Detection of intracellular phosphatidylserine in living cells

To demonstrate the intracellular phosphatidylserine (PS) distribution in neuronal cells, neuroblastoma cells and hippocampal neurons expressing green fluorescence protein (GFP)-AnnexinV were stimulated with a calcium ionophore and localization of GFP-AnnexinV was monitored by fluorescence microscopy. Initially, GFP-AnnexinV distributed evenly in the cytosol and nucleus. Raising the intracellular calcium level with ionomycin-induced translocation of cytoplasmic GFP-AnnexinV to the plasma membrane but not to the nuclear membrane, indicating that PS distributes in the cytoplasmic side of the plasma membrane. Nuclear GFP-AnnexinV subsequently translocated to the nuclear membrane, indicating PS localization in the nuclear envelope. GFP-AnnexinV also localized in a juxtanuclear organelle that was identified as the recycling endosome. However, minimal fluorescence was detected in any other subcellular organelles including mitochondria, endoplasmic reticulum, Golgi complex, and lysosomes, strongly suggesting that PS distribution in the cytoplasmic face in these organelles is negligible. Similarly, in hippocampal primary neurons PS distributed in the inner leaflet of plasma membranes of cell body and dendrites, and in the nuclear envelope. To our knowledge, this is the first demonstration of intracellular PS localization in living cells, providing an insight for specific sites of PS interaction with soluble proteins involved in signaling processes.     

Phosphatidylserine in the brain: Metabolism and function

Phosphatidylserine (PS) is the major anionic phospholipid class particularly enriched in the inner leaflet of the plasma membrane in neural tissues. PS is synthesized from phosphatidylcholine or phosphatidylethanolamine by exchanging the base head group with serine, and this reaction is catalyzed by phosphatidylserine synthase 1 and phosphatidylserine synthase 2 located in the endoplasmic reticulum. Activation of Akt, Raf-1 and protein kinase C signaling, which supports neuronal survival and differentiation, requires interaction of these proteins with PS localized in the cytoplasmic leaflet of the plasma membrane. Furthermore, neurotransmitter release by exocytosis and a number of synaptic receptors and proteins are modulated by PS present in the neuronal membranes. Brain is highly enriched with docosahexaenoic acid (DHA), and brain PS has a high DHA content. By promoting PS synthesis, DHA can uniquely expand the PS pool in neuronal membranes and thereby influence PS-dependent signaling and protein function. Ethanol decreases DHA-promoted PS synthesis and accumulation in neurons, which may contribute to the deleterious effects of ethanol intake. Improvement of some memory functions has been observed in cognitively impaired subjects as a result of PS supplementation, but the mechanism is unclear.     

Multiple cellular proteins modulate the dynamics of K-ras association with the plasma membrane

Although specific proteins have been identified that regulate the membrane association and facilitate intracellular transport of prenylated Rho- and Rab-family proteins, it is not known whether cellular proteins fulfill similar roles for other prenylated species, such as Ras-family proteins. We used a previously described method to evaluate how several cellular proteins, previously identified as potential binding partners (but not effectors) of K-ras4B, influence the dynamics of K-ras association with the plasma membrane. Overexpression of either PDEδ or PRA1 enhances, whereas knockdown of either protein reduces, the rate of dissociation of K-ras from the plasma membrane. Inhibition of calmodulin likewise reduces the rate of K-ras dissociation from the plasma membrane, in this case in a manner specific for the activated form of K-ras. By contrast, galectin-3 specifically reduces the rate of plasma membrane dissociation of activated K-ras, an effect that is blocked by the K-ras antagonist farnesylthiosalicylic acid (salirasib). Multiple cellular proteins thus control the dynamics of membrane association and intercompartmental movement of K-ras to an important degree even under basal cellular conditions.     

Role of Flippases,Scramblases and Transfer Proteins in Phosphatidylserine Subcellular Distribution

It is well known that lipids are heterogeneously distributed throughout the cell. Most lipid species are synthesized in the endoplasmic reticulum (ER) and then distributed to different cellular locations in order to create the distinct membrane compositions observed in eukaryotes. However, the mechanisms by which specific lipid species are trafficked to and maintained in specific areas of the cell are poorly understood and constitute an active area of research. Of particular interest is the distribution of phosphatidylserine (PS), an anionic lipid that is enriched in the cytosolic leaflet of the plasma membrane. PS transport occurs by both vesicular and non‐vesicular routes, with members of the oxysterol‐binding protein family (Osh6 and Osh7) recently implicated in the latter route. In addition, the flippase activity of P4‐ATPases helps build PS membrane asymmetry by preferentially translocating PS to the cytosolic leaflet. This asymmetric PS distribution can be used as a signaling device by the regulated activation of scramblases, which rapidly expose PS on the extracellular leaflet and play important roles in blood clotting and apoptosis. This review will discuss recent advances made in the study of phospholipid flippases, scramblases and PS‐specific lipid transfer proteins, as well as how these proteins contribute to subcellular PS distribution.      

Ceramide regulates interaction of Hsd17b4 with Pex5 and function of peroxisomes

The sphingolipid ceramide regulates beta-oxidation of medium and long chain fatty acids in mitochondria. It is not known whether it also regulates oxidation of very long chain fatty acids (VLCFAs) in peroxisomes. Using affinity chromatography, co-immunoprecipitation, and proximity ligation assays we discovered that ceramide interacts with Hsd17b4, an enzyme critical for peroxisomal VLCFA oxidation and docosahexaenoic acid (DHA) generation. Immunocytochemistry showed that Hsd17b4 is distributed to ceramide-enriched mitochondria-associated membranes (CEMAMs). Molecular docking and in vitro mutagenesis experiments showed that ceramide binds to the sterol carrier protein 2-like domain in Hsd17b4 adjacent to peroxisome targeting signal 1 (PTS1), the C-terminal signal for interaction with peroxisomal biogenesis factor 5 (Pex5), a peroxin mediating transport of Hsd17b4 into peroxisomes. Inhibition of ceramide biosynthesis induced translocation of Hsd17b4 from CEMAMs to peroxisomes, interaction of Hsd17b4 with Pex5, and upregulation of DHA. This data indicates a novel role of ceramide as a molecular switch regulating interaction of Hsd17b4 with Pex5 and peroxisomal function.     

Transbilayer movement of phosphatidylserine in nonhuman erythrocytes: evidence that the aminophospholipid transporter is a ubiquitous membrane protein

A 31-32-kDa integral membrane protein has been previously identified in erythrocytes as the protein most likely to be responsible for the transbilayer movement of phosphatidylserine (PS) [Connor & Schroit (1988) Biochemistry 27, 848-851]. Using similar techniques, we have identified analogous proteins of identical molecular weights in bovine, equine, ovine, porcine, canine, caprine, and rhesus red blood cells. Similar to human red blood cells, all of the mammalian cells were able to specifically transport an exogenously supplied fluorescent PS analogue from their outer-to-inner membrane leaflet. In addition, transport could be reversibly inhibited with the sulfhydryl-specific inhibitor pyridyldithioethylamine (PDA). PDA-sensitive PS transport was also observed in nucleated human and murine cell lines. Analysis of isolated plasma membranes from 125I-PDA-labeled cells revealed marked labeling of a 32,000-Da component. Attempts to inhibit PS transport by treating the cells with proteases, lectins, or antibody suggested that the 32-kDa polypeptide is an integral membrane protein that does not contain sites critical to its function at the cell surface.     

Characterization of the hypothalamic-pituitary-gonadal axis in estrogen receptor (ER) Null mice reveals hypergonadism and endocrine sex reversal in females lacking ERalpha but not ERbeta

To determine the role of each estrogen receptor (ER) form (ERalpha, ERbeta) in mediating the estrogen actions necessary to maintain proper function of the hypothalamic-pituitary-gonadal axis, we have characterized the hypothalamic-pituitary-gonadal axis in female ER knockout (ERKO) mice. Evaluation of pituitary function included gene expression assays for Gnrhr, Cga, Lhb, Fshb, and Prl. Evaluation of ovarian steroidogenic capacity included gene expression assays for the components necessary for estradiol synthesis: i.e. Star, Cyp11a, Cyp17, Cyp19, Hsd3b1, and Hsd17b1. These data were corroborated by assessing plasma levels of the respective peptide and steroid hormones. alphaERKO and alphabetaERKO females exhibited increased pituitary Cga and Lhb expression and increased plasma LH levels, whereas both were normal in betaERKO. Pituitary Fshb expression and plasma FSH were normal in all three ERKOs. In the ovary, all three ERKOs exhibited normal expression of Star, Cyp11a, and Hsd3b1. In contrast, Cyp17 and Cyp19 expression were elevated in alphaERKO but normal in betaERKO and alphabetaERKO. Plasma steroid levels in each ERKO mirrored the steroidogenic enzyme expression, with only the alphaERKO exhibiting elevated androstenedione and estradiol. Elevated plasma testosterone in alphaERKO and alphabetaERKO females was attributable to aberrant expression of Hsd17b3 in the ovary, representing a form of endocrine sex reversal, as this enzyme is unique to the testes. Enhanced steroidogenic capacity in alphaERKO ovaries was erased by treatment with a GnRH antagonist, indicating these phenotypes to be the indirect result of excess LH stimulation that follows the loss of ERalpha in the hypothalamic-pituitary axis. Overall, these findings indicate that ERalpha, but not ERbeta, is indispensable to the negative-feedback effects of estradiol that maintain proper LH secretion from the pituitary. The subsequent hypergonadism is illustrated as increased Cyp17, Cyp19, Hsd17b1, and ectopic Hsd17b3 expression in the ovary.     

Identification of a 17β-hydroxysteroid dehydrogenase type 12 pseudogene as the source of a highly restricted BALB/c Meth A tumor rejection peptide

Mass spectrometric analysis identified the peptide recognized by a cytotoxic T lymphocyte (CTL) specific for the chemically induced BALB/c Meth A sarcoma as derived from a 17β-hydroxysteroid dehydrogenase type 12 (Hsd17b12) pseudogene present in the BALB/c genome, but only expressed in Meth A sarcoma. The sequence of the peptide is TYDKIKTGL and corresponds to Hsd17b12_114–122 with threonine instead of isoleucine at codon 114 and is designated Hsd17b12^114T. Immunization of mice with an Hsd17b12^114T peptide-pulsed dendritic cell-based vaccine or a non-viral plasmid construct expressing the Hsd17b12^114T peptide protected the mice from lethal Meth A tumor challenge in tumor rejection assays. A Hsd17b12_114–122 peptide-pulsed vaccine was ineffective in inducing resistance in mice to Meth A sarcoma. These results confirm the immunogenicity of the identified tumor peptide, as well as demonstrate the efficacies of these vaccine vehicles. These findings suggest that the role of the human homolog of Hsd17b12, HSD17B12, as a potential human tumor antigen be explored.     

Inward relocation of exogenous phosphatidylserine triggered by IGF-1 in non-apoptotic C2C12 cells is concentration dependent

The plasma membrane is composed of two leaflets that are asymmetric with regard to their phospholipid composition with phosphatidylserine (PS) predominantly located within the inner leaflet whereas other phospholipids such as phosphatidylcholine (PC) are preferentially located in the outer leaflet. An intimate relationship between cellular physiology and the composition of the plasma membrane has been demonstrated, with for example apoptosis requiring PS exposure for macrophage recognition. In skeletal muscle development, differentiation also requires PS exposure in myoblasts to create cell-cell contact areas allowing the formation of multinucleate myotubes. Although it is clearly established that membrane composition/asymmetry plays an important role in cellular physiology, the role of cytokines in regulating this asymmetry is still unclear. When incubated with myoblasts, insulin-like growth factor I (IGF-1) has been shown to promote proliferation versus differentiation in a concentration dependent manner and therefore, may be a potential candidate regulating cell membrane asymmetry. We show, in non-apoptotic C2C12 cells, that relocation of an exogenous PS analogue, from the outer into the inner leaflet, is accelerated by IGF-1 in a concentration-dependent manner and that maintenance of membrane asymmetry triggered by IGF-1 is however independent of the PI3K inhibitor wortmannin.     

Cell membrane extensions, generated by mechanical constraint, are associated with a sustained lipid raft patching and an increased cell signaling

Platelet activation triggers an imbalance in plasma membrane phospholipids by a specific aminophospholipid outflux, resulting in filopodia formation. Similarly, the addition of a phospholipid excess in the outer leaflet of the plasma membrane induces cellular extensions and actin polymerization. The implication of membrane microdomains in sustaining these mechanical constraints remains, however, unknown and was investigated in human platelets and mouse fibroblasts. The disruption of lipid rafts by cholesterol depletion prevents actin polymerization and formation of cellular extensions. Phospholipid excess triggers raft patching underneath the cell extensions, recruitment of protein raft markers and increase of tyrosine phosphorylation of raft proteins. Using a mass spectrometric analysis of isolated platelet rafts, we identified tyrosine kinases and proteins implicated in the formation of cell membrane extensions, cell adhesion and motility. They are recruited to rafts in response to a mechanical constraint. Taken together, our results demonstrate that exogenous phospholipid addition causes a modulation of the lateral plasma membrane organization and an activation of the cell signaling triggering actin remodeling and the formation of cellular protrusions. Raft disruption abolishes these processes, demonstrating that their integrity is crucial for cell shape changes in response to a mechanical constraint on plasma membrane.     

Detergent-insoluble glycosphingolipid/cholesterol-rich membrane domains, lipid rafts and caveolae (review).

Within the cell membrane glycosphingolipids and cholesterol cluster together in distinct domains or lipid rafts, along with glycosyl-phosphatidylinositol (GPI)-anchored proteins in the outer leaflet and acylated proteins in the inner leaflet of the bilayer. These lipid rafts are characterized by insolubility in detergents such as Triton X-100 at 4 degrees C. Studies on model membrane systems have shown that the clustering of glycosphingolipids and GPI-anchored proteins in lipid rafts is an intrinsic property of the acyl chains of these membrane components, and that detergent extraction does not artefactually induce clustering. Cholesterol is not required for clustering in model membranes but does enhance this process. Single particle tracking, chemical cross-linking, fluorescence resonance energy transfer and immunofluorescence microscopy have been used to directly visualize lipid rafts in membranes. The sizes of the rafts observed in these studies range from 70-370 nm, and depletion of cellular cholesterol levels disrupts the rafts. Caveolae, flask-shaped invaginations of the plasma membrane, that contain the coat protein caveolin, are also enriched in cholesterol and glycosphingolipids. Although caveolae are also insoluble in Triton X-100, more selective isolation procedures indicate that caveolae do not equate with detergent-insoluble lipid rafts. Numerous proteins involved in cell signalling have been identified in caveolae, suggesting that these structures may function as signal transduction centres. Depletion of membrane cholesterol with cholesterol binding drugs or by blocking cellular cholesterol biosynthesis disrupts the formation and function of both lipid rafts and caveolae, indicating that these membrane domains are involved in a range of biological processes.     

Disruption of the Lipid-Transporting LdMT-LdRos3 Complex in Leishmania donovani Affects Membrane Lipid Asymmetry but Not Host Cell Invasion

Maintenance and regulation of the asymmetric lipid distribution across eukaryotic plasma membranes is governed by the concerted action of specific membrane proteins controlling lipid movement across the bilayer. Here, we show that the miltefosine transporter (LdMT), a member of the P4-ATPase subfamily in Leishmania donovani, and the Cdc50-like protein LdRos3 form a stable complex that plays an essential role in maintaining phospholipid asymmetry in the parasite plasma membrane. Loss of either LdMT or LdRos3 abolishes ATP-dependent transport of NBD-labelled phosphatidylethanolamine (PE) and phosphatidylcholine from the outer to the inner plasma membrane leaflet and results in an increased cell surface exposure of endogenous PE. We also find that promastigotes of L. donovani lack any detectable amount of phosphatidylserine (PS) but retain their infectivity in THP-1-derived macrophages. Likewise, infectivity was unchanged for parasites without LdMT-LdRos3 complexes. We conclude that exposure of PS and PE to the exoplasmic leaflet is not crucial for the infectivity of L. donovani promastigotes.     

Polyamine regulation of plasma membrane phospholipid flip-flop during apoptosis.

During apoptosis, phosphatidylserine (PS) is moved from the plasma membrane inner leaflet to the outer leaflet where it triggers recognition and phagocytosis of the apoptotic cell. Although the mechanisms of PS appearance during apoptosis are not well understood, it is thought that declining activity of the aminophospholipid translocase and calcium-mediated, nonspecific flip-flop of phospholipids play a role. As previous studies in the erythrocyte ghost have shown that polyamines can alter flip-flop of phospholipids, we asked whether alterations in cellular polyamines in intact cells undergoing apoptosis would affect PS appearance, either by altering aminophospholipid translocase activity or phospholipid flip-flop. Cells of the human leukemic cell line, HL-60, were incubated with or without the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and induced to undergo apoptosis by ultraviolet irradiation. Whereas DFMO treatment resulted in profound depletion of putrescine and spermidine (but not spermine), it had no effect on caspase activity, DNA fragmentation, or plasma membrane vesiculation, typical characteristics of apoptosis. Notably, DFMO treatment prior to ultraviolet irradiation did not alter the decline in PS inward movement by the aminophospholipid translocase as measured by the uptake of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS detected in the flow cytometer. Conversely, the appearance of endogenous PS in the plasma membrane outer leaflet detected with fluorescein isothiocyanate-labeled annexin V and enhanced phospholipid flip-flop detected by the uptake of 1-palmitoyl-1-[6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminocaproyl]-sn-glycero-3-phosphocholine (NBD-PC) seen during apoptosis were significantly inhibited by prior DFMO treatment. Importantly, replenishment of spermidine, by treatment with exogenous putrescine to bypass the metabolic blockade by DFMO, restored both enhanced phospholipid flip-flop and appearance of PS during apoptosis. Such restoration was seen even in the presence of cycloheximide but was not seen when polyamines were added externally just prior to assay. Taken together, these data show that intracellular polyamines can modulate PS appearance resulting from nonspecific flip-flop of phospholipids across the plasma membrane during apoptosis.     

Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.

BACKGROUND: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins. METHODS: We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis. RESULTS: Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content less, less than or approximately equal to 2.5%-8% for the membrane domains that stained with lactadherin but not annexin V. CONCLUSIONS: Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology.     

Phosphatidylserine induces apoptosis in adherent cells

Phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane in apoptotic cell death. However, the roles of PS in apoptotic signaling are still unclear. In this study, we found that exogenous PS, but not other phospholipids, induced cell death in adherent cells, but not in suspension culture. The cell death exhibited typical features of apoptosis such as cell shrinkage, nuclear fragmentation and abnormal chromatin condensation. When PS was added to CHO-K1 cells in monolayer culture, they began to show changes in cell shape and actin cytoskeleton and protein kinase C (PKC) activity, followed by cell detachment, caspase activation, cleavage of focal adhesion kinase (FAK) and finally loss of viability. These results suggested that PS causes apoptosis through actin disorganization, cell detachment and cleavage of FAK.     

Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts

The trafficking and function of cell surface proteins in eukaryotic cells may require association with detergent-resistant sphingolipid- and sterol-rich membrane domains. The aim of this work was to obtain evidence for lipid domain phenomena in plant membranes. A protocol to prepare Triton X-100 detergent-resistant membranes (DRMs) was developed using Arabidopsis (Arabidopsis thaliana) callus membranes. A comparative proteomics approach using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins. They included eight glycosylphosphatidylinositol-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins, and proteins of the stomatin/prohibitin/hypersensitive response family, suggesting that the DRMs originated from PM domains. We also identified a plant homolog of flotillin, a major mammalian DRM protein, suggesting a conserved role for this protein in lipid domain phenomena in eukaryotic cells. Lipid analysis by gas chromatography-mass spectrometry showed that the DRMs had a 4-fold higher sterol-to-protein content than the average for Arabidopsis membranes. The DRMs were also 5-fold increased in sphingolipid-to-protein ratio. Our results indicate that the preparation of DRMs can yield a very specific set of membrane proteins and suggest that the PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition. Our results also suggest a conserved role of lipid modification in targeting proteins to both the intracellular and extracellular leaflet of these domains. The proteins associated with these domains provide important new experimental avenues into understanding plant cell polarity and cell surface processes.     

Sialidase NEU3 is a peripheral membrane protein localized on the cell surface and in endosomal structures

Sialidase NEU3 is also known as the plasma-membrane-associated form of mammalian sialidases, exhibiting a high substrate specificity towards gangliosides. In this respect, sialidase NEU3 modulates cell-surface biological events and plays a pivotal role in different cellular processes, including cell adhesion, recognition and differentiation. At the moment, no detailed studies concerning the subcellular localization of NEU3 are available, and the mechanism of its association with cellular membranes is still unknown. In the present study, we have demonstrated that sialidase NEU3, besides its localization at the plasma membrane, is present in intracellular structures at least partially represented by a subset of the endosomal compartment. Moreover, we have shown that NEU3 present at the plasma membrane is internalized and locates then to the recycling endosomal compartment. The enzyme is associated with the outer leaflet of the plasma membrane, as shown by selective cell-surface protein biotinylation. This evidence is in agreement with the ability of NEU3 to degrade gangliosides inserted into the plasma membrane of adjacent cells. Moreover, the mechanism of the protein association with the lipid bilayer was elucidated by carbonate extraction. Under these experimental conditions, we have succeeded in solubilizing NEU3, thus demonstrating that the enzyme is a peripheral membrane protein. In addition, Triton X-114 phase separation demonstrates further the hydrophilic nature of the protein. Overall, these results provide important information about the biology of NEU3, the most studied member of the mammalian sialidase family.     

A putative plant aminophospholipid flippase, the Arabidopsis P4 ATPase ALA1, localizes to the plasma membrane following association with a β-subunit

Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner leaflet and sphingolipids in the outer leaflet. This unequal distribution of lipids between leaflets is, amongst several proposed functions, hypothesized to be a prerequisite for endocytosis. P4 ATPases, belonging to the P-type ATPase superfamily of pumps, are involved in establishing lipid asymmetry across plasma membranes, but P4 ATPases have not been identified in plant plasma membranes. Here we report that the plant P4 ATPase ALA1, which previously has been connected with cold tolerance of Arabidopsis thaliana, is targeted to the plasma membrane and does so following association in the endoplasmic reticulum with an ALIS protein β-subunit.     

Plasma membrane isolated with a defined orientation used to investigate protein topography

An investigation into the protein topography of tomato plasma membrane proteins was undertaken. Plasma membrane was isolated by phase partitioning to expose the extracellular leaflet, and by coating the protoplasts with silica microbeads to expose the cytosolic surface. Marker enzyme analysis indicated that both methods yielded relatively pure plasma membrane. Orientation of these plasma membrane fractions was established by investigating the latency of H⁺-ATPase activity. Triton X-100 stimulated H⁺-ATPase activity by 6-fold in the phase-partitioned plasma membrane fraction but did not stimulate this enzyme in the silica microbead-isolated plasma membrane. The impermeant photoactivable probes, 3-azido-(2,7)-naphthalene disulfonate and 5-azido-1-naphthalene monosulfonate, were used to probe the hydrophilic and hydrophobic regions of the plasma membrane, respectively. Using 5-azido-1-naphthalene monosulfonate, six proteins were labeled from the cytosolic leaflet of the plasma membrane and five proteins were labeled from the extracellular leaflet. Only two proteins were labeled by 3-azido-(2,7)-naphthalene disulfonate, and these were from the cytosolic-facing leaflet. The results indicate that these photoactive probes can be used in conjunction with aqueous two-phase partitioning and silica microbeads for transmembrane mapping of plasma membrane proteins.