Live-cell analysis of cell penetration ability and toxicity of oligo-arginines.

Cell penetrating peptides (CPPs) are useful tools to deliver low-molecular-weight cargoes into cells; however, their mode of uptake is still controversial. The most efficient CPPs belong to the group of arginine-rich peptides, but a systematic assessment of their potential toxicity is lacking. In this study we combined data on the membrane translocation abilities of oligo-arginines in living cells as a function of their chain length, concentration, stability and toxicity. Using confocal microscopy analysis of living cells we evaluated the transduction frequency of the L-isoforms of oligo-arginines and lysines and then monitored their associated toxicity by concomitant addition of propidium iodide. Whereas lysines showed virtually no transduction, the transduction ability of arginines increased with the number of consecutive residues and the peptide concentration, with L-R9 and L-R10 performing overall best. We further compared the L- and D-R9 isomers and found that the D-isoform always showed a higher transduction as compared to the L-counterpart in all cell types. Notably, the transduction difference between D- and L-forms was highly variable between cell types, emphasizing the need for protease-resistant peptides as vectors for drug delivery. Real-time kinetic analysis of the D- and L-isomers applied simultaneously to the cells revealed a much faster transduction for the D-variant. The latter underlies the fact that the isomers do not mix, and penetration of one peptide does not perturb the membrane in a way that gives access to the other peptide. Finally, we performed short- and long-term cell viability and cell cycle progression analyses with the protease-resistant D-R9. Altogether, our results identified concentration windows with low toxicity and high transduction efficiency, resulting in fully bioavailable intracellular peptides.     

Cellular internalization and distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum, and plasma membrane associated proteoglycans

The exact mechanisms by which arginine-rich cell-penetrating peptides enter cells are still the subject of debate. Here, we have analyzed in detail the effects of serum and extracellular concentration on the internalization of oligoarginines (R n; n = 4, 8, 12, 16). The presence of serum in the incubation medium had a major influence on the uptake of R12 and R16 peptides but did not affect the uptake of R4 and R8 significantly. Incubation of cells at 37 degrees C with R12 and R16 peptides in serum-containing medium showed that the majority of labeling was confined to punctate endocytic structures. Performing the same experiments in serum-free media led to a dramatic increase in cytosolic labeling, and similarly diffuse R12 and R16 labeling was observed in cells treated with peptides at 4 degrees C. This suggests, in both cases, that the peptides were entering via a nonendocytic mechanism. Further studies on R12 peptide suggest that the initiation of nonendocytic uptake and cytosolic labeling is also dependent on serum concentration and extracellular peptide concentration. At relatively low concentrations, the peptide labels endocytic structures, but upon raising the peptide concentration, the fraction labeling the cytosol increases dramatically and this accompanies a nonlinear increase in total cellular fluorescence. Membrane-associated proteoglycans also contribute to increasing the peptide concentration at the cell surface by enhancing their recruitment via electrostatic interactions. These results demonstrate that uptake mechanisms of these compounds are highly dependent on both the presence of serum and the effective extracellular peptide concentration.     

A comprehensive model for the cellular uptake of cationic cell-penetrating peptides

The plasma membrane represents an impermeable barrier for most macromolecules. Still some proteins and so-called cell-penetrating peptides enter cells efficiently. It has been shown that endocytosis contributes to the import of these molecules. However, conflicting results have been obtained concerning the nature of the endocytic process. In addition, there have been new findings for an endocytosis-independent cellular entry. In this study, we provide evidence that the Antennapedia-homeodomain-derived antennapedia (Antp) peptide, nona-arginine and the HIV-1 Tat-protein-derived Tat peptide simultaneously use three endocytic pathways: macropinocytosis, clathrin-mediated endocytosis and caveolae/lipid-raft-mediated endocytosis. Antennapedia differs from Tat and R9 by the extent by which the different import mechanisms contribute to uptake. Moreover, at higher concentrations, uptake occurs by a mechanism that originates from spatially restricted sites of the plasma membrane and leads to a rapid cytoplasmic distribution of the peptides. Endocytic vesicles could not be detected, suggesting an endocytosis-independent mode of uptake. Heparinase treatment of cells negatively affects this import, as does the protein kinase C inhibitor rottlerin, expression of dominant-negative dynamin and chlorpromazine. This mechanism of uptake was observed for a panel of different cell lines. For Antp, significantly higher peptide concentrations and inhibition of endocytosis were required to induce its uptake. The relevance of these findings for import of biologically active cargos is shown.     

Cell transduction pathways of transportans

Attempts to unravel the cell translocation mechanism of a growing number of cell-penetrating peptides (CPP) have revealed molecular determinants essential for internalization ability. The peptide sequence and the charge have been proposed to be the major factors in determining the membrane interaction mode and subsequent internalization pathway. Recent research in this field has shifted to search and design of novel CPPs with predefined vectorial properties and elucidation of the mechanism of cell entry of CPPs with high cargo delivery efficiency. Here we present a map of interaction modes with cell surface and intracellular traffic of transportan and its analogue TP10 complexed with fluorescently labeled avidin or streptavidin-gold conjugates. The protein cargo complexed with either peptide is transduced into HeLa and Bowes cells mostly in the endocytic vesicles with heterogeneous morphology and size as demonstrated by transmission electron microscopy (TEM) and confocal laser scanning fluorescence microscopy. Most of the induced vesicles are large, with 0.5-2 mum diameter, probably macropinosomes, but the complexes are present also in smaller vesicles, suggesting involvement of different pathways. Later the majority of complexes are translocated from the cell periphery into vesicles of perinuclear region and partly to lysosomes. A fraction of transportan-streptavidin complexes is present also freely in cytoplasm, both in the close vicinity of plasma membrane and more centrally, suggesting the escape from endosomal vesicles, since vesicles with discontinuous membrane were also detected by TEM. The cell-translocation process of transportan-protein complexes is temperature dependent and strongly inhibited at 8-10 degrees C and blocked at 4 degrees C when only interaction with the plasma membrane takes place.     

A critical reassessment of penetratin translocation across lipid membranes

Penetratin is a short, basic cell-penetrating peptide able to induce cellular uptake of a vast variety of large, hydrophilic cargos. We have reassessed the highly controversial issue of direct permeation of the strongly cationic peptide across negatively charged lipid membranes. Confocal laser scanning microscopy on rhodamine-labeled giant vesicles incubated with carboxyfluorescein-labeled penetratin yielded no evidence of transbilayer movement, in contradiction to previously reported results. Confocal fluorescence spectroscopy on black lipid membranes confirmed this finding, which was also not affected by application of a transmembrane electric potential difference. A novel dialysis assay based on tryptophan absorbance and fluorescence spectroscopy demonstrated that the permeability of small and large unilamellar vesicles to penetratin is <10(-13) m/s. Taken together, the results show that penetratin is not capable of overcoming model membrane systems irrespective of the bilayer curvature or the presence of a transmembrane voltage. Thus, direct translocation across the hydrophobic core of the plasma membrane cannot account for the efficient uptake of penetratin into live cells, which is in accord with recent in vitro studies underlining the importance of endocytosis in the internalization process of cationic cell-penetrating peptides.     

Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S4(13)-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S4(13)-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S4(13)-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S4(13)-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S4(13)-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S4(13)-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide-membrane interactions.     

Pathway for polyarginine entry into mammalian cells

Cationic peptides known as protein transduction domains (PTDs) provide a means to deliver molecules into mammalian cells. Here, nonaarginine (R(9)), the most efficacious of known PTDs, is used to elucidate the pathway for PTD internalization. Although R(9) is found in the cytosol as well as the nucleolus when cells are fixed, this peptide is observed only in the endocytic vesicles of live cells. Colocalization studies with vesicular markers confirm that PTDs are internalized by endocytosis rather than by crossing the plasma membrane. The inability of R(9) to enter living cells deficient in heparan sulfate (HS) suggests that binding to HS is necessary for PTD internalization. This finding is consistent with the high affinity of R(9) for heparin (K(d) = 109 nM). Finally, R(9) is shown to promote the leakage of liposomes but only at high peptide:lipid ratios. These and other data indicate that the PTD-mediated delivery of molecules into live mammalian cells involves (1) binding to cell surface HS, (2) uptake by endocytosis, (3) release upon HS degradation, and (4) leakage from endocytic vesicles.     

Molecular interplays involved in the cellular uptake of octaarginine on cell surfaces and the importance of syndecan-4 cytoplasmic V domain for the activation of protein kinase Cα

Arginine-rich cell-penetrating peptides (CPPs) are promising carriers for the intracellular delivery of various bioactive molecules. However, many ambiguities remain about the molecular interplays on cell surfaces that ultimately lead to endocytic uptake of CPPs. By treatment of cells with octaarginine (R8), enhanced clustering of syndecan-4 on plasma membranes and binding of protein kinase Cα (PKCα) to the cytoplasmic domain of syndecan-4 were observed; these events potentially lead to the macropinocytic uptake of R8. The cytoplasmic V domain of syndecan-4 made a significant contribution to the cellular uptake of R8, whereas the cytoplasmic C1 and C2 domains were not involved in the process.     

Biophysical and biological studies of end-group-modified derivatives of Pep-1

Pep-1 is a tryptophane-rich cell-penetrating peptide (CPP) that has been previously proposed to bind protein cargoes by hydrophobic assembly and translocate them across cellular membranes. To date, however, the molecular mechanisms responsible for cargo binding and translocation have not been clearly identified. This study was conducted to gain insight into the interaction between Pep-1 with its cargo and the biological membrane to identify the thereby involved structural elements crucial for translocation. We studied three peptides differing in their N- and C-termini: (i) Pep-1, carrying an acetylated N-terminus and a C-terminal cysteamine elongation, (ii) AcPepWAmide, with an acetylated N-terminus and an amidated C-terminus, and (iii) PepW, with two free termini. Thioredoxin (TRX) and beta-galactosidase were used as protein cargoes. To study CPP-membrane interactions, we performed biophysical as well as biological assays. To mimic biological membranes, we used phospholipid liposomes in a dye leakage assay and surfactant micelles for high-resolution NMR studies. In addition, membrane integrity, cell viability, and translocation efficiency were analyzed in HeLa cells. An alpha-helical structure was found for all peptides in the hydrophobic N-terminal region encompassing residues 4-13, whereas the hydrophilic region remained unstructured in the presence of micelles. Our results show that the investigated peptides interacted with the micelles as well as with the protein cargo via their tryptophan-rich domain. All peptides displayed an orientation parallel to the micelle surface. The C-terminal cysteamine group formed an additional membrane anchor, leading to more efficient translocation properties in cells. No membrane permeabilization was observed, and our data were largely compatible with an endocytic pathway for cellular uptake.     

Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S4₁₃-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S4₁₃-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S4₁₃-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S4₁₃-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S4₁₃-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S4₁₃-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide-membrane interactions.     

Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.     

Metabolic energy-independent mechanism of internalization for the cell penetrating peptide penetratin

Cellular uptake of vector peptides used for internalization of hydrophilic molecules into cells is known to follow two different pathways: direct translocation of the plasma membrane and internalization by endocytosis followed by release into the cytosol. These pathways differ in their energy dependence. The first does not need metabolic energy while the second requires metabolic energy. Herein we used erythrocytes and plasma membrane vesicles to study membrane perturbations induced by the cell penetrating peptide penetratin. The results show that cell penetrating peptides are able to be internalized by two metabolic energy-independent pathways: direct crossing of the plasma membrane and endocytosis-like mechanisms. The last mechanism involves the induction of membrane negative curvature resulting in invaginations that mimic the endosomal uptake in the absence of ATP. This new mechanism called "physical endocytosis" or "self-induced endocytosis" might explain different data concerning the independence or dependence on metabolic energy during cellular uptake and reveals the autonomous capacity of peptides to induce their internalization.     

Myristoyl-based transport of peptides into living cells

Translocation of membrane-impermeant molecules to the interior of living cells is a necessity for many biochemical investigations. Myristoylation was studied as a means to introduce peptides into living cells. Uptake of a myristoylated, fluorescent peptide was efficient in the B lymphocyte cell line BA/F3. In contrast, this cell line was resistant to uptake of a cell-penetrating peptide derived from the TAT protein. In BA/F3 cells, membrane association was shown to be rapid, reaching a maximum within 30 min. Cellular uptake of the peptide lagged the membrane association but occurred within a similar time frame. Experiments performed at 37 versus 4 degrees C demonstrated profound temperature dependence in the cellular uptake of myristoylated cargo. Myristoylated peptides with either positive or negative charge were shown to load efficiently. In contrast to TAT-conjugated cargo, pyrenebutyrate did not enhance cellular uptake of the myristoylated peptide. The myristoylated peptide did not adversely affect cell viability at concentrations up to 100 muM. This assessment of myristoyl-based transport provides fundamental data needed in understanding the intracellular delivery of myristoylated peptide cargoes for cell-based biochemical studies.     

Protein cargo delivery properties of cell-penetrating peptides. A comparative study

Application of cell-penetrating peptides for delivering various hydrophilic macromolecules with biological function into cells has gained much attention in recent years. We compared the protein transduction efficiency of four cell-penetrating peptides: penetratin, Tat peptide, transportan, and pVEC and studied the effects of various medium parameters on the uptake. Depletion of cellular energy and lowering of temperature strongly impaired the internalization of protein complexed with cell-penetrating peptides, confirming the endocytotic mechanism of peptide-mediated protein cellular transduction. Peptide-induced protein association with HeLa cells decreased 3-6-fold in energy-depleted cells. Inhibition of clathrin-dependent endocytosis by the hyperosmolar medium decreased the uptake of peptide-avidin complexes 1.5-3-fold and the removal of cholesterol from the plasma membrane 1.2-2-fold, suggesting that both clathrin-dependent and independent endocytosis were involved in peptide-induced cellular delivery of avidin. However, even under conditions of cellular energy depletion, ceasing of cellular traffic, and partial depolarization of plasma membrane, peptide-protein complexes associated with HeLa cells, as observed by FACS analysis and spectrofluorimetry. Among the studied peptides, pTat and transportan revealed higher protein transduction efficiency than penetratin or pVEC.     

Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes

Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 microm). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide.     

Fluorescent sterols monitor cell penetrating peptide Pep-1 mediated uptake and intracellular targeting of cargo protein in living cells

Although cell-penetrating peptides (CPP) facilitate endocytic uptake of proteins, little is known regarding the extent to which CPPs facilitate protein cargo exit from endocytic vesicles for targeting to other intracellular sites. Since the plasma membrane and less so intracellular membranes contain cholesterol, the fluorescent sterol analogues dansyl-cholestanol (DChol) and dehydroergosterol (DHE) were used to monitor the uptake and intracellular distribution of fluorescent-tagged acyl coenzyme A binding protein (ACBP) into COS-7 cells and rat hepatoma cells. Confocal microscopy colocalized DChol and Texas Red-ACBP (TR-ACBP) with markers for the major endocytosis pathways, especially fluorescent-labeled cholera toxin (marker of ganglioside GM1 in plasma membrane lipid rafts) and dextran (macropinocytosis marker), but less so with transferrin (clathrin-mediated endocytosis marker). These findings were confirmed by multiphoton laser scanning microscopy colocalization of TR-ACBP with DHE (naturally-fluorescent sterol) and by double immunofluorescence labeling of native endogenous ACBP. Serum greatly and Pep-1 further 2.4-fold facilitated uptake of TR-ACBP, but neither altered the relative proportion of TR-ACBP colocalized with membranes/organelles (nearly 80%) vs cytoplasm and/or nucleoplasm (20%). Interestingly, Pep-1 selectively increased TR-ACBP associated with mitochondria while concomitantly decreasing that in endoplasmic reticulum. In summary, fluorescent sterols (DChol, DHE) were useful markers for comparing the distributions of both transported and endogenous proteins. Pep-1 modestly enhanced the translocation and altered the intracellular targeting of exogenous-delivered (TR-ACBP) in living cells.     

Cellular uptake of transportan 10 and its analogs in live cells: Selectivity and structure-activity relationship studies

Transportan 10 (TP10) is an amphipathic cell-penetrating peptide with high translocation ability. In order to obtain more details of structure-activity relationship of TP10, we evaluated the effects of structure and charge on its translocation ability. Our results demonstrated that disrupting the helical structure or Arg substitution could remarkably decrease the cellular uptake of TP10. However, increasing the number of positive charge was an effective strategy to enhance translocation ability of TP10. Furthermore, the molecular dynamics simulation supported the results derived from experiments, suggesting that higher membrane disturbance leads to higher cellular uptake of peptides. In addition, our study also demonstrated TP10 and its analogs preferentially entered cancer cells rather than normal cells. The uptake selectivity toward cancer cells makes TP10 and its analogs as potent CPPs for drug delivery.     

Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.     

Existence of two parallel mechanisms for glucose uptake in heterotrophic plant cells

The implied existence of two mechanisms for glucose uptake into heterotrophic plant cells was investigated using the fluorescent glucose derivative 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), two membrane impermeable fluorescent markers (3000 mol. wt. dextran-Texas Red (d-TR) and Alexa-488), hexose carrier and endocytic inhibitors (phloridzin and wortmannin-A, respectively), and fluorescent and confocal microscopy. Both phloridzin and wortmannin-A significantly reduced the uptake of 2-NBDG into sycamore cultured cells, which was confirmed by fluorescent microscopy. Phloridzin prevented 2-NBDG uptake exclusively into the cytosol, whereas the wortmannin-A effect was more general, with 2-NBDG uptake into the vacuole being the more affected. Simultaneous incubation of cells in the membrane-impermeable fluorescent probes Alexa-488 and d-TR for 24 h resulted in co-localization of the labelling in the central vacuole and other endosomal compartments. Cytoplasts, cells devoid of vacuoles, were instrumental in demonstrating the transport of 2-NBDG by separate uptake mechanisms. In cytoplasts incubated simultaneously in 2-NBDG and d-TR for 2 h, a green fluorescent cytosol was indicative of transport of hexoses across the plasmalemma, while the co-localization of 2-NBDG and d-TR in internal vesicles demonstrated transport via an endocytic system. The absence of vesicles when cytoplasts were pre-incubated in wortmannin-A authenticated the endocytic vesicular nature of the co-shared 2-NBDG and d-TR fluorescent structures. In summary, uptake of 2-NBDG occurs by two separate mechanisms: (i) a plasmalemma-bound carrier-mediated system that facilitates 2-NBDG transport into the cytosol, and (ii) an endocytic system that transports most of 2-NBDG directly into the vacuole.     

Photolabeling of erythrocyte and adipocyte hexose transporters using a benzophenone derivative of bis(D-mannose)

The benzophenone derivative of 1,3-bis(D-mannos-4-yloxy)-2-propylamine (BB-BMPA) has been tested as an exofacial photoaffinity label for the sugar transport systems of human erythrocytes and rat adipocytes. The half-maximal inhibition constants for the reagent are 971 microM in erythrocytes and 536 microM in basal and 254 microM in insulin-treated adipocytes. The photolabelling of erythrocyte membranes is very specific for the 50 kDa transporter peptide and is completely displaced by D-glucose. The exofacial photoaffinity labelling of adipocytes also shows labelling of a 50 kDa transporter peptide, which is displaced by cytochalasin B, but extensive nonspecific labelling of a 75 kDa plasma membrane peptide occurs. The transporter is labelled in insulin-treated cells but not in basal cells which indicates that this in situ labelling technique selectively reveals only those transporters that visit and are active in the plasma membrane during the labelling period. This also indicates that in basal cells transporters do not turn over rapidly. Subcellular redistribution of transporters after the labelling period has been studied. Following incubation and washing at 37 degrees C in the presence of insulin, 30% of the transporters photolabelled at the plasma membrane are internalised and are found in the light microsome fraction of the cell. The proportion of transporter that is observed to be internalised is much greater than can be accounted for by a contamination of the light microsome fraction by plasma membrane. The labelled 50 kDa transporter peptide in the light microsomes is enriched when compared with the carry-over of the 75 kDa nonspecifically labelled plasma membrane peptide. Thus we have obtained direct evidence for transporter translocation.