Absence Of Detergent Research Articles

New insights on β-glycan synthases using in vitro GT-array (i-GT-ray) platform

Cellulose and hemicellulose are the major structural β-glycan polysaccharides in cell walls of land plants. They are characterized by a backbone of β-(1,3)- and/or β-(1,4)-linked sugars such as glucose, mannose, or xylose. The backbones of these polymers are produced by processive glycosyltransferases (GTs) called synthases having multiple transmembrane domains anchoring them to the membrane. Thus, they are among the most difficult membrane proteins to test in vitro and to purify. Recently, we developed an in vitro GT-array (i-GTray) platform and showed that non-processive type II membrane GTs could be produced via cell-free system in a soluble and active form and tested in this platform. To determine whether i-GT-ray platform is adequate for the production and testing of β-glycan synthases, we tested five synthases involved in cellulose, xyloglucan, (gluco)mannan, and β-(1,3)(1,4)-mixed-linkage glucan synthesis. Our results revealed unsuspected features of these enzymes. For example, all these synthases could be produced in a soluble and active form and are active in the absence of detergent or membrane lipids, and none of them required a primer for initiation of synthesis. All synthases produced ethanol-insoluble products that were susceptible to the appropriate hydrolases (i.e., cellulase, lichenase, mannanase). Using this platform, we showed that AtCslC4 and AtXXT1 interact directly to form an active xyloglucan synthase that produced xylosylated cello-oligosaccharides (up to three xylosyl residues) when supplied with UDP-Glc and UDP-Xyl. i-GTray platform represents a simple and powerful functional genomics tool for discovery of new insights of synthase activities and can be adapted to other enzymes.

Critical Role of Molecular Packing in Lo Phase Membrane Solubilization.

Membrane solubilization induced by Triton X-100 (TX-100) was investigated. Different membrane compositions and phase states were studied along the detergent titration. Expected solubilization profiles were obtained but new information is provided. The fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids indicates that the liquid-ordered (Lo)/liquid-disordered (Ld) phase coexistence is barely unaffected at sub-solubilizing detergent concentrations and highlights the vesicle-to-micelle transition. Moreover, the location of the NBD group in the bilayer emphasizes a detergent-membrane interaction in the case of the insoluble Lo phase membrane. It has also been shown that the molecular packing of the membrane loosens in the presence of TX-100, regardless of the solubilization profile. Motivated by studies on GPMVs, the solubilization of less ordered Lo phase membranes was considered in order to improve the effect of molecular packing on the extent of solubilization. Membranes composed of SM and Chol in an equimolar ratio doped with different amounts of PC were studied. The more ordered the Lo phase membrane is in the absence of detergent, the less likely it is to be solubilized. Furthermore, and in contrast to what is observed for membranes exhibiting an Lo/Ld phase coexistence, a very small decrease in the molecular packing of the Lo phase membrane radically modifies the extent of solubilization. These results have implications for the reliability of TX-100 insolubility as a method to detect ordered domains.

The Introduction of Detergents in Thermal Proteome Profiling Requires Lowering the Applied Temperatures for Efficient Target Protein Identification.

Although the use of detergents in thermal proteome profiling (TPP) has become a common practice to identify membrane protein targets in complex biological samples, surprisingly, there is no proteome-wide investigation into the impacts of detergent introduction on the target identification performance of TPP. In this study, we assessed the target identification performance of TPP in the presence of a commonly used non-ionic detergent or a zwitterionic detergent using a pan-kinase inhibitor staurosporine, our results showed that the addition of either of these detergents significantly impaired the identification performance of TPP at the optimal temperature for soluble target protein identification. Further investigation showed that detergents destabilized the proteome and increased protein precipitation. By lowering the applied temperature point, the target identification performance of TPP with detergents is significantly improved and is comparable to that in the absence of detergents. Our findings provide valuable insight into how to select the appropriate temperature range when detergents are used in TPP. In addition, our results also suggest that the combination of detergent and heat may serve as a novel precipitation-inducing force that can be applied for target protein identification.

The cytoplasmic N-terminal tail of Zika virus NS4A protein forms oligomers in the absence of detergent or lipids

The non-structural (NS) NS4A protein in flaviviruses has three predicted transmembrane domains, is critical for virulence and participates in membrane morphogenesis. In Dengue virus (DENV), both hydrophylic N-terminal tail and its first transmembrane domain participate in the formation of oligomers which are important for pathogenicity. However, the relative importance of the N-terminal domain in oligomerization has been under debate. In particular, since in the absence of detergent or lipids, this domain (residues 1–48) in both DENV and Zika virus (ZIKV) NS4A, was found to be disordered. Recently, however, we reported preliminary data that showed that peptide ZIKV NS4A 4–58 adopts a defined secondary structure in aqueous solution and forms oligomers, signaling its importance for full length NS4A oligomerization. Herein we have performed detailed analytical ultracentrifugation experiments to further characterize the oligomerization of this peptide and also a shorter variant (residues 4–44). In both cases, sedimentation velocity produced a single species with concentration-dependent sedimentation coefficient, consistent with a fast equilibrium between at least two species. Combining sedimentation velocity and equilibrium experiments, data is best fitted to a monomer–dimer–trimer equilibrium. Possible models of NS4A oligomers obtained with AlphaFold-2 predict the stabilizing role for residues in this N-terminal domain, such as Arg20, Asn27, Ala44 and Glu50, all at highly conserved positions in flavivirus NS4A proteins. Our results are thus consistent with N-terminal domain interactions acting as one of the driving forces for NS4A homo-oligomerization.

Detergent modulates the conformational equilibrium of SARS-CoV-2 Spike during cryo-EM structural determination

The Spike glycoprotein of SARS-CoV-2 mediates viral entry into the host cell via the interaction between its receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (ACE2). Spike RBD has been reported to adopt two primary conformations, a closed conformation in which the binding site is shielded and unable to interact with ACE2, and an open conformation that is capable of binding ACE2. Many structural studies have probed the conformational space of the homotrimeric Spike from SARS-CoV-2. However, how sample buffer conditions used during structural determination influence the Spike conformation is currently unclear. Here, we systematically explored the impact of commonly used detergents on the conformational space of Spike. We show that in the presence of detergent, the Spike glycoprotein predominantly occupies a closed conformational state during cryo-EM structural determination. However, in the absence of detergent, such conformational compaction was neither observed by cryo-EM, nor by single-molecule FRET designed to visualize the movement of RBD in solution in real-time. Our results highlight the highly sensitive nature of the Spike conformational space to buffer composition during cryo-EM structural determination, and emphasize the importance of orthogonal biophysical approaches to validate the structural models obtained.

A method for selective 19 F-labeling absent of probe sequestration (SLAPS).

Fluorine (19F) offers several distinct advantages for biomolecular nuclear magnetic resonance spectroscopy such as no background signal, 100% natural abundance, high sensitivity, and a large chemical shift range. Exogenous cysteine‐reactive 19F‐probes have proven especially indispensable for characterizing large, challenging systems that are less amenable to other isotopic labeling strategies such as G protein‐coupled receptors. As fluorine linewidths are inherently broad, limiting reactions with offsite cysteines is critical for spectral simplification and accurate deconvolution of component peaks—especially when analyzing systems with intermediate to slow timescale conformational exchange. Here, we uncovered noncovalent probe sequestration by detergent proteomicelles as a second source of offsite labeling when using the popular 19F‐probe BTFMA (2‐bromo‐N‐(4‐[trifluoromethyl]phenyl)acetamide). The chemical shift and relaxation rates of these unreacted 19F‐BTFMA molecules are insufficient to distinguish them from protein‐conjugates, but they can be easily identified using mass spectrometry. We present a simple four‐step protocol for Selective Labeling Absent of Probe Sequestration (SLAPS): physically disrupt cell membranes in the absence of detergent, incubate membranes with cysteine‐reactive 19F‐BTFMA, remove excess unreacted 19F‐BTFMA molecules via ultracentrifugation, and finally solubilize in the detergent of choice. Our approach builds upon the in‐membrane chemical modification method with the addition of one crucial step: removal of unreacted 19F‐probes by ultracentrifugation prior to detergent solubilization. SLAPS is broadly applicable to other lipophilic cysteine‐reactive probes and membrane protein classes solubilized in detergent micelles or lipid mimetics.

Molecular Basis for Membrane Binding and Lipolysis Activation by ABHD5

Triglycerides are the main reservoir for long‐term energy storage in humans and are stored intracellularly in lipid droplets. Abnormal triglyceride metabolism is a key factor leading to obesity, diabetes, fatty liver disease and cardiovascular disease. When energy is needed, triglycerides are broken down in a metabolic pathway called lipolysis. Intracellular lipolysis is initiated by adipose triglyceride lipase (ATGL), which hydrolyzes triacylglycerol (TAG) to make diacylglycerol and a free fatty acid. Alpha‐beta hydrolase domain‐containing 5 (ABHD5) is a highly conserved regulator of ATGL‐mediated lipolysis that increases ATGL activity and is necessary to maintain lipid homeostasis. Consistently, mutations in ABHD5 cause Chanarin‐Dorfman syndrome, which leads to excessive accumulation of triglycerides. Despite over two decades of studies using biochemical approaches to mouse models, the structure of ABHD5 and molecular mechanisms of regulation remained uncharacterized. To understand the mechanism, we purified full‐length human ABHD5, visualized its self‐assembled, multiple oligomeric state in the absence of detergents by cryo‐EM, and generated preliminary crystals of detergent‐solubilized, monomeric ABHD5. We found ABHD5 is phosphorylated in vitro by PKA and recruited to membranes by specific lipids. We also purified the full‐length human ATGL. To further investigate the structural and regulation mechanisms of ABHD5 and ATGL, we will utilize hydrogen deuterium exchange mass spectrometry to map ATGL and ABHD5 protein‐protein and protein‐membrane interactions, biochemical experiments to study their co‐activation mechanism and membrane interaction. This study will provide a deeper understanding for the clinically relevant mutations, membrane recruitment and lipolysis activation of these key players in lipid metabolism.

Impact of nanodisc lipid composition on cell-free expression of proton-coupled folate transporter.

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

Trimerization of the N-Terminal Tail of Zika Virus NS4A Protein: A Potential In Vitro Antiviral Screening Assay.

The nonstructural (NS) protein NS4A in flaviviruses is a membrane protein that is critical for virulence, and, among other roles, it participates in membrane morphogenesis. In dengue virus (DENV), the NS4A hydrophilic N–terminal tail, together with the first transmembrane domain, is involved in both homo-oligomerization and hetero–oligomerization with NS4B. In both DENV and Zika virus (ZIKV), this N-terminal tail (residues 1–48) forms a random coil in solution but becomes mostly α-helical upon interaction with detergents or lipid membranes. Herein, we show that a peptide from ZIKV NS4A that spans residues 4–58, which includes most of the N–terminal tail and a third of its first transmembrane domain, forms homotrimers in the absence of detergents or liposomes. After interaction with the latter, α–helical content increases, consistent with binding. The oligomeric size of NS4A is not known, as it has only been reported in SDS gels. Therefore, we propose that full-length NS4A forms homotrimers mediated by this region, and that disruption of the oligomerization of peptide ZIKV NS4A 4–58 in solution can potentially constitute the basis for an in vitro assay to discover antivirals.

Characterization of Homogeneous and Heterogeneous Amyloid-β42 Oligomer Preparations with Biochemical Methods and Infrared Spectroscopy Reveals a Correlation between Infrared Spectrum and Oligomer Size.

Soluble oligomers of the amyloid-β(1-42) (Aβ42) peptide, widely considered to be among the relevant neurotoxic species involved in Alzheimer’s disease, were characterized with a combination of biochemical and biophysical methods. Homogeneous and stable Aβ42 oligomers were prepared by treating monomeric solutions of the peptide with detergents. The prepared oligomeric solutions were analyzed with blue native and sodium dodecyl sulfate polyacrylamide gel electrophoresis, as well as with infrared (IR) spectroscopy. The IR spectra indicated a well-defined β-sheet structure of the prepared oligomers. We also found a relationship between the size/molecular weight of the Aβ42 oligomers and their IR spectra: The position of the main amide I′ band of the peptide backbone correlated with oligomer size, with larger oligomers being associated with lower wavenumbers. This relationship explained the time-dependent band shift observed in time-resolved IR studies of Aβ42 aggregation in the absence of detergents, during which the oligomer size increased. In addition, the bandwidth of the main IR band in the amide I′ region was found to become narrower with time in our time-resolved aggregation experiments, indicating a more homogeneous absorption of the β-sheets of the oligomers after several hours of aggregation. This is predominantly due to the consumption of smaller oligomers in the aggregation process.

Extraction and reconstitution of membrane proteins into lipid nanodiscs encased by zwitterionic styrene-maleic amide copolymers

Membrane proteins can be reconstituted in polymer-encased nanodiscs for studies under near-physiological conditions and in the absence of detergents, but traditional styrene-maleic acid copolymers used for this purpose suffer severely from buffer incompatibilities. We have recently introduced zwitterionic styrene-maleic amide copolymers (zSMAs) to overcome this limitation. Here, we compared the extraction and reconstitution of membrane proteins into lipid nanodiscs by a series of zSMAs with different styrene:maleic amide molar ratios, chain sizes, and molecular weight distributions. These copolymers solubilize, stabilize, and support membrane proteins in nanodiscs with different efficiencies depending on both the structure of the copolymers and the membrane proteins.

Distinct host-immune response toward species related intracellular mycobacterial killing: A transcriptomic study

ABSTRACT The comparison of the host immune response when challenged with pathogenic and nonpathogenic species of mycobacteria can provide answers to the unresolved question of how pathogens subvert or inhibit an effective response. We infected human monocyte derived macrophages (hMDMs) with different species of mycobacteria, in increasing order of pathogenicity, i.e. M. smegmatis, M. bovis BCG, and M. tuberculosis R179 that had been cultured in the absence of detergents. RNA was isolated post-infection and transcriptomic analysis using amplicons (Ampliseq) revealed 274 differentially expressed genes (DEGs) across three species, out of which we selected 19 DEGs for further validation. We used qRT-PCR to confirm the differential expression of 19 DEGs. We studied biological network through Ingenuity Pathway Analysis® (IPA) which revealed up-regulated pathways of the interferon and interleukin family related to the killing of M. smegmatis. Apart from interferon and interleukin family, we found one up-regulated (EIF2AK2) and two down-regulated (MT1A and TRIB3) genes as unique potential targets found by Ampliseq and qRT-PCR which may be involved in the intracellular mycobacterial killing. The roles of these genes have not previously been described in tuberculosis. Multiplex ELISA of culture supernatants showed increased host immune response toward M. smegmatis as compared to M. bovis BCG and M.tb R179. These results enhance our understanding of host immune response against M.tb infection.

Profiling the Escherichia coli membrane protein interactome captured in Peptidisc libraries.

Protein-correlation-profiling (PCP), in combination with quantitative proteomics, has emerged as a high-throughput method for the rapid identification of dynamic protein complexes in native conditions. While PCP has been successfully applied to soluble proteomes, characterization of the membrane interactome has lagged, partly due to the necessary use of detergents to maintain protein solubility. Here, we apply the peptidisc, a 'one-size fits all' membrane mimetic, for the capture of the Escherichia coli cell envelope proteome and its high-resolution fractionation in the absence of detergent. Analysis of the SILAC-labeled peptidisc library via PCP allows generation of over 4900 possible binary interactions out of >700,000 random associations. Using well-characterized membrane protein systems such as the SecY translocon, the Bam complex and the MetNI transporter, we demonstrate that our dataset is a useful resource for identifying transient and surprisingly novel protein interactions. For example, we discover a trans-periplasmic supercomplex comprising subunits of the Bam and Sec machineries, including membrane-bound chaperones YfgM and PpiD. We identify RcsF and OmpA as bone fide interactors of BamA, and we show that MetQ association with the ABC transporter MetNI depends on its N-terminal lipid anchor. We also discover NlpA as a novel interactor of MetNI complex. Most of these interactions are largely undetected by standard detergent-based purification. Together, the peptidisc workflow applied to the proteomic field is emerging as a promising novel approach to characterize membrane protein interactions under native expression conditions and without genetic manipulation.

Evidence to Suggest Bacterial Lipoprotein Diacylglyceryl Transferase (Lgt) is a Weakly Associated Inner Membrane Protein.

The unique and ubiquitous bacterial lipoprotein biosynthesis pathway is an attractive new antibiotic target. Crystal structures of its three biosynthetic enzymes have been solved recently. The first enzyme, Phosphatidylglycerol:proLipoprotein diacylglyceryl Transferase (Lgt), which initiates the post-translational modification at the metabolic interface of protein biosynthesis, phospholipid biosynthesis, protein secretion and lipid modification was reported to be a seven-transmembrane helical structure with a catalytic periplasmic head. Its complete solubilization in water or mild detergent in a fully active state, its chromatographic behaviour as an active monomer in the absence of detergent and recovery of active whole-length protein after proteolytic treatment of spheroplasts cast serious doubts about its proposed membrane association and orientation. Rather, it could be a seven-helical bundle partially embedded in the inner membrane's inner leaflet aided by hydrophobic interaction. In fact, there are examples where originally reported seven-transmembrane proteins were later shown to be seven-helical peripheral membrane proteins based on solubilization criterion and re-analysis. Validated computational tool, Membrane Optimal Docking Area (MODA), also predicted a weaker association of Lgt's helices with the membrane compared to typical transmembrane proteins. This insight is crucial to Lgt-based antibiotic design.

Self-Assembly of Polymer-Encased Lipid Nanodiscs and Membrane Protein Reconstitution.

The absence of detergent and curvature makes nanodiscs excellent membrane mimetics. The lack of structural and mechanistic model of polymer-encapsulated lipid nanodiscs limits their use in the study of the structure, dynamics, and functions of membrane proteins. In this study, we parameterized and optimized the coarse-graining (CG) bead mapping for two differently charged and functionalized copolymers, containing styrene-maleic acid (SMAEA) and polymethacrylate (PMAQA), for the Martini force-field framework and showed nanodisc formation (<8 nm diameter) on a time scale of tens of microseconds using molecular dynamics (MD) simulations. Structural models of ∼2.0 or 4.8 kDa PMAQA and ∼2.2 kDa SMAEA polymer-based lipid nanodiscs highlight the importance of the polymer chemical structure, size, and polymer-lipid ratio in the optimization of the nanodisc structure. The ideal spatial arrangement of polymers in nanodiscs, nanodisc size, and thermal stability obtained from our MD simulation correlates well with the experimental observations. The polymer-nanodiscs were tested for the reconstitution of single-pass or multipass transmembrane proteins. We expect this study to be useful in the development of novel polymer-based lipid nanodiscs and for the structural studies of membrane proteins.

Reconstitution of a Tail‐anchored Mitochondrial Membrane Protein

Tail‐anchored (TA) proteins have a single transmembrane domain (TMD) located on the C‐terminus with the bulk of the protein facing the cytosol; in contrast with the conventional membrane protein that is translated directly into the membrane of interest. TA proteins are involved in organelle division, vesicle trafficking, and apoptosis. TA proteins lack an N‐terminal signal sequence and exhibit wide sequence variation in the TMD. Thus, TA proteins are likely inserted into the membrane post translationally. We asked if we can reconstitute Fis1, a mitochondrial outer membrane TA protein, in a detergent micelle to determine its structure. We were able to purify soluble recombinant Fis1 in the absence of detergent. Preliminary results suggest that in the absence of detergent, Fis1 exists in a soluble aggregate that can be disrupted by addition of detergent. This soluble aggregate is likely facilitated by the TMD of Fis1. We further show that in the detergent micelle Fis1 is oriented such that the cytoplasmic domain faces the solvent, reminiscent of the orientation on the mitochondrial surface. Using properly reconstituted Fis1 in detergent micelles, we collected CD, DLS, NMR, and EPR data with the goal of determining a high‐resolution structure of Fis1 in the membrane, which is an outstanding question in the mitochondrial fission field. More generally, this work has many implications for TA membrane protein reconstitution.Support or Funding InformationNIH R01‐GM067180This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of an 8R-Lipoxygenase From the Coral Plexaura homomalla.

Methods are presented for the use of the coral 8R-lipoxygenase from the Caribbean sea whip coral Plexaura homomalla as a model enzyme for structural studies of animal lipoxygenases. The 8R-lipoxygenase is remarkably stable and can be stored at 4°C for 3 months with virtually no loss of activity. In addition, an engineered "pseudo wild-type" enzyme is soluble in the absence of detergents, which helps facilitate the preparation of enzyme:substrate complexes.

Constructing cellular niche properties by localized presentation of Wnt proteins on synthetic surfaces.

Wnt signaling is crucial during embryonic development and for the maintenance of adult tissues. Depending on the tissue type, the Wnt pathway can promote stem cell self-renewal and/or direct lineage commitment. Wnt proteins are subject to lipid modification, often restricting them to act in a localized manner on responsive cells. Most methods for inducing Wnt signaling in stem cell cultures do not control the spatial presentation of the protein. To recreate the local presentation of Wnt proteins often seen in vivo, we previously developed a method to immobilize the protein onto synthetic surfaces. Here we describe a detailed protocol based on covalent binding of nucleophilic groups on Wnt proteins to activated carboxylic acid (COOH) or glutaraldehyde (COH) groups functionalized on synthetic surfaces. As an example, we describe how this method can be used to covalently immobilize Wnt3a proteins on microbeads or a glass surface. This procedure requires ∼3 h and allows for the hydrophobic protein to be stored in the absence of detergent. The immobilization efficiency of active Wnt proteins can be assessed using different T-cell factor (TCF) reporter assays as a readout for Wnt/β-catenin-dependent transcription. Immobilization efficiency can be measured 12-18 h after seeding the cells and takes 2-4 h. The covalent immobilization of Wnt proteins can also be used for single-cell analysis using Wnt-coated microbeads (12-18 h of live imaging) and to create a Wnt platform on a glass surface for stem cell maintenance and cell population analysis (3 d). The simple chemistry used for Wnt immobilization allows for adaptation to new materials and other developmental signals. Therefore, this method can also be incorporated into tissue engineering platforms in which depletion of the stem cell pool restricts the complexity and maturity of the tissue developed.

Substrate-Induced Conformational Change in LeuT

LeuT is a prokaryotic amino acid transporter that has been used extensively as a model for neurotransmitter transport. We recently demonstrated that the conformational change induced by Na+ ions requires the Na2 site observed in LeuT crystal structures. We observed this conformational change using LeuT in E. coli membranes, in the absence of detergent, by a decrease in reactivity of a single cysteine (Y265C) in the cytoplasmic permeation pathway. We now show that this effect of Na+ is observed whether K+ or NMDG+ is used as a control ion. In the presence of Na+, addition of alanine, a substrate, induces the reverse conformational change, opening the cytoplasmic pathway and increasing Cys-265 reactivity. Three mutations in the substrate binding site each altered the affinity of LeuT for leucine and alanine, but did not interfere with the conformational change induced by Na+. One of the mutations also blocked the substrate-dependent conformational change. The results suggest a mechanism by which substrate interactions with the central binding site of LeuT reverse the ability of Na+ to stabilize outward-facing conformations of this model transporter.

Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase.

Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.