Mechanical and chemical injury to thylakoid membranes during freezing in vitro

The relative contributions of membrane rupture due to osmotic stress and of chemical membrane damage due to the accumulation of cryotoxic solutes to cryoinjury was investigated using thylakoid membranes as a model system. When thylakoid suspensions were subjected to a freeze-thaw cycle in the presence of different molar ratios of NaCl as the cryotoxic solute and sucrose as the cryoprotective solute, membrane survival first increased linearly with the osmolality of the solutions used to suspend the membranes, regardless of the molar ratio of salt to sucrose. It subsequently decreased when the ratio of sucrose to salt was not sufficiently high for complete cryopreservation by sucrose. There was an optimum of cryopreservation at intermediate osmolalities (approx. 0.1 osmol/kg). This optimum of cryopreservation at a given sucrose concentration could be shifted to lower solute concentration, if mixtures of NaCl and NaBr were used instead of NaCl alone. At suboptimal initial osmolalities, damage is attributed mainly to membrane rupture. Under these conditions, cryopreservation is not influenced by the chaotropicity of the suspending medium. At supraoptimal initial solute concentrations, solute (i.e., chemical) effects determine membrane survival. Under these conditions, increased ratios of sugar to salt increased cryoprotection. In mixtures of NaCl and NaBr at constant molar ratios of salt to sucrose, chemical membrane damage was quantitatively related to the lyotropic properties of the ions used. The degree of chemical damage becomes more pronounced with rising osmolalities of the suspending media. With NaF as the cryotoxic solute, damage was more severe than should be expected from its lyotropic properties. This may reflect a specific interaction of fluoride with the membranes. Protein release from the membranes during freezing in the presence of different anions was qualitatively comparable at identical ratios of sugar to salt. However, the total amount of protein released was correlated linearly with membrane inactivation, even when different anions acted on the membranes. Gel electrophoretic analysis of proteins released from thylakoid membranes during freezing revealed discrete bands indicative of mechanical and chemical damage, respectively.     

Cryoprotection by glucose, sucrose, and raffinose to chloroplast thylakoids

Differential cryoprotection is afforded to chloroplast thylakoids against freeze-induced uncoupling of cyclic photophosphorylation by equimolar concentrations of glucose, sucrose, and raffinose. This differential protective effect appears to be due to nonideal activity-concentration profiles exhibited by the sugars during freezing. When cryoprotection is analyzed as a function of the mole fraction of NaCl to which the membranes are exposed during freezing, the pattern of protection to cyclic photophosphorylation and its component reactions is not dependent upon the chemical identity of the protective solute. Cryoprotective efficiency of glucose, sucrose, and raffinose can be accounted for by proposing an activity dependent alteration in the freezing environment rather than specific solute-membrane interactions.     

Cryoprotective leaf proteins.

Leaves of frost-resistant plants contain a number of soluble proteins which are capable of protecting isolated biomembranes against inactivation during freezing. Such proteins have not been found in non-hardy summer material. The pattern of protective proteins was not uniform in hardy material of different origin and appeared to change with the season. Cryoprotective proteins were isolated by preparative gel electrophoresis. Molecular weights of different proteins as determined by their electrophoretic mobility in sodium dodecyl sulfate gels were between 10000 and 20000. Circular dichroism measurements failed to indicate helical structures. The amino acid composition of 2 active proteins revealed a high content of polar amino acids. The proteins were heat-stable. They were, on a molar basis, more than 1000 times as effective in protecting thylakoid membranes against freezing damage as low-molecular-weight cryoprotectants such as sucrose, glycerol or dimethylsulfoxide. Very low concentrations of the proteins increased cryoprotection provided by sucrose. Of a number of oligopeptides of known composition, only a few were cryoprotective. Their activity was very small as compared with that of the active proteins. The concentration of the cryoprotective proteins in hardy leaves appeared to be high enough for a significant contribution of the proteins to the frost tolerance of resistant plants.     

Flow cytometry of spinach chloroplasts : determination of intactness and lectin-binding properties of the envelope and the thylakoid membranes

Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly “intact” and “disrupted” chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right-side-out vesicles of the thylakoid membranes.     

High concentrations of the compatible solute glycinebetaine destabilize model membranes under stress conditions

Compatible solutes are accumulated by diverse organisms in response to environmental stresses such as drought, salt, or cold. Glycinebetaine (Bet) is such a solute that is accumulated by many plants and microorganisms to high concentrations under stress conditions. It is an osmoprotectant in bacteria and stabilizes both soluble and peripherally membrane-bound proteins in vitro. Here, the effects of Bet on the stability of model lipid membranes are compared to the effects of two other compatible solutes, sucrose and trehalose. Both in the presence of 1M NaCl and during freezing to -20 degrees C, Bet is highly destabilizing to liposomes containing nonbilayer lipids, while the disaccharides are either protective or, in some cases, much less destabilizing. The destabilizing effect of Bet is more pronounced in membranes containing the nonbilayer galactolipid monogalactosyldiacylglycerol from plant chloroplasts than in membranes containing the nonbilayer phospholipid phosphatidylethanolamine. The most dramatic differences between the sugars and Bet were observed in liposomes made from a combination of lipids resembling plant chloroplast thylakoid membranes. Measurements with the dye merocyanine 540 indicate that the water-membrane interface was affected in opposite directions by the presence of high concentrations of sucrose or Bet. The dynamics of the lipids, however, were not differentially affected by the solutes, making direct solute-lipid interactions an unlikely explanation for the different effects on stability. The data offer an explanation, why Bet at high concentrations achieved during exogenous feeding of leaf tissues can be detrimental to cellular stability and survival under stress, while bacterial membranes that contain phosphatidylethanolamine instead of monogalactosyldiacylglycerol, or cyanobacteria that contain highly saturated monogalactosyldiacylglycerol are less susceptible.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Evidence that Envelope and Thylakoid Membranes from Pea Chloroplasts Lack Glycoproteins

Envelope and thylakoid membranes from pea (Pisum sativum var. Laxton's Progress No. 9) chloroplasts were analyzed for the presence of glycoproteins using two different approaches. First, the sugar composition of delipidated membrane polypeptides was measured directly using gas chromatographic analysis. The virtual absence of sugars suggests that plastid membranes lack glycoproteins. Second, membrane polypeptides separated by sodium dodecyl sulfate gel electrophoresis were tested for reactivity toward three different lectins: Concanavalin A, Ricinus communis agglutinin, and wheat germ agglutinin. In each case, there was no reactivity between any of the lectins and the plastid polypeptides. Microsomal membranes from pea tissues were used as a positive control. Glycoproteins were readily detectable in microsomal membranes using either of the two techniques. From these results it was concluded that pea chloroplast membranes do not contain glycosylated polypeptides.     

NaCl-induced phosphorylation of light harvesting chlorophyll a/b proteins in thylakoid membranes from the halotolerant green alga, Dunaliella salina

Light could induce phosphorylation of light harvesting chlorophyll a/b binding proteins (LHCII) in Dunaliella salina and spinach thylakoid membranes. We found that neither phosphorylation was affected by glycerol, whereas treatment with NaCl significantly enhanced light-induced LHCII phosphorylation in D. salina thylakoid membranes and inhibited that in spinach. Furthermore, even in the absence of light, NaCl and several other salts induced LHCII phosphorylation in D. salina thylakoid membranes, but not in spinach thylakoid membranes. In addition, hypertonic shock induced LHCII phosphorylation in intact D. salina under dark conditions and cells adapted to different NaCl concentrations exhibited similar LHCII phosphorylation levels. Taken together, these results show for the first time that while LHCII phosphorylation of D. salina thylakoid membranes resembles that of spinach thylakoid membranes in terms of light-mediated control, the two differ with respect to NaCl sensitivity under light and dark conditions.     

Cryoprotectin protects thylakoids during a freeze-thaw cycle by a mechanism involving stable membrane binding

Chloroplast thylakoid membranes of higher plants are damaged by freezing both in vivo and in vitro. The resulting inactivation of photosynthetic electron transport has been related to transient membrane rupture, leading to the loss of soluble electron transport proteins and osmotically active solutes from the thylakoid lumen. We have recently purified and sequenced a protein from cold acclimated cabbage, that protects thylakoids from this freeze-thaw damage. The protein belongs to the WAX9 family of nonspecific lipid transfer proteins, but has no detectable lipid transfer activity. Conversely, other transport-active lipid transfer proteins show no cryoprotective activity. We show here that cryoprotectin binds to thylakoid membranes. Both cryoprotective activity and membrane binding were inhibited in the presence of specific sugars, most effectively by Glc-6-S. The binding of cryoprotectin to thylakoids reduced the fluidity of the membrane lipids close to the membrane/solution interface, but not in the hydrophobic core region. Using immobilized liposomes we could show that cryoprotectin was able to bind to pure lipid membranes.     

Colligative and non-colligative freezing damage to thylakoid membranes

When spinach thylakoid membranes were frozen in vitro in solutions containing constant molar ratios of cryotoxic to cryoprotective solute, maintenance of functional integrity strongly depended on initial osmolarities. Optimum cryopreservation of cyclic photophosphorylation was observed when the membranes were suspended in solutions of intermediate osmolarities (approx. 50–100 mM NaCl, 75–150 mM sucrose). Both higher and lower initial osmolarities were found to result in decreased cryopreservation. In the absence of added salt, more than 100 mM sucrose were needed for full cryopreservation of the membranes. When thylakoids were frozen in solutions containing low concentrations of NaCl (2 mM), the ratio of sucrose to salt necessary to give full protection was high (up to 50). When the salt concentration was about 60 mM, ratios as low as 1.5 were sufficient for maintaining membrane integrity. This ratio increased again, as the initial NaCl concentration was increased beyond 60 mM. During freezing, proteins dissociated from the membranes, and the amount of the released proteins was correlated linearly with inactivation of photophosphorylation. The gel electrophoretic pattern of proteins released at low initial osmolarities differed from that of proteins released at high initial osmolarities. Cryopreservation was also found to depend on membrane concentration. Concentrated membrane suspensions suffered less inactivation than dilute suspensions. The protective effect of high membrane concentrations was particularly pronounced at high initial solute concentrations. It is proposed that damage at low initial osmolarities is caused predominantly by mechanical stress and by osmotic contraction/expansion. Damage at high initial osmolarities is thought to be caused mainly by solute effects. Under these conditions, both the final volume of the unfrozen solution in coexistence with ice and the membrane concentration affect membrane survival by influencing the extent of the loss of membrane components through dissociation reactions. Membrane protection by sugars is caused by colligative action under these circumstances.     

Antifreeze proteins differentially affect model membranes during freezing

Over the past decade antifreeze proteins from polar fish have been shown either to stabilize or disrupt membrane structure during low temperature and freezing stress. However, there has been no systematic study on how membrane composition affects the interaction of antifreeze proteins with membranes under stress conditions. Therefore, it is not possible at present to predict which antifreeze proteins will protect, and which will damage a particular membrane during chilling or freezing. Here, we analyze the effects of freezing on spinach thylakoid membranes and on model membranes of varying lipid composition in the presence of antifreeze protein type I (AFP I) and specific fractions of antifreeze glycoproteins (AFGP). We find that the addition of galactolipids to phospholipid model membranes changes the effect each protein has on the membrane during freezing. However, the greatest differences observed in this study are between the different types of antifreeze proteins. We find that AFP type I and the largest molecular weight fractions of AFGP induce concentration dependent leakage from, and are fusogenic to the liposomes. This is the first report that an antifreeze protein induces membrane fusion. In contrast, the smallest fraction of AFGP offers a limited degree of protection during freezing and does not induce membrane fusion at concentrations up to 10 mg/ml.     

Import of proteins into chloroplasts. Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates

Many of the thylakoid membrane proteins of plant and algal chloroplasts are synthesized in the cytosol as soluble, higher molecular weight precursors. These precursors are post-translationally imported into chloroplasts, incorporated into the thylakoids, and proteolytically processed to mature size. In the present study, the process by which precursors are incorporated into thylakoids was reconstituted in chloroplast lysates using the precursor to the light-harvesting chlorophyll a/b protein (preLHCP) as a model. PreLHCP inserted into thylakoid membranes, but not envelope membranes, if ATP was present in the reaction mixture. Correct integration into the bilayer was verified by previously documented criteria. Integration could also be reconstituted with purified thylakoid membranes if reaction mixtures were supplemented with a soluble extract of chloroplasts. Several other thylakoid precursor proteins in addition to preLHCP, but no stromal precursor proteins, were incorporated into thylakoids under the described assay conditions. These results suggest that the observed in vitro activity represents in vivo events during the biogenesis of thylakoid proteins.     

Proteomic analysis of the oxygen-evolving complex of photosystem II under biotec stress: Studies on Nicotiana benthamiana infected with tobamoviruses

We have previously shown that tobamovirus infection induces an inhibition of photosystem II electron transport, disturbing the oxygen-evolving complex (OEC). In the infected plants, the OEC polypeptide pattern was modified when compared to healthy plants, the levels of the PsbP and PsbQ extrinsic proteins being lowered to different extents. In this work we have further investigated by two-dimensional polyacrylamide gel electrophoresis (2-DE) the changes on the OEC protein pattern of thylakoid membranes isolated from Nicotiana benthamiana Domin plants infected with the Spanish strain of pepper mild mottle virus. When the thylakoid membranes from healthy plants were analyzed for the presence of PsbO and PsbP proteins by 2-DE (pI range 4-7) and further immunoassayed by using specific-antisera against these two proteins, it was observed that four polypeptides cross-reacted with each antiserum. These data, along with the N-terminal amino acid sequence determined for the eight polypeptides, indicate that the N. benthamiana PsbO and PsbP proteins correspond to protein families. In the silver-stained 2-DE gels of thylakoid membranes isolated at different days postinoculation from virus-infected plants, it was observed that the content of PsbP polypeptides decreased dramatically with respect to those of PsbO, during the progress of the infection. Interestingly, there was a differential decrease of the different PsbP proteins, indicative of a distinct regulation of their expression.     

ADP-ribosylation of thylakoid membrane polypeptides by cholera toxin is correlated with inhibition of thylakoid GTPase activity and protein phosphorylation

Incubation of pea thylakoid membranes with [32P]-NAD+ in the presence of cholera toxin resulted in the [32P]-ADP-ribosylation of a 60 kDa thylakoid membrane polypeptide. When ATP was included in the incubation mixture, a 29 kDa polypeptide was also labelled. In the absence of electron transfer cofactors or inhibitors, the extent of labelling depended on whether the membranes were preincubated in the light or dark and also on the developmental stage of the leaves used for thylakoid isolation. Irrespective of the latter, the strongest labelling was observed when DCMU was present in the light. After pretreatment of the thylakoid membranes with cholera toxin plus NAD+ under the same conditions, light-stimulated GTPase activity and protein phosphorylation were inhibited. The extent of inhibition for both processes appeared to be correlated with the amount of [32P]-ADP-ribosylation found when [32P]-NAD+ was included in the pretreatment mixture. The data presented are fully consistent with the 60 and 29 kDa polypeptides functioning as thylakoid membrane associated guanine nucleotide binding regulatory proteins.     

Glycoproteins of the Chromaffin Granule Membrane: Separation by Two-Dimensional Electrophoresis and Identification by Lectin Binding

The proteins of highly purified chromaffin-granule membranes were separated by one- or two-dimensional electrophoresis, then transferred to nitrocellulose sheets; glycosylation was investigated by binding of several different radioiodinated lectins. Over 20 different glycosylated components were identified; comparison with mitochondrial and microsomal fractions suggested that most of the major glycoproteins are genuine components of the chromaffin granule membrane, rather than contaminants originating in other organelles. Two-dimensional electrophoresis revealed heterogeneity within several of the glycoproteins, and this is ascribed to differences in the state of glycosylation, on the basis of shifts in electrophoretic mobility produced by treatment with neuraminidase. Neuraminidase treatment of chromaffin granule membranes also enhances the binding of many lectins. The identities of the lectin-binding bands are discussed: neither cytochrome b561 nor the F1-like ATPase appears to be glycosylated. Chromogranin A, although a glycoprotein, does not bind any of the lectins tested, but a number of concanavalin-A binding proteins, as well as dopamine beta-hydroxylase, are present in the chromaffin granule lysate.     

Localization of NADPH-protochlorophyllide reductase in plastids of barley at different greening stages

The localization of protochorophyllide (Pchlide) and of NADPH-protochlorophyllide oxidoreductase (POR, EC 1.6.99.1) within (etio)chloroplasts has been investigated at selected stages of greening of barley seedlings. Pchlide pigment and POR protein contents were evaluated in different plastid membrane fractions by fluorescence spectroscopy and immunoblot analysis using a monospecific polyclonal antibody raised against the purified enzyme. Fluorescence analysis showed the presence of Pchlide in both the envelope and thylakoid membranes. During greening, the Pchlide content, expressed on a total protein basis, decreased in thylakoid membranes, whereas it increased in the envelope membranes. POR proteins were detected mainly in thylakoid membranes at early greening stages. In contrast, the weak amount of POR proteins was associated more specifically with envelope membranes of mature chloroplasts. Whatever the greening stage, thylakoid-bound Pchlide and POR proteins were more abundant in the thylakoid regions which remained unsolubilized after mild Triton treatment used as standard procedure to prepare PS II particles. This suggests the preferential association of Pchlide and POR to the appressed regions of thylakoids. This revised version was published online in June 2006 with corrections to the Cover Date.     

A mutant small heat shock protein with increased thylakoid association provides an elevated resistance against UV-B damage in synechocystis 6803

Besides acting as molecular chaperones, the amphitropic small heat shock proteins (sHsps) are suggested to play an additional role in membrane quality control. We investigated sHsp membrane function in the model cyanobacterium Synechocystis sp. PPC 6803 using mutants of the single sHsp from this organism, Hsp17. We examined mutants in the N-terminal arm, L9P and Q16R, for altered interaction with thylakoid and lipid membranes and examined the effects of these mutations on thylakoid functions. These mutants are unusual in that they retain their oligomeric state and chaperone activity in vitro but fail to confer thermotolerance in vivo. We found that both mutant proteins had dramatically altered membrane/lipid interaction properties. Whereas L9P showed strongly reduced binding to thylakoid and model membranes, Q16R was almost exclusively membrane-associated, properties that may be the cause of reduced heat tolerance of cells carrying these mutations. Among the lipid classes tested, Q16R displayed the highest interaction with negatively charged SQDG. In Q16R cells a specific alteration of the thylakoid-embedded Photosystem II (PSII) complex was observed. Namely, the binding of plastoquinone and quinone analogue acceptors to the Q(B) site was modified. In addition, the presence of Q16R dramatically reduced UV-B damage of PSII activity because of enhanced PSII repair. We suggest these effects occur at least partly because of increased interaction of Q16R with SQDG in the PSII complex. Our findings further support the model that membrane association is a functional property of sHsps and suggest sHsps as a possible biotechnological tool to enhance UV protection of photosynthetic organisms.     

Formation of cross-linking between photosystem 2 proteins during irradiation of thylakoid membranes at high temperature

Irradiation of thylakoid membranes at 40 °C resulted in complete inhibition of photosystem (PS) 2 activity measured as 2,6-dichlorophenol indophenol (DCIP) photoreduction either in the absence or presence of 1,5-diphenylcarbazide (DPC). Concomitant with the inactivation of PS2 activity, several thylakoid proteins were lost and high molecular mass cross-linking products appeared that cross-reacted with antibodies against proteins of PS2 but not with antibodies against proteins of other three complexes PS1, ATP synthase, and cytochrome b₆f. Irradiation of thylakoid membranes suspended in buffer of basic pH or high concentration of Tris at 25 °C resulted in the formation of cross-linking products similar to those in thylakoid membranes irradiated at 40 °C. Presence of radical scavengers and DPC during the high temperature treatment prevented the formation of cross-linking products. These results suggest the involvement of oxygen evolving co mplex (OEC) in the formation of cross-linking between PS2 proteins in thylakoid membrane irradiated at high temperature. This revised version was published online in September 2006 with corrections to the Cover Date.     

Lipid composition determines the effects of arbutin on the stability of membranes.

Arbutin (hydroquinone-beta-D-glucopyranoside) is an abundant solute in the leaves of many freezing- or desiccation-tolerant plants. Its physiological role in plants, however, is not known. Here we show that arbutin protects isolated spinach (Spinacia oleracea L.) thylakoid membranes from freeze-thaw damage. During freezing of liposomes, the presence of only 20 mM arbutin led to complete leakage of a soluble marker from egg PC (EPC) liposomes. When the nonbilayer-forming chloroplast lipid monogalactosyldiacylglycerol (MGDG) was included in the membranes, this leakage was prevented. Inclusion of more than 15% MGDG into the membranes led to a strong destabilization of liposomes during freezing. Under these conditions arbutin became a cryoprotectant, as only 5 mM arbutin reduced leakage from 75% to 20%. The nonbilayer lipid egg phosphatidylethanolamine (EPE) had an effect similar to that of MGDG, but was much less effective, even at concentrations up to 80% in EPC membranes. Arbutin-induced leakage during freezing was accompanied by massive bilayer fusion in EPC and EPC/EPE membranes. Twenty percent MGDG in EPC bilayers completely inhibited the fusogenic effect of arbutin. The membrane surface probes merocyanine 540 and 2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosph ocholi ne (NBD-C(6)-HPC) revealed that arbutin reduced the ability of both probes to partition into the membranes. Steady-state anisotropy measurements with probes that localize at different positions in the membranes showed that headgroup mobility was increased in the presence of arbutin, whereas the mobility of the fatty acyl chains close to the glycerol backbone was reduced. This reduction, however, was not seen in membranes containing 20% MGDG. The effect of arbutin on lipid order was limited to the interfacial region of the membranes and was not evident in the hydrophobic core region. From these data we were able to derive a physical model of the perturbing or nonperturbing interactions of arbutin with lipid bilayers.