On the Role of the Difference in Surface Tensions Involved in the Allosteric Regulation of NHE-1 Induced by Low to Mild Osmotic Pressure,Membrane Tension and Lipid Asymmetry

The sodium-proton exchanger 1 (NHE-1) is a membrane transporter that exchanges Na⁺ for H⁺ ion across the membrane of eukaryotic cells. It is cooperatively activated by intracellular protons, and this allosteric regulation is modulated by the biophysical properties of the plasma membrane and related lipid environment. Consequently, NHE-1 is a mechanosensitive transporter that responds to osmotic pressure, and changes in membrane composition. The purpose of this study was to develop the relationship between membrane surface tension, and the allosteric balance of a mechanosensitive transporter such as NHE-1. In eukaryotes, the asymmetric composition of membrane leaflets results in a difference in surface tensions that is involved in the creation of a reservoir of intracellular vesicles and membrane buds contributing to buffer mechanical constraints. Therefore, we took this phenomenon into account in this study and developed a set of relations between the mean surface tension, membrane asymmetry, fluid phase endocytosis and the allosteric equilibrium constant of the transporter. We then used the experimental data published on the effects of osmotic pressure and membrane modification on the NHE-1 allosteric constant to fit these equations. We show here that NHE-1 mechanosensitivity is more based on its high sensitivity towards the asymmetry between the bilayer leaflets compared to mean global membrane tension. This compliance to membrane asymmetry is physiologically relevant as with their slower transport rates than ion channels, transporters cannot respond as high pressure-high conductance fast-gating emergency valves.     

Transferrin and iron distribution in subcellular fractions of K562 cells in the early stages of transferrin endocytosis.

Iron distribution in subcellular fractions was investigated at different times after a single cohort of 59Fe-125 I-labeled transferrin (Tf) endocytosis in K562 cells. Cell homogenates prepared by hypotonic lysis and deoxyribonuclease (DNAase) treatment were fractionated on Percoll density gradients. Iron-containing components in the postmitochondrial supernatant were further fractionated according to their molecular weight using gel chromatography and membrane filtration. In the initial phases of endocytosis, both iron and Tf were found in the light vesicular fraction. After 3 min the labels diverged, with iron appearing in the postmitochondrial supernatant and Tf in the heavy fraction containing mitochondria, lysosomes and nuclei. Iron released from Tf-containing vesicles appeared both in low- and high-molecular-weight fractions in the postmitochondrial supernatant. After 5 min of endocytosis 59Fe activity in the low-molecular-weight fraction remained constant and 59Fe accumulated in a high-molecular-weight fraction susceptible to desferrioxamine chelation. After 10 min, 59Fe radioactivity in this fraction decreased and a majority of cytosolic 59Fe was found in ferritin. These results do not support the concept of the cytosolic low-molecular-weight iron pool as a kinetic intermediate between transferrin and ferritin iron in K562 cells.     

OVCAR-3 cells internalize TAT-peptide modified liposomes by endocytosis

For cytosolic delivery of liposomes containing macromolecular drugs, such as proteins or nucleic acids, it would be beneficial to bypass endocytosis to prevent degradation in the lysosomes. Recent reports pointed to the possibility that coupling of TAT-peptides to the outer surface of liposome particles would enable translocation over the cellular plasma membrane. Here, we demonstrate that cellular uptake of TAT-liposomes occurs via endocytosis rather than plasma membrane translocation. The coupling of HIV-1 derived TAT-peptide to liposomes enhances their binding to ovarian carcinoma cells. The binding was inhibited by the presence of heparin or dextran sulfate, indicating that cell surface proteoglycans are involved in the binding interaction. Furthermore, living confocal microscopy studies revealed that binding of the TAT-liposomes to the plasma membrane is followed by intracellular uptake in vesicular structures. Staining the endosomes and lysosomes demonstrated that fluorescent liposomal labels are present within the endosomal and lysosomal compartments. Furthermore, incubation at low temperature or addition of a metabolic or an endocytosis inhibitor blocked cellular uptake. In conclusion, coupling TAT-peptide to the outer surface of liposomes leads to enhanced endocytosis of the liposomes by ovarian carcinoma cells, rather than direct cytosolic delivery by plasma membrane translocation.     

Architectural remodeling of the tonoplast during fluid-phase endocytosis

During fluid phase endocytosis (FPE) in plant storage cells, the vacuole receives a considerable amount of membrane and fluid contents. If allowed to accumulate over a period of time, the enlarging tonoplast and increase in fluids would invariably disrupt the structural equilibrium of the mature cells. Therefore, a membrane retrieval process must exist that will guarantee membrane homeostasis in light of tonoplast expansion by membrane addition during FPE. We examined the morphological changes to the vacuolar structure during endocytosis in red beet hypocotyl tissue using scanning laser confocal microscopy and immunohistochemistry. The heavily pigmented storage vacuole allowed us to visualize all architectural transformations during treatment. When red beet tissue was incubated in 200 mM sucrose, a portion of the sucrose accumulated entered the cell by means of FPE. The accumulation process was accompanied by the development of vacuole-derived vesicles which transiently counterbalanced the addition of surplus endocytic membrane during rapid rates of endocytosis. Topographic fluorescent confocal micrographs showed an ensuing reduction in the size of the vacuole-derived vesicles and further suggest their reincorporation into the vacuole to maintain vacuolar unity and solute concentration.     

Membrane Expansion Increases Endocytosis Rate during Mitosis

Mitosis in mammalian cells is accompanied by a dramatic inhibition of endocytosis. We have found that the addition of amphyphilic compounds to metaphase cells increases the endocytosis rate even to interphase levels. Detergents and solvents all increased endocytosis rate, and the extent of increase was in direct proportion to the concentration added. Although the compounds could produce a variety of different effects, we have found a strong correlation with a physical alteration in the membrane tension as measured by the laser tweezers. Plasma membrane tethers formed by latex beads pull back on the beads with a force that was related to the in-plane bilayer tension and membrane– cytoskeletal adhesion. We found that as cells enter mitosis, the membrane tension rises as the endocytosis rate decreases; and as cells exited mitosis, the endocytosis rate increased as the membrane tension decreased. The addition of amphyphilic compounds decreased membrane tension and increased the endocytosis rate. With the detergent, deoxycholate, the endocytosis rate was restored to interphase levels when the membrane tension was restored to interphase levels. Although biochemical factors are clearly involved in the alterations in mitosis, we suggest that endocytosis is blocked primarily by the increase in apparent plasma membrane tension. Higher tensions inhibit both the binding of the endocytic complex to the membrane and mechanical deformation of the membrane during invagination. We suggest that membrane tension is an important regulator of the endocytosis rate and alteration of tension is sufficient to modify endocytosis rates during mitosis. Further, we postulate that the rise in membrane tension causes cell rounding and the inhibition of motility, characteristic of mitosis.     

Vesicular ATPase inserted into the plasma membrane of motor terminals by exocytosis alkalinizes cytosolic pH and facilitates endocytosis

Key components of vesicular neurotransmitter release, such as Ca(2+) influx and membrane recycling, are affected by cytosolic pH. We measured the pH-sensitive fluorescence of Yellow Fluorescent Protein transgenically expressed in mouse motor nerve terminals, and report that Ca(2+) influx elicited by action potential trains (12.5-100 Hz) evokes a biphasic pH change: a brief acidification (～ 13 nM average peak increase in [H(+)]), followed by a prolonged alkalinization (～ 30 nM peak decrease in [H(+)]) that outlasts the stimulation train. The alkalinization is selectively eliminated by blocking vesicular exocytosis with botulinum neurotoxins, and is prolonged by the endocytosis-inhibitor dynasore. Blocking H(+) pumping by vesicular H(+)-ATPase (with folimycin or bafilomycin) suppresses stimulation-induced alkalinization and reduces endocytotic uptake of FM1-43. These results suggest that H(+)-ATPase, known to transfer cytosolic H(+) into prefused vesicles, continues to extrude cytosolic H(+) after being exocytotically incorporated into the plasma membrane. The resulting cytosolic alkalinization may facilitate vesicular endocytosis.     

Voltage-dependent calcium channels at the plasma membrane, but not vesicular channels, couple exocytosis to endocytosis

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis.     

Low cytoplasmic pH inhibits endocytosis and transport from the trans- Golgi network to the cell surface

A fibroblast mutant cell line lacking the Na+/H+ antiporter was used to study the influence of low cytoplasmic pH on membrane transport in the endocytic and exocytic pathways. After being loaded with protons, the mutant cells were acidified at pH 6.2 to 6.8 for 20 min while the parent cells regulated their pH within 1 min. Cytoplasmic acidification did not affect the level of intracellular ATP or the number of clathrin-coated pits at the cell surface. However, cytosolic acidification below pH 6.8 blocked the uptake of two fluid phase markers, Lucifer Yellow and horseradish peroxidase, as well as the internalization and the recycling of transferrin. When the cytoplasmic pH was reversed to physiological values, both fluid phase endocytosis and receptor-mediated endocytosis resumed with identical kinetics. Low cytoplasmic pH also inhibited the rate of intracellular transport from the Golgi complex to the plasma membrane. This was shown in cells infected by the temperature-sensitive mutant ts 045 of the vesicular stomatitis virus (VSV) using as a marker of transport the mutated viral membrane glycoprotein (VSV-G protein). The VSV-G protein was accumulated in the trans-Golgi network (TGN) by an incubation at 19.5 degrees C and was transported to the cell surface upon shifting the temperature to 31 degrees C. This transport was arrested in acidified cells maintained at low cytosolic pH and resumed during the recovery phase of the cytosolic pH. Electron microscopy performed on epon and cryo-sections of mutant cells acidified below pH 6.8 showed that the VSV-G protein was present in the TGN. These results indicate that acidification of the cytosol to a pH less than 6.8 inhibits reversibly membrane transport in both endocytic and exocytic pathways. In all likelihood, the clathrin and nonclathrin coated vesicles that are involved in endo- and exocytosis cannot pinch off from the cell surface or from the TGN below this critical value of internal pH.     

Distinct apical and basolateral membrane requirements for stretch-induced membrane traffic at the apical surface of bladder umbrella cells

Epithelial cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are initiated and coordinated and the mechanotransduction pathways involved are not well understood. We observed that in response to a dynamic mechanical environment, increased apical membrane tension, but not pressure, stimulated apical membrane exocytosis and ion transport in bladder umbrella cells. The exocytic response was independent of temperature but required the cytoskeleton and the activity of a nonselective cation channel and the epithelial sodium channel. The subsequent increase in basolateral membrane tension had the opposite effect and triggered the compensatory endocytosis of added apical membrane, which was modulated by opening of basolateral K⁺ channels. Our results indicate that during the dynamic processes of bladder filling and voiding apical membrane dynamics depend on sequential and coordinated mechanotransduction events at both membrane domains of the umbrella cell.     

The pathway and targeting signal for delivery of the integral membrane glycoprotein LEP100 to lysosomes

A complete set of chimeras was made between the lysosomal membrane glycoprotein LEP100 and the plasma membrane-directed vesicular stomatitis virus G protein, combining a glycosylated lumenal or ectodomain, a single transmembrane domain, and a cytosolic carboxyl-terminal domain. These chimeras, the parent molecules, and a truncated form of LEP100 lacking the transmembrane and cytosolic domains were expressed in mouse L cells. Only LEP100 and chimeras that included the cytosolic 11 amino acid carboxyl terminus of LEP100 were targeted to lysosomes. The other chimeras accumulated in the plasma membrane, and truncated LEP100 was secreted. Chimeras that included the extracellular domain of vesicular stomatitis G protein and the carboxyl terminus of LEP100 were targeted to lysosomes and very rapidly degraded. Therefore, in chimera-expressing cells, virtually all the chimeric molecules were newly synthesized and still in the biosynthesis and lysosomal targeting pathways. The behavior of one of these chimeras was studied in detail. After its processing in the Golgi apparatus, the chimera entered the plasma membrane/endosome compartment and rapidly cycled between the plasma membrane and endosomes before going to lysosomes. In pulse-expression experiments, a large population of chimeric molecules was observed to appear transiently in the plasma membrane by immunofluorescence microscopy. Soon after protein synthesis was inhibited, this surface population disappeared. When lysosomal proteolysis was inhibited, chimeric molecules accumulated in lysosomes. These data suggest that the plasma membrane/early endosome compartment is on the pathway to the lysosomal membrane. This explains why mutations that block endocytosis result in the accumulation of lysosomal membrane proteins in the plasma membrane.     

Loss of membrane phospholipid asymmetry during activation of blood platelets and sickled red cells; mechanisms and physiological significance

Membrane phospholipid asymmetry is considered to be a general property of biological membranes. Detailed information is presently available on the non-random orientation of phospholipids in red cell- and platelet membranes. The outer leaflet of the lipid bilayer membrane is rich in choline-phospholipids, whereas amino-phospholipids are abundant in the inner leaflet. Studies with blood platelets have shown that these asymmetries are not maintained when the cells are activated in various ways. Undoing the normal asymmetry of membrane phospholipids in activated blood cells is presumably mediated by increased transbilayer movement of phospholipids. This process, which leads to increased exposure of negatively charged phosphatidylserine at the outer surface, plays an important physiological role in local blood clotting reactions. A similar phenomenon occurs in sickled red cells. Phospholipid vesicles breaking off from reversibly sickled cells contribute similarly to intravascular clotting in the crisis phase of sickle cell disease.The loss of membrane phospholipid asymmetry in activated platelets seems to be strictly correlated with degradation of cytoskeletal proteins by endogenous calpain. It is remarkable that membrane phospholipid asymmetry can be (partly) restored when activated platelets are treated with reducing agents. This leads to disappearance of phosphatidylserine from the outer leaflet where it was previously exposed during cell activation. These observations will be discussed in relation to two mechanisms which have been recognized to play a role in the regulation of membrane phospholipid asymmetry; i.e. the interaction of aminophospholipids to cytoskeletal proteins, and the involvement of a phospholipid-translocase catalyzing outward-inward transbilayer movement of amino-phospholipids.     

Is lipid translocation involved during endo- and exocytosis?

Stimulation of the aminophospholipid translocase, responsible for the transport of phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet of the plasma membrane, provokes endocytic-like vesicles in erythrocytes and stimulates endocytosis in K562 cells. In this article arguments are given which support the idea that the active transport of lipids could be the driving force involved in membrane folding during the early step of endocytosis. The model is sustained by experiments on shape changes of pure lipid vesicles triggered by a change in the proportion of inner and outer lipids. It is shown that the formation of microvesicles with a diameter of 100-200 nm caused by the translocation of plasma membrane lipids implies a surface tension in the whole membrane. It is likely that cytoskeleton proteins and inner organelles prevent a real cell from undergoing overall shape changes of the type seen with giant unilamellar vesicles. Another hypothesis put forward in this article is the possible implication of the phospholipid 'scramblase' during exocytosis which could favor the unfolding of microvesicles.     

Micropipette aspiration of human erythrocytes induces echinocytes via membrane phospholipid translocation.

When a discocytic erythrocyte (RBC) was partially aspirated into a 1.5-microns glass pipette with a high negative aspiration pressure (delta P = -3.9 kPa), held in the pipette for 30 s (holding time, th), and then released, it underwent a discocyte-echinocyte shape transformation. The degree of shape transformation increased with an increase in th. The echinocytes recovered spontaneously to discocytes in approximately 10 min, and there was no significant difference in recovery time at 20.9 degrees C, 29.5 degrees C, and 37.4 degrees C, respectively. At 11 degrees C the recovery time was significantly elevated to 40.1 +/- 6.7 min. At 20.9 degrees C the shape recovery time varied directly with the isotropic RBC tension induced by the pipetting. Sodium orthovanadate (vanadate, 200 microM), which inhibits the phospholipid translocase, blocks the shape recovery. Chlorpromazine (CP, 25 microM) reversed the pipette-induced echinocytic shape to discocytic in < 2 min, and the RBC became a spherostomatocyte-II after another 30 min. It was hypothesized that the increase in cytosolic pressure during the pipette aspiration induced an isotropic tension in the RBC membrane followed by a net inside-to-outside membrane lipid translocation. After a sudden release of the aspiration pressure the cytosolic pressure and the membrane tension normalized immediately, but the translocated phospholipids remained temporarily "trapped" in the outer layer, causing an area excess and hence the echinocytic shape. The phospholipid translocase activity, when not inhibited by vanadate, caused a gradual return of the translocated phospholipids to the inner layer, and the RBC shape recovered with time.     

Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation

Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7 M serine and 0.3 M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays.     

Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosylphosphorylcholine (C(6)-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca(2+) ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-beta-cyclodextrin, which is indicative of a loss of tight SM-cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C(6)- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.     

Editorial

Stimulated secretion in endocrine cells and neuronal synapses causes a rise in endocytosis rates to recover the added membrane. The endocytic process involves the mechanical deformation of the membrane to produce an invagination. Studies of osmotic swelling effects on endocytosis indicate that the increased surface tension is tightly correlated to a significant decrease of endocytosis. When rat basophilic leukemia (RBL) cells are stimulated to secrete, there is a dramatic drop in the membrane tension and only small changes in membrane bending stiffness. Neither the shape change that normally accompanies secretion nor the binding of ligand without secretion causes a drop in tension. Further, tension decreases within 6 s, preceding shape change and measurable changes in endocytosis. After secretion stops, tension recovers. On the basis of these results we suggest that the physical parameter of membrane tension is a major regulator of endocytic rate in RBL cells. Low tensions would stimulate endocytosis and high tensions would stall the endocytic machinery.     

Role of HRB in clathrin-dependent endocytosis

Human immunodeficiency virus Rev-binding protein (HRB), also called human Rev-interacting protein (hRIP) or Rev/Rex activation domain binding (RAB) is a partner of the tyrosine kinase substrate EPS15, and it has been recovered in the AP-2 interactome. EPS15 and AP-2 are involved in endocytosis, but the function of HRB in this process is still unknown. Here we identified HRB as a partner of the vesicular SNARE tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) in yeast two-hybrid screens and using biochemical assays. In HeLa cells, HRB localized both in the nucleus and in the cytoplasm. In the cytoplasm, HRB colocalized with clathrin-, AP-2-, EPS15-, and transferrin receptor-containing vesicles. We did not see significant colocalization between HRB and TI-VAMP in HeLa cells, and we saw partial colocalization with green fluorescent protein-TI-VAMP in stably expressing Madin-Darby canine kidney cells. Nevertheless using a pHLuorin-tagged TI-VAMP construct, we found that HRB and TI-VAMP colocalize close to the plasma membrane after 5 min of anti-green fluorescent protein antibody uptake. These results suggest that TI-VAMP and HRB may interact only during the early stages of endocytosis. Furthermore uptake experiments followed by fluorescence-activated cell sorting showed that the endocytosis of fluorescent transferrin and pHLuorin-TI-VAMP is strongly reduced in HRB knockdown cells. Altogether these results suggest that HRB is involved in clathrin-dependent endocytosis and recruits TI-VAMP in this process.     

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.      

Regulation of caveolar endocytosis by syntaxin 6-dependent delivery of membrane components to the cell surface

Caveolar endocytosis has an important function in the cellular uptake of some bacterial toxins, viruses and circulating proteins. However, the molecular machinery involved in regulating caveolar uptake is poorly defined. Here, we demonstrate that caveolar endocytosis is regulated by syntaxin 6, a target membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) involved in membrane fusion events along the secretory pathway. When syntaxin 6 function was inhibited, internalization through caveolae was dramatically reduced, whereas other endocytic mechanisms were unaffected. Syntaxin 6 inhibition also reduced the presence of caveolin-1 and caveolae at the plasma membrane. In addition, syntaxin 6 inhibition decreased the delivery of GM1 ganglioside (GM1) and glycosylphosphatidylinositol (GPI)-GFP (but not vesicular stomatitis virus-glycoprotein G; VSV-G) protein from the Golgi complex to the plasma membrane. Addition of GM1 to syntaxin 6-inhibited cells resulted in the reappearance of caveolin-1 and caveolae at the plasma membrane, and restored caveolar uptake. These results suggest that syntaxin 6 regulates the delivery of microdomain-associated lipids and proteins to the cell surface, which are required for caveolar endocytosis.