Acidification of endocytic compartments and the intracellular pathways of ligands and receptors

Several hormones, serum proteins, toxins, and viruses are brought into the cell by receptor-mediated endocytosis. Initially, many of these molecules and particles are internalized into a common endocytic compartment via the clathrin-coated pit pathway. Subsequently, the ligands and receptors are routed to several destinations, including lysosomes, the cytosol, or the plasma membrane. We have examined the mechanism by which sorting of internalized molecules occurs. A key step in the process is the rapid acidification of endocytic vesicles to a pH of 5.0-5.5 This acidification allows dissociation of several ligands from their receptors, the release of iron from transferrin, and the penetration of diphtheria toxin and some viral nucleocapsids into the cytoplasm. Transferrin, a ligand that cycles through the cell with its receptor, has been used as a marker for the recycling receptor pathway. We have found that in Chinese hamster ovary (CHO) cells transferrin is rapidly segregated from other ligands and is routed to a complex of small vesicles and/or tubules near the Golgi apparatus. The pH of the transferrin-containing compartment is approximately 6.4, indicating that it is not in continuity with the more acidic endocytic vesicles which contain ligands destined to be degraded in lysosomes.     

Analysis of intracellular receptor/ligand sorting in endosomes

After binding to specific cell surface receptors, many extracellular ligand molecules are internalized via the process termed receptor-mediated endocytosis. Within the cell, in endosomes, a sorting process occurs: receptors and ligands are directed along various intracellular pathways. The extent of this intracellular separation of receptors from ligands has been shown experimentally to vary with receptor and ligand properties such as binding affinity and valency. In this paper, we propose and analyze a simple model mechanism for the sorting process based on binding and dissociation kinetics along with diffusive molecular transport. We show that the outcome of the sorting process can be directly linked to measurable parameters such as the intrinsic rate constants for the binding to, dissociation from, and crosslinking of receptors by ligands. We further show that this mechanism is able to account for the wide range of reported experimental observations. Manipulation of ligand and receptor properties guided by the results presented here may enable the outcome of the sorting process to be controlled.     

Molecular and cellular mechanisms of receptor-mediated endocytosis.

In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors and ligands are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of certain viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Although endocytosis is common to all nucleated eukaryotic cells, the factors that regulate these receptor-mediated endocytic pathways are not fully understood. Defective receptors that are not capable of undergoing normal endocytosis can lead to certain disease states, as in the case of familial hypercholesteremia (FH). This review has three objectives: (i) to describe the different routes that receptors and ligands follow after internaliation; (ii) to describe the potential mechanisms which regulate the initiation and subsequent sorting of receptors and ligands so they reach their final destination; and (iii) to describe the potential functions of receptor-mediated endocytosis.     

Cohort movement of different ligands and receptors in the intracellular endocytic pathway of alveolar macrophages

The rate of movement of different receptors and ligands through the intracellular endocytic apparatus was studied in alveolar macrophages. Cells were exposed to iodinated alpha-macroglobulin-protease complexes, mannose terminal glycoproteins, diferric transferrin, and maleylated proteins. By use of the diaminobenzidine density shift procedure, we demonstrated that these ligands were internalized into the same endocytic vesicle. We then compared the rates of transfer to the lysosome or recycling to the cell surface of different ligands/receptors contained in the same endosome. We found that although the rate constant for degradation was ligand specific, the lag time prior to the initiation of degradation was the same for all three ligands. We also found that molecules taken up nonspecifically by fluid-phase pinocytosis had the same lag time prior to degradation as ligands internalized via receptor-mediated endocytosis. These data suggest that different molecules within the same endocytic compartment are transferred to the lysosome (or degradative compartment) at the same rate. We measured the rate of return of receptors to the cell surface by either inactivating surface receptors by protease treatment at 0 degrees C, or by incubating cells with saturating amounts of nonradioactive ligand at 37 degrees C. We then measured the rate of appearance of "new" receptors on the cell surface. Using these approaches, we found that three different receptors were transferred from internal pools to the cell surface at the same rate. The rate of transfer was independent of whether receptors were initially occupied or unoccupied. Our observations indicate that receptor/ligands, once inside alveolar macrophages, are transported by vesicles which transfer their contents as a cohort from one compartment to another. The rate of movement of these receptors is determined by the movement of vesicles and is independent of their content.     

SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors

Endocytic sorting of signalling receptors between recycling and degradative pathways is a key cellular process controlling the surface complement of receptors and, accordingly, the cell's ability to respond to specific extracellular stimuli. The β2 adrenergic receptor (β2AR) is a prototypical seven-transmembrane signalling receptor that recycles rapidly and efficiently to the plasma membrane after ligand-induced endocytosis. β2AR recycling is dependent on the receptor's carboxy-terminal PDZ ligand and Rab4. This active sorting process is required for functional resensitization of β2AR-mediated signalling. Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4. Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule. SNX27 does not seem to directly interact with the retromer core complex, but does interact with the retromer-associated Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.     

HVEM tomography of the trans-Golgi network: structural insights and identification of a lace-like vesicle coat

High voltage electron microscopy and computer axial tomography have been used to study the 3-D structure of trans-Golgi cisternae and trans- Golgi networks (TGNs) in NRK cells. Both structures were specifically labeled by photoconversion of a fluorescent analogue of ceramide using a modification of the techique of Pagano et al. (J. Cell Biol. 1991. 113: 1267-1279). Regions of the Golgi ribbon in fixed, stained cells were cut in 250-nm sections and analyzed by tilt series microscopy and subsequent tomographic reconstruction. Resolution of the reconstructions ranged from 6 to 10 nm. The size and structure of the TGN varied considerably throughout the Golgi ribbon; all reconstructions were made from regions with pronounced TGN. Most regions analyzed contained multiple (2-4) Golgi cisternae that stain with ceramide. These "peel off" from the closely stacked cisternae and are continuous at their ends with tubules that contribute to the TGN. Most vesicular profiles visualized in the TGN are connected to TGN tubules. The budding of vesicles appears to occur synchronously along the length of a TGN tubule. Two distinct coats were visualized on budding vesicles: clathrin cages and a novel, lace-like structure. Individual TGN tubules produce vesicles of only one coat type. These observations lead to the following predictions: (a) sorting of molecules must occur prior to the formation of TGN tubules; (b) vesicle formation takes place almost synchronously along a given TGN tubule; and (c) lace-like coats form an exocytic vesicles.     

Membranes of sorting organelles display lateral heterogeneity in receptor distribution

This study describes the distribution of an intrinsic membrane protein, the asialoglycoprotein receptor (ASGP-R) in the trans-Golgi reticulum and compartment of uncoupling receptor and ligand (CURL) of rat liver cells. Using quantitative immunogold electron microscopy and membrane length measurements, we showed lateral nonhomogeneity of receptors in the membranes of trans-Golgi reticulum and CURL, in particular in the membranes of secretory vesicles (identified by their content of albumin and very low density lipoprotein particles) and of CURL vesicles (endosomes), including multivesicular bodies. The characteristic tubulovesicular morphology of both sorting organelles defines the transition of receptor-rich tubular membrane and the receptor-poor limiting membrane of the attached vesicles. There was a direct relationship between the size of the secretory and CURL vesicles and the density of ASGP-Rs in their membranes. Receptor density in the smallest vesicles was similar to that found in adjacent continuous tubules. The larger the vesicles, the less receptor was detectable in their membranes. We propose that the receptor molecules are excluded from the vesicle membranes by dynamic lateral redistribution. Nonrandom receptor distribution in the CURL vesicle membranes was present even at the multivesicular body stage. These observations strongly suggest the existence of barriers to ASGP-R diffusion at the junctions of tubules and vesicles. In addition, our observations suggest that ASGP-Rs are transported to the plasma membrane via a mechanism other than the normal secretory pathway.     

Quantitative ultrastructural, autoradiographic evidence for the magnitude and early involvement of the Golgi apparatus complex in the endocytosis of wheat germ agglutinin by cultured neuroblastoma

Several ligands undergo endocytosis into the Golgi apparatus. We have examined with a quantitative ultrastructural, autoradiographic method the sequential endocytosis of tritiated wheat germ agglutinin (3H-WGA) by cultured murine neuroblastoma cells. Cells were incubated with 3H-WGA for 1 hour at 4 degrees C, washed, and incubated in complete medium without ligand at 37 degrees C for 5, 15, 30, and 120 minutes. At 5 minutes, the optimized sources/micron 2 of neuroblastoma cell area, which represented the grain density of each compartment, were as follows: smooth vesicles and tubules, 1.03 +/- 0.88; Golgi-associated vesicles, i.e., clusters of vesicles within a 1 micron radius of the Golgi cisterns, 1.03 +/- 0.31; Golgi cisterns, less than 0.01; and lysosomes, 0.26 +/- 0.16. At 15 minutes grain densities were: smooth vesicles and tubules, 0.9 +/- 0.34; Golgi-associated vesicles, 1.41 +/- 0.28; Golgi cisterns, 0.73 +/- 0.41; and lysosomes, 0.1 +/- 0.09. At 30 minutes grain densities were: smooth vesicles and tubules, 0.46 +/- 0.46; Golgi-associated vesicles, 1.78 +/- 0.34; Golgi cisterns, 0.89 +/- 0.78; and lysosomes, 0.39 +/- 0.14. At 2 hours, smooth vesicles, tubules, and Golgi cisterns were not labeled, Golgi-associated vesicles were still labeled (0.71 +/- 0.1), and lysosomes were heavily labeled (2.17 +/- 0.22). These results are consistent with the hypotheses that either the Golgi complex (cisterns and associated vesicles) is an early and intermediate step of the endocytosis of 3H-WGA into lysosomes or that it constitutes part of a separate and quantitatively significant pathway of endocytosis of this ligand.     

Induction of mutant dynamin specifically blocks endocytic coated vesicle formation

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline- inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.     

Recycling of the asialoglycoprotein receptor: biochemical and immunocytochemical evidence

One of the best documented systems of receptor-mediated endocytosis is the clearance of asialoglycoproteins (ASGP) from the blood plasma by liver parenchymal cells. There are 200 000-500 000 ligand binding sites per cell, which makes this system favourable for molecular studies of receptor function. By using both biochemical and immunocytochemical approaches, we have obtained evidence for receptor recycling. We have also localized the intracellular site at which the endocytosed receptor and ligand dissociate. The human hepatoma cell Hep G2 contains abundant ASGP receptors (approximately 225 000 per cell). In growing cells approximately 85% of the functional receptors are on the cell surface and the remaining 15% are internal. The maximal rate of ligand uptake in this cell system at 37 degrees C is approximately 30 000 molecules per cell per minute. Each functional receptor can therefore bind and internalize more than 50 ligand molecules during a 6 h period (in the absence of new receptor synthesis), or one ligand each 8 min. To follow both ligand and receptor during their common endocytosis and to visualize the compartment in which the dissociation of ligand from receptor occurs, we have used our recently developed double-labelling immunocytochemical electron microscopic techniques with purified antibodies against ASGP ligand and ASGP receptor. In normal rat hepatocytes, both ligand and receptor are taken up from the sinusoidal cell surface in clathrin-coated vesicles. Both receptor and ligand are associated with the membrane of small clathrin-coated vesicles close to the cell surface. Larger vesicles, farther removed from the surface, contain ligand accumulated within the lumen. The membranes of these larger vesicles contain little receptor, but receptor was concentrated in detached vesiculotubular extensions, which were largely free of ligand. These vesicles represent the compartment of uncoupling of receptor and ligand (CURL) during their common endocytosis. Ligand contained within the vesicle lumen is then transferred to multivesicular bodies and lysosomes; the tubular extensions may carry receptor back to the cell surface.     

A "release" protocol for isothermal titration calorimetry

Isothermal titration calorimetry (ITC) has become a standard method for investigating the binding of ligands to receptor molecules or the partitioning of solutes between water and lipid vesicles. Accordingly, solutes are mixed with membranes (or ligands with receptors), and the subsequent heats of incorporation (or binding) are measured. In this paper we derive a general formula for modeling ITC titration heats in both binding and partitioning systems that allows for the modeling of the classic incorporation or binding protocols, as well as of new protocols assessing the release of solute from previously solute-loaded vesicles (or the dissociation of ligand/receptor complexes) upon dilution. One major advantage of a simultaneous application of the incorporation/binding and release protocols is that it allows for the determination of whether a ligand is able to access the vesicle interior within the time scale of the ITC experiment. This information cannot be obtained from a classical partitioning experiment, but it must be known to determine the partition coefficient (or binding constant and stochiometry) and the transfer enthalpy. The approach is presented using the partitioning of the nonionic detergent C12EO7 to palmitoyloleoylphosphatidylcholine vesicles. The release protocol could also be advantageous in the case of receptors that are more stable in the ligand-saturated rather than the ligand-depleted state.     

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of beta2-adrenergic receptors

Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated beta(2)-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate beta(2)-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the beta-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of beta(2)-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of beta(2)-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of beta(2)-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.     

A Cholesterol‐Dependent Endocytic Mechanism Generates Midbody Tubules During Cytokinesis

Cytokinesis is the final stage of cell division and produces two independent daughter cells. Vesicles derived from internal membrane stores, such as the Golgi, lysosomes, and early and recycling endosomes accumulate at the intracellular bridge (ICB) during cytokinesis. Here, we use electron tomography to show that many ICB vesicles are not independent but connected, forming a newly described ICB vesicular structure – narrow tubules that are often branched. These ‘midbody tubules’ labelled with horseradish peroxidase (HRP) within 10 min after addition to the surrounding medium demonstrating that they are derived from endocytosis. HRP‐labelled vesicles and tubules were observed at the rim of the ICB after only 1 min, suggesting that midbody tubules are likely to be generated by local endocytosis occurring at the ICB rim. Indeed, at least one tubule was open to the extracellular space, indicative of a local origin within the ICB. Inhibition of cholesterol‐dependent endocytosis by exposure to methyl‐β‐cyclodextrin and filipin reduced formation of HRP‐labelled midbody tubules, and induced multinucleation following ICB formation. In contrast, dynamin inhibitors, which block clathrin‐mediated endocytosis, induced multinucleation but had no effect on the formation of HRP‐labelled midbody tubules. Therefore, our data reveal the existence of a cholesterol‐dependent endocytic pathway occurring locally at the ICB, which contributes to the accumulation of vesicles and tubules that contribute to the completion of cytokinesis.      

Modeling the effect of glutamate diffusion and uptake on NMDA and non-NMDA receptor saturation.

One- and two-dimensional models of glutamate diffusion, uptake, and binding in the synaptic cleft were developed to determine if the release of single vesicles of glutamate would saturate NMDA and non-NMDA receptors. Ranges of parameter values were used in the simulations to determine the conditions when saturation could occur. Single vesicles of glutamate did not saturate NMDA receptors unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. However, the release of eight vesicles at 400 Hz caused NMDA receptor saturation for all parameter values tested. Glutamate uptake was found to reduce NMDA receptor saturation, but the effect was smaller than that of changes in the diffusion coefficient or in the number of glutamate molecules in a vesicle. Non-NMDA receptors were not saturated unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. The release of eight vesicles at 400 Hz caused significant non-NMDA receptor desensitization. The results suggest that NMDA and non-NMDA receptors are not saturated by single vesicles of glutamate under usual conditions, and that tetanic input, of the type typically used to induce long-term potentiation, will increase calcium influx by increasing receptor binding as well as by reducing voltage-dependent block of NMDA receptors.     

Intracellular receptor sorting during endocytosis: comparative immunoelectron microscopy of multiple receptors in rat liver

Using double-label quantitative immunoelectron microscopy on ultrathin cryosections of rat liver, we have compared the endocytotic pathways of the receptors for asialoglycoprotein (ASGP-R), mannose-6-phosphate ligands (MP-R), and polymeric IgA (IgA-R). All three were found within the Golgi complex, along the entire plasma membrane, in coated pits and vesicles, and within a compartment of uncoupling of receptors and ligand ( CURL ). The receptors occurred randomly at the cell surface, in coated pits and vesicles. Within CURL tubules ASGP-R and MP-R were colocalized , but IgA-R and ASGP-R displayed dramatic microheterogeneity. Thus, in addition to its role in uncoupling and sorting recycling receptor from ligand, CURL serves as a compartment to segregate recycling receptor (e.g. ASGP-R) from receptor involved in transcytosis (e.g. IgA-R).     

Morphological analysis of ligand uptake and processing: the role of multivesicular endosomes and CURL in receptor-ligand processing

The receptor-mediated endocytosis and intracellular processing of transferrin and mannose receptor ligands were investigated in bone marrow-derived macrophages, fibroblasts and reticulocytes. Mannosylated bovine serum albumin (BSA) conjugated to colloidal gold (Au-man-BSA) or colloidal gold-transferrin (AuTf) were used to trace ligand processing in these cells. These ligands appeared to be processed by mechanisms similar to those observed previously with other mannose receptor and galactose receptor ligand probes. After uptake via coated pits and coated vesicles, Au-man-BSA appeared in small uncoated vesicles and tubular structures and was transferred to large, sometimes multivesicular endosomes (MVEs), which sometimes had arm-like protrusions reminiscent of CURL (compartment of uncoupling of receptor and ligand) [10, 11]. Initially these structures became increasingly multivesicular, but during longer incubations the inclusion vesicles appeared to disintegrate to leave a denser, amorphous lumen. Inclusion vesicle disintegration may result from the introduction of lysosomal enzymes into these structures. These results suggest a model for differential receptor-ligand and ligand-ligand sorting. As suggested [10, 11] membrane constituents may be recycled to the plasma membrane from the arms of CURL. Receptor-bound ligands, such as transferrin, would also recycle. The luminal contents, including dissociated ligands, other soluble proteins and inclusion vesicles (containing some membrane proteins), would target to lysosomes. This would result in the lysosomal degradation of any membrane proteins that were incorporated in the inclusion vesicle membranes.     

Coated endosomal vesicles: sorting and recycling compartment for transferrin in BHK cells.

We have investigated receptor-mediated endocytosis of transferrin (Tf) in baby hamster kidney (BHK) cells, using fluorescence and electron microscopy, and by carrying out colocalization experiments with clathrin antibodies and a fluorescently tagged glycolipid. Early during internalization, Tf was found in small vesicles (100-150 nm in diameter) located at the cell periphery. The ligand remained associated with such vesicles when the latter concentrated towards the cell center, before ending up in the juxtanuclear area. Throughout this vesicular trafficking pathway, clathrin colocalized with Tf. We conclude that Tf is processed intracellularly via small coated endosomal vesicles (CEV) and is not delivered into large tubular endosomes (CURL; compartment for uncoupling receptors and ligands), typical for ligand trafficking to lysosomes. By determining the kinetics of Tf internalization and by comparing the flow of Tf to that of a fluorescent glycolipid, it can also be concluded that CEVs display sorting and recycling properties, implying that small vesicles can be shed from or fuse with CEVs. Acidic pH does not prevent the formation of CEVs, but their intracellular movement, towards the cell center, is impeded.     

Insulin and alpha 2-macroglobulin-methylamine undergo endocytosis by different mechanisms in rat adipocytes: II. Comparison of intracellular events

A previous ultrastructural study showed that gold-labeled insulin (Au-I) and the non-hormonal ligand gold-labeled alpha-2-macroglobulin-methylamine (Au-alpha 2MGMA) underwent endocytosis by dissimilar cell surface structures on rat adipocytes. The present ultrastructural study compared the intracellular routes taken by these two ligands in adipocytes. Intracellular Au-alpha 2MGMA was initially found within apparent coated vesicles but Au-I was not, consistent with the previous demonstration that Au-alpha 2MGMA underwent endocytosis by coated pits whereas Au-I was internalized by uncoated micropinocytotic invaginations. Early in the endocytic pathway, the two ligands were segregated within separate small vesicles and tubulovesicles. Au-alpha 2MGMA was concentrated in a small number of these structures whereas Au-I was sparsely distributed among a relatively large number. Subsequently, the two endocytic pathways converged as the ligands intermingled within pale multivesicular bodies and lysosome-like structures. Au-I was less efficiently transferred to lysosomes than Au-alpha 2MGMA since a greater proportion of intracellular Au-I remained associated with small vesicles and tubulovesicles. This study indicates that early intracellular events in the endocytic pathways of insulin and alpha 2MGMA are distinct. These findings are discussed in light of the fundamentally dissimilar biological roles of these two molecules and the possible involvement of the endocytic pathway in the insulin signaling mechanism.     

Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells

Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double- immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed.     

Lipid metabolism and vesicle trafficking: more than just greasing the transport machinery.

The movement of lipids from their sites of synthesis to ultimate intracellular destinations must be coordinated with lipid metabolic pathways to ensure overall lipid homeostasis is maintained. Thus, lipids would be predicted to play regulatory roles in the movement of vesicles within cells. Recent work has highlighted how specific lipid metabolic events can affect distinct vesicle trafficking steps and has resulted in our first glimpses of how alterations in lipid metabolism participate in the regulation of intracellular vesicles. Specifically, (i) alterations in sphingolipid metabolism affect the ability of SNAREs to fuse membranes, (ii) sterols are required for efficient endocytosis, (iii) glycerophospholipids and phosphorylated phosphatidylinositols regulate Golgi-mediated vesicle transport, (iv) lipid acylation is required for efficient vesicle transport mediated membrane fission, and (v) the addition of glycosylphosphatidylinositol lipid anchors to proteins orders them into distinct domains that result in their preferential sorting from other vesicle destined protein components in the endoplasmic reticulum. This review describes the experimental evidence that demonstrates a role for lipid metabolism in the regulation of specific vesicle transport events.