchange (Kee), and that maximum zero-trans efflux (Vzt21) is about 4-times greater than maximum zero-trans influx (Vzt12) in outdated cells (directional asymmetry), whereas they are about the same in fresh red cells. At 25 degrees C, the nucleoside-loaded carrier of fresh cells moves on the average 6-times more rapidly than the empty carrier, whereas the differential mobility of loaded and empty carrier from outdated cells is about 15-fold. Our results also show that greater efflux than influx in outdated cells is not due to a general leakiness of outdated cells, that the differences in kinetic properties of the transporter developed during the first two weeks of blood storage and that the differences are greatly amplified when transport is measured at 5 degrees C rather than 25 degrees C. At 5 degrees C, the loaded carrier from outdated red cells moves about 325-times more rapidly than the empty carrier and maximum zero-trans efflux exceeds maximum zero-trans influx about 14-times, whereas the transport of fresh cells exhibits directional symmetry just as at 25 degrees C. The changes in kinetic properties of transport induced by temperature and storage are probably related to structural alterations in the plasma membrane and suggest that the operation of carrier is subject to modification by the membrane environment. Other results show that the kinetics of the sugar transport of human red cells is not affected in the same manner by blood storage as those of the nucleoside transporter.     

Temperature dependence of the viscoelastic recovery of red cell membrane.

The time-dependent recovery of an elongated red cell is studied as a function of temperature. Before release, the elongated cell is in static equilibrium where external forces are balanced by surface elastic force resultants. Upon release, the cell recovers its initial shape with a time-dependent exponential behavior characteristic of a viscoelastic solid material undergoing large ("finite") deformation. The recovery process is characterized by a time constant, tc, that decreases from approximately 0.27 s at 6 degrees C to 0.06 s at 37 degrees C. From this measurement of the time constant and an independent measurement of the shear modulus of surface elasticity for red cell membrane, the value for the membrane surface viscosity as a function of temperature can be calculated.     

On cell membrane lipid fluidity and plant lectin agglutinability. A spin label study of mouse ascites tumor cells

The fluidity of the plasma membrane of Sarcoma 180 mouse ascites tumor cells has been studied in viable cells using fatty acid spin labels. The order parameter was found to vary from 0.61, approximately four carbon bond lengths removed from the membrane surface, to 0.47 approximately eleven bond lengths removed at 22 degrees C and from 0.55 to 0.33 at 37 degrees C. Thus these cells show similar membrane fluidity to that found in other mammalian cells with the exception of human erythrocytes which are less fluid. The concanavalin A mediated agglutinability of Sarcoma 180 cells was altered by the addition of cytochalasin B and the fluidity was found to be the same as in unaltered cells.     

Effects of temperature on the yeast cell cycle analyzed by flow cytometry

The effects of temperature (in the range 15-36 degrees C) on growth and the nuclear and budding cycle have been studied in populations of the yeast Saccharomyces cerevisiae exponentially growing in batch on yeast nitrogen base (YNB) glucose medium. The maximal rate of exponential growth is achieved at 30 degrees C, and a transition point is apparent at about 20 degrees C. At all tested temperatures DNA replication begins when cells are still unbudded and both the budded period and the postreplicative period have the same temperature dependence. A temperature compensatory mechanism seems to operate in S phase, during which duration remains relatively constant, in the range 21-36 degrees C, while duration of G2+ M phases shows a much more pronounced temperature dependence. The results are discussed in terms of a cell-cycle model for budding yeast.     

Isolation and characterization of large (0.5 - 1.0 micron) cytoskeleton-free vesicles from human and rabbit erythrocytes

Large (0.5 - 1.0 micron) cytoskeleton-free vesicles were obtained, by 'budding', from fresh human and rabbit erythrocytes incubated at 45 degrees C and titrated with EDTA and CaCl2. This process occurs without hemolysis. The isolated vesicles maintain their cytoplasmic integrity and normal membrane orientation, and are resistant to hemolysis over the pH range 5.0 - 11.0 and temperature range 4-50 degrees C. The only membrane proteins detected in vesicles from human erythrocytes were band 3 region polypeptides and bands PAS-1, PAS-2 and PAS-3. Vesicles obtained from rabbit erythrocytes were similarly simple. Because of their size and stability these vesicles are amenable to both kinetic and quantitative analysis using the same experimental techniques employed in studies of synthetic lipid membranes. The results obtained in this study indicate that these vesicles are essentially markedly simplified biological cells, and thus may be useful as a biologically relevant model membrane system for examining the molecular interactions which occur within, across and between cell membranes.     

The titration of tetanus antitoxin I. Factors affecting the sensitivity of the indirect haemagglutination test

Various factors affecting the indirect HA test for the titration of tetanus antitoxin have been evaluated with a view to obtaining maximum sensitivity in tests using unfixed sheep erythrocytes and sheep erythrocytes fixed with glutaraldehyde, formaldehyde and pyruvic aldehyde. The optimal concentration of tannic acid has been found to be 1/40 000 for tanning both fixed and unfixed sheep erythrocytes. Tanned sheep erythrocytes sensitized with 50 Lf/ml of tetanus toxoid at pH 7.2 for one hour were the most sensitive. Although the optimal temperature of sensitization was found to be 56 degrees C, unfixed cells tended to clump and lyse at this temperature. Thus a temperature of 37 degrees C was used to sensitize unfixed sheep erythrocytes. Sheep erythrocytes from different animals and the final concentration of sensitized sheep erythrocytes both had great effects on sensitivity. A final concentration of 0.5% of sensitized sheep erythrocytes was found suitable as a compromise between sensitivity and readability. The loss of sensitivity of fixed and sensitized erythrocytes was investigated by storing these cells at 4-8 degrees C for six to nine months.     

Influence of malaria parasite (P. vivax) on erythrocyte aggregation: a study based on dynamic imaging and analysis

Malaria due to P. vivax (PV) is prevalent in many countries. The present work is aimed to determine the cell-cell interaction through formation of aggregates under dynamic conditions. Blood samples are obtained from patients (n=11) suffering from PV malaria, and the normal subjects (n=10) in test tubes containing citrate phosphate dextrose (10:1.4), as an anticoagulant. The signature analysis of infected erythrocytes shows significant alterations in their shape and membrane. For aggregation analysis, erythrocyte suspension in plasma at hematocrit 5%, was placed in a glass chamber and mounted vertically on the stage of the video-microscope system. The aggregate images thus acquired show erythrocytes adhering with each other to form mash-like structures. With increase in parasitaemia, the erythrocytes show hyper-aggregation compared to that of normal cells. By processing of the sequence of recorded images during sedimentation, the various aggregation parameters are obtained. These parameters show that the formed aggregates are compact which produce distinct changes in sedimentation pattern with significantly higher sedimentation velocity compared to that in healthy blood samples. These changes in malaria could partly be responsible for alteration in blood flow through microcirculatory system.     

Temperature dependence of the energy-linked monosaccharide transport across the cell membrane of Rhodotorula gracilis.

The temperature dependence of the active monosaccharide transport across the cell membrane of the yeast Rhodotorula gracilis has been studied between 0 and 55 degrees C with D-xylose as the transported substrate: (i) Between 0 and 10 degrees C there is virtually no transport. (ii) The initial velocity of transport increases exponentially from 15 to 30 degrees C (deltaE equal to 32 plus or minus 2 kcal/mol). (iii) At 30 degrees C a sharp "break" occurs in the Arrhenius plot and with increasing temperature the transport becomes inactivated, with a positive slope of the corresponding straight line ("deltaE equal to minus 15 kcal/mol"). (iv) In the temperature range of 50-55 degrees C, both the transport and the metabolic activity cease. In order to account for the abrupt changes of the membrane permeability, we attempted to ascribe them to phase transitions in the membrane structure: the first one, between 10 and 15 degrees C, to the crystalline: liquid-crystalline phase change; the second one, around 30 degrees C, to a change from highly ordered (low entropy) to less ordered (high entropy) membrane structure. Whereas the former phase transition is reversible, the latter appears to be irreversible. Arrhenius plots of the cell respiration exhibit a "break" at 30 degrees C, as well. However, at higher temperatures there is no thermal inactivation of the respiratory activity. The importance of a proper organization of the cell membrane constituents for the efficient transport function is discussed.     

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.     

Multicopy suppressors of phenotypes resulting from the absence of yeast VDAC encode a VDAC-like protein.

The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (delta por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37 degrees C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of delta por1 yeast grown on glycerol at 37 degrees C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in delta por1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30 degrees C, albeit more slowly than delta por1 single mutants. Like delta por1 single mutants, they could not grow on glycerol at 37 degrees C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, delta por1 cells, delta por1 delta por2 cells, and delta por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in delta por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37 degrees C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.