Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast

Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.     

受体介导式入胞的分子机理(2)

早期内体除了接受来自细胞膜的囊泡之外,还接受来自高尔基体和晚期内体的囊泡,并且将内体中的物质以囊泡的形式重新分配到细胞膜、高尔基体和晚期内体(如图1,见本刊第11期第13页),这种重新分配也称分选。例如,细胞膜上的脂质和膜蛋白能够被早期内体分选,一部分回到胞膜重新利用,另一部分被转运到溶酶体降解。     

The receptor-mediated endocytosis of nonspherical particles

Enveloped viruses and nanosized biomimetic particles for drug and gene delivery enter target cells mainly through receptor-mediated endocytosis. A few models have been presented to elucidate the mechanics of particle engulfment by the cell membrane, showing how size and surface chemico-physical properties favor or oppose internalization. In this work, the effect of particle nonsphericity is addressed considering elliptical cylindrical particles with aspect ratio Γ. Using a continuum energetic approach, three different conditions have been identified: for sufficiently small Γ, the particle is not even wrapped by the cell membrane; for sufficiently large Γ, the particle is partially wrapped (“frustrated endocytosis”); and for intermediate values of Γ, the particle is fully wrapped and eventually internalized. Given the pleomorphism of viruses and the broad spectrum of shapes for nanosized biomimetic particles, the results presented may be of interest to virologists, pharmacologists, toxicologists, and nanotechnologists.     

Role for phospholipase D in receptor-mediated endocytosis

In response to epidermal growth factor (EGF), the EGF receptor is endocytosed and degraded. A substantial lag period exists between endocytosis and degradation, suggesting that endocytosis is more than a simple negative feedback. Phospholipase D (PLD), which has been implicated in vesicle formation in the Golgi, is activated in response to EGF and other growth factors. We report here that EGF receptor endocytosis is dependent upon PLD and the PLD1 regulators, protein kinase C alpha and RalA. EGF-induced receptor degradation is accelerated by overexpression of either wild-type PLD1 or PLD2 and retarded by overexpression of catalytically inactive mutants of either PLD1 or PLD2. EGF-induced activation of mitogen-activated protein kinase, which is dependent upon receptor endocytosis, is also dependent upon PLD. These data suggest a role for PLD in signaling that facilitates receptor endocytosis.     

Inhibitory role of endophilin 3 in receptor-mediated endocytosis

Endophilin 1 (Endo1) participates in synaptic vesicle biogenesis through interactions of its Src homology 3 domain with the polyphosphoinositide phosphatase Synaptojanin and the GTPase Dynamin. Endo1 has also been reported to affect endocytosis by converting membrane curvature via its lysophosphatidic acid acyltransferase activity. Here we report that a closely related isoform of Endo1, Endo3, inhibits clathrin-mediated endocytosis. Mutational analyses showed that the variable region of Endo3 is important in regulating transferrin endocytosis. In the brain, Endo3 is co-localized with dopamine D2 receptor in olfactory nerve terminals and inhibits its clathrin-mediated endocytosis in COS-7 cells. Furthermore, overexpression of Endo3 in an olfactory epithelium-derived cell line suppressed dopamine D2 receptor-mediated endocytosis and therefore accelerated its dopamine-induced differentiation. These results indicate that Endo3 may act as a negative regulator of clathrin-mediated endocytosis in brain neurons.     

Regulation of Wnt signalling by receptor-mediated endocytosis

Wnts compromise a large family of secreted and hydrophobic glycoproteins that control a variety of developmental and adult processes in all metazoan organisms. Recent advances in the field of Wnt signalling have revealed that Wnt activates multiple intracellular cascades, resulting in the regulation of cellular proliferation, differentiation, migration and polarity. However, it is not clear how Wnt activates these pathways after it binds to the receptors. It has been shown that Wnt and its antagonist Dickkopf are internalized with their receptors. This review highlights distinct endocytic pathways correlate with specificity of Wnt signalling events.     

Clathrin-mediated endocytosis in budding yeast

Clathrin-mediated endocytosis in the budding yeast Saccharomyces cerevisiae involves the ordered recruitment, activity and disassembly of nearly 60 proteins at distinct sites on the plasma membrane. Two-color live-cell fluorescence microscopy has proven to be invaluable for in vivo analysis of endocytic proteins: identifying new components, determining the order of protein arrival and dissociation, and revealing even very subtle mutant phenotypes. Yeast genetics and functional genomics facilitate identification of complex interaction networks between endocytic proteins and their regulators. Quantitative datasets produced by these various analyses have made theoretical modeling possible. Here, we discuss recent findings on budding yeast endocytosis that have advanced our knowledge of how -60 endocytic proteins are recruited, perform their functions, are regulated by lipid and protein modifications, and are disassembled, all with remarkable regularity.     

Coated vesicles participate in the receptor-mediated endocytosis of insulin

We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the high-affinity insulin receptor present in a variety of tissues. We isolated liver coated vesicles from rats which had received injections of 125I-insulin in the hepatic portal vein. We found that insulin administered in this fashion was rapidly and specifically taken up by liver coated vesicles. Taken together, these data are compatible with a functional role for coated vesicles in the receptor-mediated endocytosis of insulin.     

The crossroads of receptor-mediated signaling and endocytosis in plants

Plants deploy numerous plasma membrane receptors to sense and rapidly react to environmental changes. Correct localization and adequate protein levels of the cell-surface receptors are critical for signaling activation and modulation of plant development and defense against pathogens. After ligand binding, receptors are internalized for degradation and signaling attenuation. However, one emerging notion is that the ligand-induced endocytosis of receptor complexes is important for the signal duration, ampli tude, and specificity. Recently, mutants of major endocytosis players, including clathrin and dynamin have been shown to display defects in activation of a subset of signal transduction pathways, implying that signaling in plants might not be solely restricted to the plasma membrane. Here, we summarize the up-to-date knowledge of receptor complex endocytosis and its effect on the signaling outcome, in the context of plant development and immunity.     

An enzymatic method to determine receptor-mediated endocytosis

Many studies have measured receptor-mediated endocytosis using radiolabeled ligands or antibodies. Upon ligation and cross-linking, the labeled ligand or antibody is endocytosed and the internalization of the radioisotope is assayed after stripping the uninternalized ligand from the cell membrane. This study reports on an enzymatic assay to measure receptor-mediated endocytosis and compares it with the radioactive method. The results show that receptor-mediated endocytosis measured using the peroxidase conjugated antibody is two fold higher than that measured with a radiolabeled antibody. Thus, approximately 38% endocytosis of CD3 is measured using an ¹²⁵I-labeled antibody, whereas approximately 79% endocytosis is detected by peroxidase conjugated antibody method. Similar increases are also found with CD2 receptor-mediated endocytosis. Our study has demonstrated that the enzymatic method could be employed in determining receptor-mediated endocytosis. In addition to increased sensitivity, the enzymatic assay eliminates the use of radioactive materials.     

Monensin inhibits receptor-mediated endocytosis of asialoglycoproteins in rat hepatocytes

Isolated rat liver parenchymal cells incubated in the presence of monensin exhibited a reduced uptake of ¹²⁵I-asialofetuin (¹²⁵I-AF). Binding studies indicated that the effect was due to a rapid reduction in the number of active surface receptors for the asialoglycoprotein. Monensin had no effect on receptor internalization, but apparently interrupted the recycling of receptors back to the cell surface. Monensin also inhibited the degradation of ¹²⁵I-AF previously bound to the cells; this inhibition was probably not due to a direct effect on intralysosomal proteolysis, as no lysosomal accumulation of undegraded ligand could be demonstrated in subcellular fractionation studies by means of sucrose gradients. It is more likely that monensin inhibits transfer of the labelled ligand from endocytic vesicles to lysosomes, as indicated by the accumulation of radioactivity in the former and by the ability of monensin to prevent the normally observed time-dependent increase in the buoyant density of endocytic vesicles. Whereas the effect of monensin on binding and uptake of asialofetuin was reversible, the effect on asialofetuin degradation could not be reversed.     

Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes.

Endocytosis may originate both in coated pits and in uncoated regions of the plasma membrane. In hepatocytes it has been shown that fluid phase endocytosis (here defined as 'pinocytosis') is unaffected by treatments that arrest coated pit-mediated endocytosis, indicating that pinocytosis is primarily a clathrin-independent process. In this study we have tried to determine possible connections between pinocytosis and clathrin-dependent endocytosis in rat hepatocytes by means of subcellular fractionation, electron microscopy, and by assessing the influence of inhibitors of clathrin-dependent endocytosis on pinocytosis. As marker for clathrin-dependent endocytosis was used asialoorosomucoid (AOM) labelled with [(125)I]tyramine cellobiose ([(125)I]TC). [(125)I]TC-labelled bovine serum albumin ([(125)I]TC-BSA) was found to be a useful marker for pinocytosis. Its uptake in the cells is not saturable, and any remnants of [(125)I]TC-BSA associated with the cell surface could be removed by incubating the cells with 0.3% pronase at 0 degrees C for 60 min. The data obtained by electron microscopy and by subcellular fractionation suggested that early after initiation of uptake (<15 min) [(125)I]TC-BSA and [(125)I]TC-AOM were present in different endocytic vesicles. The two probes probably join prior to their entrance in the lysosomal compartment. The relation between endocytosis via coated pits and pinocytosis was also studied with techniques that induced a selective density shift either in the clathrin-dependent pathway (by AOM-HRP) or in the pinocytic pathway (by allowing uptake of AuBSA). Both treatments indicated that the two probes ([(125)I]TC-AOM and [(125)I]TC-BSA) were early after uptake, at least partly, in separate endocytic compartments. The different distribution of the fluid phase marker and the ligand (internalised via coated pits) was not due to a difference in the rate at which they enter a later compartment, since a lowering of the incubation temperature to 18 degrees C, which should keep the probes in the early endosomes, did not affect their early density distribution. Incubation of cells in a hypertonic medium reduced uptake both of [(125)I]TC-AOM and [(125)I]TC-BSA; the uptake of [(125)I]TC-AOM was, however, reduced much more than that of the fluid phase marker. This finding supports the notion that the two probes enter the cells via different routes.     

ATP is required for receptor-mediated endocytosis in intact cells

We have demonstrated a requirement for cellular ATP in the receptor- mediated endocytosis of transferrin. This has been accomplished using a novel assay for endocytosis based on acquisition of resistance to the membrane impermeable reducing agent, glutathione (GSH). Diferric- transferrin was conjugated to biotin via a cleavable disulfide bond and iodinated. Internalization of 125I-biotin-S-S-transferrin (125I-BSST) was quantitated by adsorption to avidin-Sepharose after treatment of cells with GSH. Receptor-mediated endocytosis of 125I-BSST was severely inhibited in ATP-depleted cells. Similar results were obtained when ATP was depleted by incubation of cells either under a N2-atmosphere or in the presence of NaN3 and NaF. The latter treatment, alone, also resulted in a loss of surface transferrin receptors which could not be correlated to reductions in cellular ATP. In contrast to the acquisition of GSH resistance, the apparent internalization of 125I- BSST as assessed by inaccessibility to antitransferrin antibodies reached control levels in ATP-depleted cells. Our biochemical and morphological data suggested that, although ATP is required for receptor-mediated endocytosis, in ATP-depleted cells ligands can become efficiently sequestered into deeply invaginated pits that are inaccessible to large probes such as antibodies, but remain accessible to small molecules such as GSH.     

Lipophorin receptor-mediated lipoprotein endocytosis in insect fat body cells

High-density lipophorin (HDLp) in the circulation of insects is able to selectively deliver lipids to target tissues in a nonendocytic manner. In Locusta migratoria, a member of the LDL receptor family has been identified and shown to mediate endocytosis of HDLp in mammalian cells transfected with the cDNA of this receptor. This insect lipophorin receptor (iLR) is temporally expressed in fat body tissue of young adult as well as larval locusts, as shown by Western blot analysis. Fluorescence microscopy revealed that fat body cells internalize fluorescently labeled HDLp and human receptor-associated protein only when iLR is expressed. Expression of iLR is down-regulated on Day 4 after an ecdysis. Consequently, HDLp is no longer internalized. By starving adult locusts immediately after ecdysis, we were able to prolong iLR expression. In addition, expression of the receptor was induced by starving adults after down-regulation of iLR. These results suggest that iLR mediates endocytosis of HDLp in fat body cells, and that expression of iLR is regulated by the demand of fat body tissue for lipids.     

The proteasome regulates receptor-mediated endocytosis of interleukin-2

Recent studies have increasingly implicated the proteasome in the regulation of cell surface receptors. In the present study, we investigated the role of the proteasome for ligand-dependent endocytosis and degradation of the interleukin-2 (IL-2)-interleukin-2 receptor (IL-2R) complex. Proteasome inhibitors impaired internalization of IL-2.IL-2R and prevented the lysosomal degradation of this cytokine. Based on time-course studies, proteasome activity is primarily required after initial endocytosis of the IL-2.IL-2R. Proteasome function was also necessary for the lysosomal degradation of IL-2 internalized by IL-2R that were comprised of cytoplasmic tailless beta- or gamma c-subunits, suggesting that the target protein for the proteasome is independent of either the cytoplasmic tail of the IL-2R beta- or gamma c-subunits and their associated signaling components. Therefore, a functional proteasome is required for optimal endocytosis of the IL-2R/ligand complex and is essential for the subsequent lysosomal degradation of IL-2, possibly by regulating trafficking to the lysosome.     

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.     

In vitro fusion of endosomes following receptor-mediated endocytosis

Receptor-mediated endocytosis and receptor recycling involve a series of intracellular membrane fusion events that appear to play an important role in the regulation of the overall rate and efficiency of the process. An endosome-endosome fusion assay is described using two ligands that (i) rapidly and efficiently enter the endosomal compartment via the macrophage mannose receptor and (ii) that mutually recognize each other. Dinitrophenol-derivatized beta-glucuronidase (DNP-beta-glucuronidase), a ligand for the mannose receptor, was endocytosed by one population of J774 E clone cells, and mannose-derivatized monoclonal anti-DNP IgG (Man-IgG) was internalized by a second set of cells. Both ligands were localized in endosomes as determined by fractionation on Percoll gradients. Incubation of vesicles prepared from the two set of cells resulted in vesicle fusion as indicated by the formation of DNP-beta-glucuronidase-Man-IgG complexes. Under standard conditions, fusion was time-, ATP-, and temperature-dependent. KCl was required for fusion. Fusion required both cytosolic- and membrane-associated proteins. N-Ethylmaleimide treatment of cytosol inhibited fusion. Proton ionophores and amines had no effect on the fusion reaction. ATP-dependent fusion was only observed between early endocytic compartments. While in the presence of a Ca2+ chelator fusion was ATP-dependent, in its absence fusion was also observed in an ATP-independent fashion.     

Cell-free assays and the mechanism of receptor-mediated endocytosis

The increasing ease with which complex cellular functions can be observed and cell-free systems is making it possible to unravel the mechanism of receptor-mediated endocytosis.