Single Protein Tracking of P450-Reductase in an Endoplasmic Reticulum Biomimetic Reveals a NADPH Dependent Interaction with the Membrane (original) (raw)
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Journal of the American Chemical Society, 2017
Cytochrome P450-reductase (CPR) is a versatile NADPH-dependent electron donor located in the cytoplasmic side of the endoplasmic reticulum. It is an electron transferase that is able to deliver electrons to a variety of membrane-bound oxidative partners, including the drug-metabolizing enzymes of the cytochrome P450s (P450). CPR is also stoichiometrically limited compared to its oxidative counterparts, and hypotheses have arisen about possible models that can overcome the stoichiometric imbalance, including quaternary organization of P450 and diffusion-limited models. Described here are results from a single-protein tracking study of fluorescently labeled CPR and cytochrome P450 2C9 (CYP2C9) molecules in which stochastic analysis was used to determine the dissociation constants of CPR/CYP2C9 complexes in a lipid bilayer membrane for the first time. Single-protein trajectories demonstrate the transient nature of these CPR-CYP2C9 interactions, and the measured Kd values are highly dep...
Metabolic activity induces membrane phase separation in endoplasmic reticulum
Metabolic activity induces membrane phase separation in endoplasmic reticulum, 2017
Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However , the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell. lipid metabolism | membrane phase | endoplasmic reticulum | Raman imaging | fatty acid L ipid bilayers enclose the boundary of life, and their diverse chemical compositions together with physical phases underlie membrane organizations and biological functions (1). Studies of in vitro model membranes have shown that, depending on chemical composition, mixture of lipids in a bilayer could either blend into a uniform phase or phase-separate into coexisting domains due to immiscibility. Three major phases have been observed in these studies: liquid disordered (L d), liquid ordered (L o), and solid ordered (S o), each representing a distinct type of bilayer organization (1, 2). However, biological membranes are fundamentally different from in vitro model systems. As a manifestation, liquid phase separation gives rise to static microdomains that can be readily observed in model membrane, yet the existence of raft domains (L o-like) in live cell has been a debate for decades due to much smaller domain size and faster dynamics (3). Many factors have been proposed to account for the unique aspects of biological membranes. One important factor is attributed to lipid–protein interaction, including the crowding effect of membrane protein as well as the intimate regulation by cytoskeleton (3, 4). Another less-considered but more fundamental factor concerns the thermodynamic state of the membrane. The living cell is an in-trinsically nonequilibrium system that constantly undergoes lipid turnover and membrane trafficking. This is especially relevant and important for the endoplasmic reticulum (ER), the largest membrane system as well as the lipid metabolic center in cell. Indeed, the ER is subject to the influx of lipid molecules synthesized from fatty acids, the efflux due to vesicle budding and secretion, and the exchange of lipids with other organelles via extensive and dynamic physical contacts (1). All of the above contribute to a fast turnover rate of ER lipids (half-life of ∼100 h or one cell division) (5, 6). With rich metabolic activity, ER membrane is easily perturbed from equilibrium by dynamic influx of nutrients. We hence hypothesize that the nonequilibrium factor, such as lipid synthesis in the ER, is capable of driving ER membrane from its poised L d phase to phase separation. Our hypothesis is motivated by recent studies and findings. Experimentally, fatty acids such as palmi-tate have been reported to promote saturated lipid synthesis and accumulation in the ER, which subsequently impair ER structural integrity (7–9). However, the ER phase behavior was not examined there. Theoretical study has also alluded to local phase separation when patches of membrane with different composition are delivered to a bilayer (10). This could be relevant to the condition of active lipid synthesis occurring in the ER. Toward testing this hypothesis, our study creates a non-equilibrium condition with the presence of lipid synthesis in ER membrane of living cells. To examine the spatial heterogeneity of membrane and interrogate its biophysical status, we employed the emerging vibrational imaging technique, that is, stimulated Raman scattering microscopy, and correlated it with lipidomics and fluorescence imaging. We found that ER membrane phase is susceptible to the modulation by metabolic activity. Remarkably, lipid synthesis derived from palmitate, a prevalent saturated fatty acid (SFA), even drives formation of solid-like domains, which Significance Membranes can adopt distinct phases. The endoplasmic reticulum (ER) is the largest membrane system inside cells and also harbors the richest metabolic activity including lipid synthesis. Unlike plasma membrane where separated " lipid raft " domains have been predicted and observed, ER membrane is thought to be uniformly fluidic. However, such understanding is based on bio-physical studies of model membrane under thermodynamic equilibrium. It remains unclear whether and how lipid synthesis activity perturbs the equilibrium and promotes phase segregation in ER membrane. Herein, we utilized coherent Raman imaging technique to track lipid synthesis and surprisingly revealed solid-like domains emerging from liquid ER membrane. Interestingly, this phenomenon can be tuned by the incoming nutrient source, demonstrating the susceptibility of ER membrane to nonequilibrium modulation.
Introducing a fluorescence-based standard to quantify protein partitioning into membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015
The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (K P). Although several methods to determine K P are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining K P , and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His 6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate K P. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive K P. Both modes were compared by determining the affinity of our standard, resulting in equivalent K P values. As observed in other systems, eGFP-His 6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His 6 standard. Hence, we determined the K P of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure K P and understand it in terms of molecules per lipid surface.
Biochemistry, 2005
The effect of nonlamellar-prone lipids, diacylglycerol (DG) and phosphatidylethanolamine (PE), on the stability of human cytochrome P450 1A2 (CYP1A2) was examined. When 100% phosphatidylcholine (PC) in standard vesicles was gradually replaced with either DG or PE, the stability of CYP1A2 increased; the incubation time-dependent destruction of spectrally detectable P450, decrease of catalytic activity, reduction of intrinsic fluorescence, and increased sensitivity to trypsin digestion were significantly alleviated. The ternary system of PC/PE/DG increased the stability of CYP1A2 more, even at lower concentrations of each nonlamellar-prone lipid, than that of the binary lipid mixture (PC/nonlamellar lipid). By incorporating the nonlamellar-prone lipids, the CYP1A2-induced increase of the surface pressure of the lipid monolayer was much higher compared to that for 100% PC. Increased surface pressure indicates a deep insertion of the protein into lipid monolayers. Nonlamellar lipids also increased the transition temperature of CYP1A2 in thermal unfolding and reduced the incubation time-dependent detachment of membrane-bound CYP1A2 from vesicles. Taken together, these results suggest that nonlamellar lipids per se and/or the phase properties of the membrane containing these lipids are important in the enhanced stability of CYP1A2 and the concomitant maintenance of catalytic activity of the protein.
Membrane protein dynamics: limited lipid control
Pmc Biophysics, 2009
Correlation of lipid disorder with membrane protein dynamics has been studied with infrared spectroscopy, by combining data characterizing lipid phase, protein structure and, via hydrogen-deuterium (H/D) exchange, protein dynamics. The key element was a new measuring scheme, by which the combined effects of time and temperature on the H/D exchange could be separated. Cyanobacterial and plant thylakoid membranes, mammalian mitochondria
Assessing the Role of Lipids in the Molecular Mechanism of Membrane Proteins
International Journal of Molecular Sciences, 2021
Membrane proteins have evolved to work optimally within the complex environment of the biological membrane. Consequently, interactions with surrounding lipids are part of their molecular mechanism. Yet, the identification of lipid–protein interactions and the assessment of their molecular role is an experimental challenge. Recently, biophysical approaches have emerged that are compatible with the study of membrane proteins in an environment closer to the biological membrane. These novel approaches revealed specific mechanisms of regulation of membrane protein function. Lipids have been shown to play a role in oligomerization, conformational transitions or allosteric coupling. In this review, we summarize the recent biophysical approaches, or combination thereof, that allow to decipher the role of lipid–protein interactions in the mechanism of membrane proteins.
Determinants of specificity at the protein–lipid interface in membranes
FEBS Letters, 2010
The complexity of pro-and eukaryotic lipidomes is increasingly appreciated mainly owing to the advance of mass spectrometric methods. Biophysical approaches have revealed that the large number of lipid classes and molecular species detected have implications for the self-organizing potential of biological membranes, resulting in the formation of lateral heterogeneous phases. How membrane proteins are able to adapt specifically to their surrounding heterogeneous matrix, and whether this environment affects protein targeting and function, is therefore a matter of particular interest. Here, we review specific protein-lipid interactions, focusing on the molecular mechanisms that determine specificity at the protein-lipid interface, and on membrane proteins that require lipids as cofactors for their architecture and function.
Archives of Biochemistry and Biophysics, 2009
Cytochrome P450 46A1 (CYP46A1) and NADPH-cytochrome P450 oxidoreductase (CPR) are the components of the brain microsomal mixed-function monooxygenase system that catalyzes the conversion of cholesterol to 24-hydroxycholesterol. Both CYP46A1 and CPR are monotopic membrane proteins that are anchored to the endoplasmic reticulum via the N-terminal transmembrane domain. The exact mode of peripheral association of CYP46A1 and CPR with the membrane is unknown. Therefore, we studied their membrane topology by using an approach in which solutionexposed portion of heterologously expressed membrane-bound CYP46A1 or CPR was removed by digestion with either trypsin or chymotrypsin followed by extraction of the residual peptides and their identification by mass spectrometry. The identified putative membrane-interacting peptides were mapped onto available crystal structures of CYP46A1 and CPR and the proteins were positioned in the membrane considering spatial location of the missed cleavage sites located within these peptide as well as the flanking residues whose cleavage produced these peptides. Experiments were then carried out to validate the inference from our studies that the substrate, cholesterol, enters CYP46A1 from the membrane. As for CPR, its putative membrane topology indicates that the Q153R and R316W missense mutations found in patients with disordered steroidogenesis are located within the membrane-associated regions. This information may provide insight in the deleterious nature of these mutations.
A new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress
Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work provided insights into the subcellular distribution of lipids, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remained insufficiently characterized. Here we describe a method for purifying organellar membranes from yeast, MemPrep. We demonstrate the purity of our ER preparations by quantitative proteomics and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings have important implications for understanding the role of lipids in membrane protein insertion, folding, and their sorting along the secretory pathway. Application of...