The molecular structures of the various intrinsic lipids in membranes regulate lipid-protein interactions. These different lipid structures with unique volumes produce different lipid molecular packing stresses/lateral stresses in lipid membranes. Most studies examining lipid packing effects have used phosphatidylcholine and phosphatidylethanolamine (PE), which are the main phospholipids of eukaryotic cell membranes. In contrast, Gram-negative or Gram-positive bacterial membranes are composed primarily of phosphatidylglycerol (PG) and PE, and the physical and thermodynamic properties of each acyl chain in PG at the molecular level remain unresolved. In this study, we used 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG, 16:0-18:1 PG) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (PAPG, 16:0-20:4 PG) to prepare lipid bilayers (liposome) with the rod-type fluorescence probe DPH. We measured the lipid packing conditions by determining the rotational freedom of DPH in POPG or PAPG bilayers. Furthermore, we investigated the effect of different monoacyl chains on a K+ channel (KcsA) structure when embedded in POPG or PAPG membranes. The results revealed that differences in the number of double bonds and carbon chain length in the monoacyl chain at sn-2 affected the physicochemical properties of the membrane and the structure and orientation of KcsA.

We investigated the effects of hydrophobic thickness on rates of G-protein binding in membranes consisting of 14:0,14:1 PC (hydrophobic thickness = 21.4 angstroms) and 18:0,18:1 PC (hydrophobic thickness = 29.2 angstroms). Purified bovine rhodopsin was reconstituted with and without transducin (Gt) in large unilamellar liposomes consisting of 14:0,14:1 PC and 18:0,18:1 PC at a lipid:protein ratio of 200. Kinetics of formation of the signaling competent state MII and MII-Gt complex formation where measured with flash photolysis, and bilayer properties were assessed via time-resolved fluorescence anisotropy decay measurements of diphenylhexatriene (DPH). All samples were examined from 20 to 40°C. The equilibrium concentration of MII was higher in 18:0, 18:1 PC by about a factor of 2 at all temperatures. MII formation kinetics was analyzed in terms of a model with 2 pathways to MII. In the absence of Gt the rate of MII formation was higher in 18:0,18:1 PC at all temperatures by a factor of 2 to 3. Analysis of the temperature dependence of the kinetics in terms of reaction rate theory showed this was chiefly due to increased activation enthalpy for two of the forward rates; Lumi to MI-380 and MI-480 to MII. All 6 of the microscopic rate constants for the conversion of lumirhodopsin to MII were accelerated in the thicker bilayer. At 30°C in 18:0,18:1 PC, MII formed with a time constant of 1.2 ms and the MII-Gt complex formed in 1.4 ms. Formation of MII-Gt rapidly after MII formation has also been observed for rhodopsin in the native membrane. In 14:0,14:1 PC at 30°C MII formed in 2.9 ms and MII-G complex formed in 19.2 ms. This long lag between appearance of MII and Gt binding demonstrates that hydrophobic mismatch slows the time course of G protein-coupled signaling.

The preferential association of cholesterol with saturated phospholipids, especially sphingomyelins, is responsible for the formation of coexisting fluid phases in lipid bilayers, and is believed to play a critical role in the existence of more ordered membrane domains in biological systems, sometimes termed lipid rafts. The coexistence of separate fluid phases in model bilayer constructs must be an equilibrium phenomenon, and titration of bilayer cholesterol content allows for thermodynamic characterization of cholesterol partitioning. To achieve a more complete understanding of the parameters governing phase separation, high sensitivity isothermal titration calorimetry was employed to investigate the effects of degree of unsaturation at the sn-2 position of a series of biologically relevant phospholipids. Cholesterol was either extracted from or added to bilayers of a single phospholipid component, allowing determination of a partition constant and enthalpy of transfer. By varying the relative cholesterol content of the bilayer, the non-ideal partitioning behavior was also investigated. It was found that the relative affinity of cholesterol for the bilayer decreased in a generally monotonic manner in response to increasing unsaturation, with the exception that both 20:4n6 and 22:6n3 acyl chains at the sn-2 position showed similar partition constants and enthalpies of transfer. The partitioning of cholesterol was also measured in membranes composed of mixtures of 18:0,22:6 PC and sphingomyelin. Values of both the partition constant and enthalpy of transfer extrapolated to pure sphingomyelin matched those previously reported for mixtures of 16:0,18:1 PC and sphingomyelin. This result suggests that the association of cholesterol and sphingomyelin is largely unaffected by other phospholipids in the bilayer.

Phospholipid membranes segregate into lateral domains of liquid ordered (lo) and liquid disordered (ld) phases when cholesterol and mixed species of lipids with saturated and unsaturated acyl chains are present. To examine membrane permeability and detergent solubility in pure lo, pure ld, and mixed lo/ld phases, LUVs were prepared based on the ternary phase diagram of POPC, sphingomyelin, and cholesterol. These LUVs were loaded with 2mM carboxyfluorescein (CF) and formed by extrusion at 70C. using a stopped-flow fluorometer, changes in CF fluorescence were measured when LUVs were exposed to sudden osmotic gradients, pH gradients, or 0.1% Triton. Acyl chain and phospholipid headgroup packing were assessed in all compositions with time-resolved measurements of DPH fluorescence lifetime and anisotropy decay. Water permeability was highest in the pure ld phase, and a factor of more than 100 lower in the pure lo phase. In the lo/ld coexistence region water permeability decreased approximately exponentially with increasing percent lo phase. Proton permeability was lowest in the pure ld phase, increasing linearly with increasing percent lo up to the “percolation point” (connected phase switches from lo to ld) at which point it remained approximately the same with increased lo. The rate of membrane solubilization was highest in the pure ld phase and did not decrease substantially until the percolation point was reached, and decreased by a factor of 40 in the pure lo phase. Water permeability was found to correlate approximately exponentially with acyl chain packing, decreasing with increased membrane order. Proton permeability increased linearly with increasing membrane order.

Ternary mixtures of POPC, sphingomyelin (SM) and cholesterol (Chol) form coexisting liquid ordered and liquid disordered fluid phases over a wide range of molar ratios. We examined the effects of polyunsaturated acyl chains in this ternary system by incrementally exchanging SDPC (18:0,22:6 PC) for POPC. All measurements were performed on extruded LUVs formed above the main phase transition of the SM. Changes in bilayer properties of the two phases were monitored with NBD linked to di-16:0 PE, which partitions into the liquid ordered phase, and NBD linked to di-18:1 PE which partitions into the liquid disordered phase. Fluorescence lifetime and anisotropy decay dynamics of both fluorescent probes were assessed via frequency-domain measurements collected from 5 to 300 MHz. In both phases the exchange of SDPC for POPC reduced the average fluorescence lifetime of the two probes from the value observed in 1/1/1 POPC/SM/chol. In both phases the lifetime was reduced to a value similar to that observed in a ternary mixture consisting of 8/1/1 POPC/SM/chol. In the liquid disordered phase the addition of SDPC had essentially no influence on probe dynamics, while in the liquid ordered phase SDPC caused a significant acceleration of probe rotational motion. This result was unexpected since it was assumed that the SDPC would be present only in the liquid disordered phase. FRET measurements using di-16:0-PE labeled with NBD and di-18:1 PE labeled with rhodamine were performed in order to monitor changes possible changes in domain size induced by the exchange of POPC for SDPC. Initial results show similar transfer efficiency between these two probes in 1/1/1 POPC/SM/chol and in 1/2/3/3 POPC/SDPC/SM/chol, suggesting the presence of SDPC does not greatly perturb domain size and structure.

Docosahexaenoic acid (DHA) disrupts the size and order of plasma membrane lipid microdomains in vitro and in vivo. However, it is unknown how the highly disordered structure of DHA mechanistically adapts to increase the order of tightly packed lipid microdomains. Therefore, we studied a novel DHA-Bodipy fluorescent probe to address this issue. We first determined if the DHA-Bodipy probe localized to the plasma membrane of primary B and immortal EL4 cells. Image analysis revealed that DHA-Bodipy localized into the plasma membrane of primary B cells more efficiently than EL4 cells. We then determined if the probe detected changes in plasma membrane order. Quantitative analysis of time-lapse movies established that DHA-Bodipy was sensitive to membrane molecular order. This allowed us to investigate how DHA-Bodipy physically adapted to ordered lipid microdomains. To accomplish this, we employed steady-state and time-resolved fluorescence anisotropy measurements in lipid vesicles of varying composition. Similar to cell culture studies, the probe was highly sensitive to membrane order in lipid vesicles. Moreover, these experiments revealed, relative to controls, that upon incorporation into highly ordered microdomains, DHA-Bodipy underwent an increase in its fluorescence lifetime and molecular order. In addition, the probe displayed a significant reduction in its rotational diffusion compared to controls. Altogether, DHA-Bodipy was highly sensitive to membrane order and revealed for the first time that DHA, despite its flexibility, could become ordered with less rotational motion inside ordered lipid microdomains. Mechanistically, this explains how DHA acyl chains can increase order upon formation of lipid microdomains in vivo.

Solubilization of lipid bilayers by the nonionic detergent OG has been well characterized in one and two component lipid systems, but has not been extensively studied in ternary membranes containing mixed fluid phases. In order to understand the effects of membrane order on solubilization by OG, LUVs of pure POPC were compared to those composed of POPC plus varying fractions of sphingomyelin (SM) and cholesterol. Detergent partitioning from the aqueous phase into the bilayer and membrane solubilization were monitored by ITC. The fluorescent membrane probe, DPH, was observed by time-resolved fluorescence intensity and polarization decay in the presence of increasing detergent concentrations to determine the effects on membrane order and dynamics. The results of partitioning experiments showed approximately a 50% decrease in the partition constant between pure POPC and the most ordered samples. ITC solubilization experiments showed clear boundaries for the micelle-bilayer mixed aggregate coexistence region in samples of low initial order, but the distinct thermodynamic signature associated with this coexistence region was not present in the cholesterol and SM rich samples. The average excited state lifetime of DPH, an indicator of water penetration, in vesicles rich in POPC showed a rapid increase at the onset of solubilization, whereas vesicles rich in SM and cholesterol showed a corresponding rapid decrease. Dynamic fluorescence depolarization data analyzed in terms of a Brownian rotational diffusion model revealed an increase in the occupancy of the bilayer midplane by DPH prior to solubilization in less ordered membranes coupled with a constant rotational correlation time. Membranes with higher initial order showed little increase in midplane occupancy and increasing rotational correlation time in the same detergent concentration range, suggesting that two modes of membrane disruption are possible and depend on the lipid makeup of the bilayer.

Detailed investigations of membrane protein folding present a number of serious technical challenges. Most studies addressing this subject have emphasized aspects of protein amino acid sequence and structure. While it is generally accepted that the interplay between proteins and lipids plays an important role in membrane protein folding, the role(s) played by membrane lipids in this process have only recently been explored in any detail. This review is intended to summarize recent studies in which particular lipids or membrane physical properties have been shown to play a role in the folding of intact, functionally competent integral membrane proteins. This article is part of a Special Issue entitled: Protein Folding in Membranes.

A range of evidence from animal, clinical and epidemiological studies indicates that highly polyunsaturated acyl chains play important roles in development, cognition, vision and other aspects of neurological function. In a number of these studies n3 polyunsaturated fatty acids (PUFAs) appear to be more efficacious than n6 PUFAs. In a previous study of retinal rod outer segments obtained from rats raised on either an n3 adequate or deficient diet, we demonstrated that the replacement of 22:6n3 by 22:5n6 in the n3 deficient rats led to functional deficits in each step in the visual signaling process (Niu et al., 2004). In this study, we examined rhodopsin and phosphodiesterase function and acyl chain packing properties in membranes consisting of phosphatidylcholines with sn-1=18:0, and sn-2=22:6n3, 22:5n6, or 22:5n3 in order to determine if differences in function are due to the loss of one double bond or due to differences in double bond location. At 37°C the n6 lipid shifted the equilibrium between the active metarhodopsin II (MII) state and inactive metarhodopsin I (MI) state towards MI. In addition, 22:5n6 reduced the rates of MII formation and MII-transducin complex formation by 2- and 6-fold, respectively. At a physiologically relevant level of rhodopsin light stimulation, the activity of phosphodiesterase was reduced by 50% in the 22:5n6 membrane, relative to either of the n3 membranes. Activity levels in the two n3 membranes were essentially identical. Ensemble acyl chain order was assessed with time-resolved fluorescence measurements of the membrane probe diphenylhexatriene (DPH). Analysis in terms of the orientational distribution of DPH showed that acyl chain packing in the two n3 membranes is quite similar, while in the 22:5n6 membrane there was considerably less packing disorder in the bilayer midplane. These results demonstrate that the n3 bond configuration uniquely optimizes the early steps in signaling via a mechanism which may involve acyl chain packing deep in the bilayer.

A recent study demonstrated that rhodopsin in 14:0,14:1 PC (hydrophobic thickness = 21.4 angstroms) has 8% less helical content than rhodopsin in 18:0,18:1 PC (hydrophobic thickness = 29.2 angstroms). We investigated the effects of hydrophobic thickness on rates of MII and transducin (Gt) binding and phospholipid dynamics and packing order. Purified rhodopsin was reconstituted in liposomes consisting of 14:0,14:1 PC and 18:0,18:1 PC at a lipid:protein ratio of 200. Kinetics of MII formation and Gt binding where measured with flash photolysis, and membrane properties were assessed via time-resolved fluorescence anisotropy decay measurements of diphenylhexatriene (DPH). MII formation was analyzed in terms of the square model. Analysis of the DPH anisotropy decay data in terms of the P2-P4 model showed that lipid dynamics and fractional free volume (fv) were higher in the 14:0,14:1 PC membrane. Previous studies demonstrate that an increase in these two bilayer properties is associated with enhanced MII formation, but in this case equilibrium concentration of MII and the rate of MII formation was higher in 18:0,18:1 PC at all temperatures. Analysis of the temperature dependence of the kinetics in terms of reaction rate theory showed this was chiefly due to increased activation enthalpy for two of the forward rates; Lumi to MI-380 and MI-480 to MII. At 30 oC in 18:0,18:1 PC, MII formed with a time constant of 0.69 ms and the MII-Gt complex formed in 0.79 ms. This near-immediate formation of MII-Gt following MII is similar to what is observed for rhodopsin in the native membrane. In 14:0,14:1 PC MII formed in 5.43 ms and MII-G complex formed in 37.6 ms. This long lag between appearance of MII and Gt binding demonstrates that hydrophobic mismatch has deleterious consequences for G protein-coupled signaling.

In the retina, rhodopsin is densely packed in a rod outer segment disk membrane rich in phospholipids with PC and PE headgroups. Increasing rhodopsin packing density in model PC membranes has been shown to alter metarhodopsin-II (MII) formation. This observation is deemed to be the consequence of rhodopsin association promoted by non-specific properties of the membrane. Here, we studied the effect of rhodopsin packing density on MII formation in membranes characterized by different intrinsic curvature and different interfacial hydrogen bonding propensity. Rhodopsin was reconstituted into a series of POPC bilayers doped with DOPC, di-and mono-methylated DOPE or DOPE at rhodopsin/lipid ratios ranging from 1:250 to 1:70. The level of rhodopsin activation and rate of MII formation were determined by steady-state and time-resolved UV/vis spectroscopy. In PC membranes, lower rhodopsin concentrations shift the MI/MII equilibrium towards MII and result in a faster rate of MII formation, in agreement with previous findings. On the contrary, in membranes rich in lipids with PE headgroups, the MII concentration is independent of rhodopsin packing density and rates of MII formation, while reduced, show only a slight dependence on rhodopsin crowding. In addition, at low or high protein density, the amount of MII formed depends to a larger extent on the ability of the annular PE headgroups to establish hydrogen bonds with the MII state than on changes in membrane curvature elasticity. These results show clearly that MII formation and interaction between rhodopsin molecules depend strongly on interactions between annular lipids and rhodopsin, highlighting the fundamental role of the first layer of lipids surrounding the protein.

We considered the issue of whether shifts in the metarhodopsin I (MI)-metarhodopsin II (MII) equilibrium from lipid composition are fully explicable by differences in bilayer curvature elastic stress. A series of six lipids with known spontaneous radii of monolayer curvature and bending elastic moduli were added at increasing concentrations to the matrix lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the MI-MII equilibrium measured by flash photolysis followed by recording UV-vis spectra. The average area-per-lipid molecule and the membrane hydrophobic thickness were derived from measurements of the 2H NMR order parameter profile of the palmitic acid chain in POPC. For the series of ethanolamines with different levels of headgroup methylation, shifts in the MI-MII equilibrium correlated with changes in membrane elastic properties as expressed by the product of spontaneous radius of monolayer curvature, bending elastic modulus, and lateral area per molecule. However, for the entire series of lipids, elastic energy explained the shifts only partially. Additional contributions correlated with the capability of the ethanolamine headgroups to engage in hydrogen bonding with the protein, independent of the state of ethanolamine methylation, with introduction of polyunsaturated sn-2 hydrocarbon chains, and with replacement of the palmitic acid sn-1 chains by oleic acid. The experiments point to the importance of interactions of rhodopsin with particular lipid species in the first layer of lipids surrounding the protein as well as to membrane elastic stress in the lipid-protein domain.

Membrane composition strongly modulates the ability of photo-activated rhodopsin to achieve the G-protein binding competent metarhodopsin II conformation (MII). In particular, MII concentration increases linearly with the bilayer concentration of non-bilayer forming PE lipids. This observation has prompted the membrane curvature hypothesis, which states that the continuum elastic properties of the lipid matrix play a dominant role on MII formation. Here, we aimed to separate the effect of membrane curvature elasticity from specific interactions between rhodopsin and PE headgroups. In a series of rhodopsin-containing proteoliposomes of different intrinsic curvature the level of rhodopsin activation was determined by steady-state and time-resolved UV/vis spectroscopy, membrane order and dynamics parameters were probed by 2H NMR and 13C MAS nuclear relaxation, and the structural response of rhodopsin to changes in membrane composition was followed by circular dichroism (CD). MII formation was increased by 18:0-22:6 PC and 18:0-22:6 PE, agents promoting negative curvature and decreased by lysophosphatidylcholine which promotes positive curvature, in agreement with the membrane curvature hypothesis. However, MII formation was also augmented by curvature-neutral lysophosphatidylethanolamine. In parallel, significant changes in helical content were observed by CD. Our results suggest that the structure and function of rhodopsin are modulated not only by membrane curvature elasticity, but also by specific interactions between rhodopsin and PE headgroups. The role of headgroup hydration, cation-pi interactions or salt bridge formation between rhodopsin and the annular lipids will be discussed on the basis of NMR experiments.

In a normal, healthy retinal rod outer segment 40% to 50% of the phospholipid acyl chains consist of docosahexaenoic acid (DHA, 22:6n-3). Diets that are deficient in n-3, or ω-3, fatty acids lead to the replacement of 22:6n-3 with 22:5n6. Dietary n-3 deficiency leads to a spectrum of developmental disorders associated with learning, memory, intelligence, and visual function. We examined rhodopsin, transducin (Gt) and phosphodiesterase (PDE) function and acyl chain packing in large unilamellar proteoliposomes consisting of phosphatidylcholines with sn-1 = 18:0, and sn-2 = 22:6n-3, 22:5n-6 or 22:5n-3. Rhodopsin activation and binding to Gt was assayed with steady-state and time-resolved UV/vis spectroscopy, acyl chain packing was assessed via time-resolved fluorescence of diphenylhexatriene (DPH) and PDE activity was determined from the change in pH due to hydrolysis of cyclic GMP. The motion of DPH in the membrane was slower in 22:5n-6 than in 22:6n-3 and overall acyl chain packing was more constrained. The most significant structural difference between the 22:5n-6 containing bilayer and bilayers containing both n-3 polyunsaturates was in the bilayer mid plane where 22:5n-6 produced much higher DPH orientational order. At physiological temperature the formation of both the active metarhodopsin II conformation (MII) and the MII-Gt complex was much slower in 18:0,22:5n-6 PC than in 18:0,22:6n-3 PC and the equilibrium amount of MII formed was 50% higher in 18:0,22:6n-3 PC. In 18:0,22:5n-6 PC PDE activity at a physiologically relevant level of rhodopsin activation is only about 60% of that observed in either 18:0,22:6n-3 PC or 18:0,22:5n-3 PC. Taken together, these results demonstrate that the subtle change in bond configuration from 22:6n-3 to 22:5n-6 produces more structured acyl chain packing in the bilayer midplane, leading to delayed and reduced MII-Gt interaction and PDE function.

We examined functional and structural roles for the bacteriorhodopsin (bR) carboxyl-terminus. The extramembranous and intracellular carboxyl-terminus was deleted by insertion of premature translation stop codons. Deletion of the carboxyl-terminus had no effect on purple membrane (PM) lattice dimensions, sheet size, or the electrogenic environment of the ground-state chromophore. Removal of the distal half of the carboxyl-terminus had no effect on light-activated proton pumping, however, truncation of the entire carboxyl-terminus accelerated the rates of M-state decay and proton uptake approximately 3.7-fold and severely distorted the kinetics of proton uptake. Differential scanning calorimetry (DSC) and SDS denaturation demonstrated that removal of the carboxyl-terminus decreased protein stability. The DSC melting temperature was lowered by 6 degrees C and the calorimetric enthalpy reduced by 50% following removal of the carboxyl-terminus. Over the time range of milliseconds to hours at least 3 phases were required to describe the SDS denaturation kinetics for each bR construction. The fastest phases were indistinguishable for all bR's, and reflected PM solubilization. At pH 7.4, 20 degrees C, and in 0.3% SDS (w/v) the half-times of bR denaturation were 19.2 min for the wild-type, 12.0 min for the half-truncation and 3.6 min for the full-truncation. Taken together the results of this study suggest that the bR ground state exhibits two "domains" of stability: (1) a core chromophore binding pocket domain that is insensitive to carboxyl-terminal interactions and (2) the surrounding helical bundle whose contributions to protein stability and proton pumping are influenced by long-range interactions with the extramembranous carboxyl-terminus.

A variety of techniques are currently in use for preparing protein-containing lipid vesicles known as proteoliposomes. However, the functionality of membrane protein in proteoliposomes prepared by various techniques has rarely been evaluated directly. We prepared rhodopsin-containing proteoliposomes consisting of asolectin or native retinal rod outer segment disk lipids using n-octyl beta-d-glucopyranoside and the detergent dialysis (DD) and rapid dilution (RD) techniques and measured the activity of rhodopsin using equilibrium UV/vis and flash photolysis spectroscopy. A significant difference in rhodopsin activity was observed in proteoliposomes prepared by these techniques. The equilibrium constant of metarhodopsin I-metarhodopsin II is 30-45% higher, and the apparent rate constant of MII formation is up to 3-fold faster in proteoliposomes prepared by RD vs DD. The DD technique produced larger yet more heterogeneous vesicles, while the RD technique yielded smaller and more homogeneous vesicles, as determined by electron microscopy and isopicnic centrifugation. Both proteoliposomes and empty lipid vesicles lacking rhodopsin were formed in the DD preparation, while only proteoliposomes were formed in the RD preparation. Under identical conditions, proteoliposomes prepared by RD have a higher L/P ratio, which is consistent with the higher level of rhodopsin activity in RD proteoliposomes. Overall, the results presented here suggest that the RD technique has an advantage over the DD technique in terms of preserving optimal rhodopsin activity and controlling the lipid to protein ratio in the final proteoliposomes.

Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30mol % cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature (Tm) of rhodopsin was scan rate-dependent, demonstrating the presence of a rate-limited component of denaturation. The activation energy of this kinetically controlled process (Ea) was determined from DSC thermograms by four separate methods. Both Tm and Ea varied with bilayer composition. Cholesterol increased the Tm both the presence and absence of docosahexaenoic acid acyl chains (DHA). In contrast, cholesterol lowered Ea in the absence of DHA, but raised Ea in the presence of 20mol % DHA-containing phospholipid. The relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. The Tm for thermal unfolding was inversely related to acyl chain packing order. Rhodopsin kinetic stability (Ea) was reduced in highly ordered or disordered membranes. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing, such as DHA and cholesterol, may be required for maximum protein stability.

Mitochondrial dysfunction plays a central role in the selective vulnerability of dopaminergic neurons in Parkinson's disease (PD) and is influenced by both environmental and genetic factors. Expression of the PD protein α-synuclein or its familial mutants often sensitizes neurons to oxidative stress and to damage by mitochondrial toxins. This effect is thought to be indirect, since little evidence physically linking α-synuclein to mitochondria has been reported. Here, we show that the distribution of α-synuclein within neuronal and non-neuronal cells is dependent on intracellular pH. Cytosolic acidification induces translocation of α-synuclein from the cytosol onto the surface of mitochondria. Translocation occurs rapidly under artificially-induced low pH conditions and as a result of pH changes during oxidative or metabolic stress. Binding is likely facilitated by low pH-induced exposure of the mitochondria-specific lipid cardiolipin. These results imply a direct role for α-synuclein in mitochondrial physiology, especially under pathological conditions, and in principle, link α-synuclein to other PD genes in regulating mitochondrial homeostasis.