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1.
Bretscher (1983) has shown that on uniformly spread giant HeLa cells, the receptors for low density lipoprotein (LDL) and transferrin are concentrated toward the periphery of the cells. To explain these nonuniform distributions, he proposed that on giant HeLa cells, recycling receptors return to the cell surface at the cell's leading edge. Since the distribution of coated pits on these cells is uniform, Bretscher and Thomson (1983) proposed that there is a bulk membrane flow toward the cell centers. Here we present a mathematical model that allows us to predict the distribution of cell surface proteins on a thin circular cell, when exocytosis occurs at the cell periphery and endocytosis occurs uniformly over the cell surface. We show that on such a cell, a bulk membrane flow will be generated, whose average velocity is zero at the cell center and increases linearly with the distance from the cell center. Our model predicts that proteins that aggregate in coated pits will have concentrations that are maximal at the cell periphery. We fit our theory to the data of Bretscher and Thomson (1983) on the distribution of ferritin receptors for the following cases: the receptors move by diffusion alone; they move by bulk membrane flow alone; they move by a combination of diffusion and bulk membrane flow. From our fits we show that tau m greater than 3.5 tau p, where tau m and tau p are the lifetimes of the membrane and the ferritin receptor on the cell surface, and that tau pD less than 6.9 X 10(-7) cm2, where D is the ferritin receptor diffusion coefficient. Surprisingly, we obtain the best fits to the data when we neglect membrane flow. Our model predicts that for proteins that are excluded from coated pits, the protein concentration will be Gaussian, being maximal at the cell center and decreasing with the distance from the cell center. If on giant HeLa cells a protein with such a distribution could be found, it would strongly support Bretcher's proposal that there is an inward membrane flow.  相似文献   

2.
Earlier studies have shown that transferrin binds to specific receptors on the reticulocyte surface, clusters in coated pits and is then internalized via endocytic vesicles. Guinea-pig reticulocytes also have specific receptors for ferritin. In this paper ferritin and transferrin endocytosis by guinea-pig reticulocytes was studied by electron microscopy using the natural electron density of ferritin and colloidal gold-transferrin (AuTf). At 4 degrees C both ligands bound to the cell surface. At 37 degrees C progressive uptake occurred by endocytosis. AuTf and ferritin clustered in the same coated pits and small intracellular vesicles. After 60 min incubations the ligands colocalized to large multivesicular endosomes (MVE), still membrane-bound. MVE subsequently fused with the plasma membrane and released AuTf, ferritin and inclusions by exocytosis. All endocytic structures labelled with AuTf contained ferritin, but 23 to 35% of ferritin-labelled endocytic structures contained no AuTf. These data suggest that ferritin and transferrin are internalized through the same pathway involving receptors, coated pits and vesicles, but that these proteins are recycled only partly in common.  相似文献   

3.
An isolated perfused liver system was used to study the distribution of asialoglycoprotein (ASGP) binding sites on rat hepatocyte cell surfaces. The number of surface receptors was quantitated by monitoring clearance of 125I-labeled ligands from the perfusate medium under two conditions that blocked their internalization: low temperature (less than 5 degrees C) or brief formaldehyde fixation. The cell surface distribution of binding sites was visualized in the electron microscope with either asialoorosomucoid covalently coupled to horseradish peroxidase (ASOR-HRP) or lactosaminated ferritin (Lac-Fer), both of which were bound with similar kinetics and to similar extents as ASOR itself. At low temperature or after prefixation, ASGP binding sites were present over much of the sinusoidal cell surface, but were concentrated most heavily over coated pits. Quantitation of ligand distribution at 4 degrees C with Lac-Fer gave an approximately 70-fold greater density of ferritin particles over coated membrane than over uncoated regions. We obtained no evidence for gradual movement of ASGP receptors into or out of coated pits within the time-course of our experiments. Finally, the number and distribution of cell surface binding sites was unaffected by previous exposure to ASOR or by inhibition of endocytic vesicle-lysosome fusion and ASOR degradation at 16 degrees C.  相似文献   

4.
Summary Using a direct conjugate of urokinase and ferritin, the binding has been followed at the plasma membrane and the internalization of urokinase into BALB/C-3T3 fibroblasts, cultured in plasminogen-free conditions. At 0° C, the conjugate was observed bound on both coated and uncoated cell surface regions as singlets, and small and large clusters. No binding was observed in the presence of excess native urokinase. The binding was impaired by preincubation of the conjugate with a competitive inhibitor of the catalytic site, suggesting an interaction between the receptor and the catalytic site of the enzyme.Within 1 min at 37° C, urokinase clustered on coated regions of the plasma membrane. At 5 min after warming, ferritin was found on deeply indented coated pits and in both coated and uncoated vesicles close to the cell surface. By 10 min at 37° C, ferritin particles were present in uncoated endosomes and in multivesicular bodies in the Golgi area. Within 10 min, the receptors on the surface strongly decreased. New receptors were observed on the membrane after 20 min at 37° C. At this time, ferritin was observed both in endosomes or multivesicular bodies and in vesicles close to the plasma membrane.  相似文献   

5.
Concentrative receptor-mediated endocytosis of many specific ligands by cultured fibroblasts occurs through the coated pit-receptosome pathway. The formation of receptosomes was studied using two impermeant electron-dense labels for the cell surface, ruthenium red and concanavalin A-horseradish peroxidase. These studies show that at 4 degrees C, virtually all coated structures near the plasma membrane are in communication with the cell surface, and are not isolated coated vesicles. On warming cells to 37 degrees C for only 1 minute, a major portion of these structures become cryptic, that is, not labeled by these surface markers. However, on cooling cells immediately back to 4 degrees C, virtually all of these structures are again in communication with the surface. Many images showed that membrane of these cryptic pits to be continuous with the cell surface when caught in the appropriate plane of section; often there was a very narrow entrance that excluded extracellular label. At 37 degrees C, receptosomes could be occasionally seen forming as an invagination of membrane adjacent to the coated region. Mechanisms by which receptosomes may form and other evidence demonstrating the failure of coated pits to pinch off to form isolated coated vesicles during endocytosis are discussed.  相似文献   

6.
Transferrin receptor and its recycling in HeLa cells.   总被引:44,自引:14,他引:30       下载免费PDF全文
The transferrin receptor is a 180 000-dalton protein which can be dissociated to two 90 000-dalton polypeptides under reducing conditions. It can be labelled by lactoperoxidase-catalysed iodination on the cell surface at 0 degree C. Trypsin digestion of labelled cells at 0 degree C can be used to degrade those receptors on the cell surface; they release a 70 000-dalton soluble fragment which binds to transferrin. When cells are labelled at 0 degree C, then warmed to 37 degrees C, the labelled receptors enter the cells and become trypsin resistant. These receptors enter the cells, probably via coated pits, with a half-life of approximately 5 min. Since there is about three times as much receptor inside cells as on the surface, this means that transit through the cell to the cell surface takes approximately 21 min, if all receptors are on the same cycling pathway.  相似文献   

7.
Coated pits trap cell surface receptors and mediate their internalization. Once internalized, many receptors recycle back to the cell surface. When recycled receptors are inserted into the plasma membrane, they move until they are again trapped in coated pits. The mechanisms for moving receptors from their insertion sites to coated pits are unknown. Unaided diffusion as the transport mechanism is consistent with the observed kinetics of receptor recycling. Another candidate for the transport mechanism is convection. For receptors that recycle to random positions on the cell surface, or to restricted regions about coated pits, we assess the importance of convective flow in the transport of receptors to coated pits. First we consider local flows set up by the formation of coated pits and their transformation into coated vesicles. As coated pits form and round into coated vesicles, surrounding membrane is drawn inward, creating flows directed toward the coated pit centers. We show that unless the lifetime of a coated pit is very short, 10 s or less, such local flows have a negligible effect on the time it takes receptors to reach coated pits. We also show that they are unlikely to be the mechanism that keeps receptors that have reached coated pits trapped within coated pits until they are internalized. Finally we calculate the mean time tau for a diffusing receptor to reach a coated pit in the presence of membrane flow that is constant in magnitude and direction, as may occur on moving cells. We show that for typical membrane flow velocities, tau can be reduced significantly from its value in the absence of flow. For example, a velocity v = 2.8 micron/min cuts the mean transport time in half.  相似文献   

8.
We have examined the shape and distribution of clathrin-coated pits in Swiss 3T3 cells at 4 or 37 degrees C using electron microscopy with serial sections and immunofluorescence light microscopy. Both groups were fixed in glutaraldehyde and preserved further using a membrane contrast enhancement technique consisting of sequential osmium-ferrocyanide, thiocarbohydrazide and osmium-ferrocyanide treatment in situ. Concanavalin A-horseradish peroxidase (conA-HRP) was used to identify these structures participating in endocytosis. Two hundred twenty-two clathrin-coated structures were analysed; 126 from cells fixed at 4 degrees C, and 96 from cells fixed after a 3 min warm-up to 37 degrees C. All coated structures labeled with conA-HRP had demonstrable connections to the plasma membrane. These coated structures were morphologically classified into three categories: (a) flat pits; (b) curved pits; and (c) pits with narrow-neck connections to the plasma membrane. At 37 degrees C, 27% of coated pits had narrow neck connections to the plasma membrane whereas at 4 degrees C only 1% had such connections. Receptosomes (endosomes) labeled with conA-HRP were found only after incubation at 37 degrees C, indicating that active endocytosis was occurring in cells at 37 degrees C, but not at 4 degrees C. Immunofluorescence with anti-clathrin antibody was used to quantitate the number of clathrin-coated pits in Swiss 3T3 cells incubated at 4 and 37 degrees C prior to fixation. No difference was detected. There were 426 +/- 122 pits per cell at 37 degrees C and 441 +/- 106 at 4 degrees C. These results support the hypothesis that formation of a narrow neck connected a coated pit to the cell surface is an early step in the mechanism of receptor-mediated endocytosis.  相似文献   

9.
When human erythroleukemic cells are induced to differentiate, they produce globin and redistribute glycophorin and spectrin to one pole of the cell. This process was accompanied by an alteration in the clathrin-coated pits at the cell surface. In nondifferentiating cells, receptors for Concanavalin A have been shown, using electron microscopy, to be concentrated into coated pits and rapidly internalized. Glycophorin was also internalized via coated pits, but was not greatly concentrated into these portions of the surface membrane. Ligands attached to glycophorin were, therefore, cleared from the cell surface more slowly than Concanavalin A. In nondifferentiating cells, immunoelectron microscopy showed that spectrin is largely excluded from coated pits. After erythroid differentiation proceeded for several days, glycophorin was totally excluded from the coated pits along with spectrin. This did not reflect a general cessation of endocytosis, however, because Concanavalin A receptors continued to be internalized. It is possible that the specific exclusion of glycophorin from coated pits is part of the remodeling process that occurs when the precursor cell membrane differentiates into that of the mature erythrocyte.  相似文献   

10.
Recent experiments suggest that low density lipoprotein (LDL) receptors on human fibroblasts are not inserted into the plasma membrane uniformly, as earlier experiments indicated, but are inserted into specialized regions, called plaques, where coated pits form. If the consequent reduction in the time required for LDL receptors to diffuse to coated pits were significant, this could alter conclusions drawn from previous calculations based on the assumption that LDL receptors are inserted uniformly. In particular, the conclusion could be wrong that diffusion of LDL receptors to coated pits is the rate limiting step in the interaction of cell surface LDL receptors with coated pits. Here we calculate the extent of the reduction in mean travel time of an LDL receptor to a coated pit, as a function of the plaque radius. We find that only if LDL receptor insertion is limited to a very small portion of the plasma membrane near coated pit sites is there a substantial decrease in the average time it would take an LDL receptor to diffuse to a coated pit. In order for preferential insertion of LDL receptors into plaques to cut the mean receptor travel time in half, plaques would have to take up no more than 10% of the cell surface area; to reduce the travel time by a factor of 10, plaques would have to cover only 2% of the cell surface, approximately twice the area covered by coated pits at 37 degrees C.  相似文献   

11.
Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.  相似文献   

12.
A variety of receptors are known to aggregate in specialized cell surface structures called coated pits, prior to being internalized when the coated pits close off. At 37 degrees C on human fibroblasts, as well as on other cell types, a recycling process maintains a constant number of coated pits on the cell surface. In this paper, we explore implications for receptor aggregation and internalization of the two types of recycling models that have been proposed for the maintenance of the coated pit concentration. In one model, coated pits alternate between accessible and inaccessible states at fixed locations on the cell surface, while in the other model, coated pits recycle to random locations on the cell surface. We consider receptors that are randomly inserted in the membrane, move by pure diffusion with diffusion coefficient D, and are instantly and irreversibly trapped when they reach a coated pit boundary (the diffusion limit). For such receptors, we calculate for each of the two models: the mean time tau to reach a coated pit, the forward rate constant k+ for the interaction of a receptor with a coated pit, and the fraction phi of receptors aggregated in coated pits. We show that for the parameters that characterize coated pits on human fibroblasts, the way in which coated pits return to the surface has a negligible effect on the values of tau, k+, and phi for mobile receptors, D greater than or equal to 1.0 X 10(-11) cm2/s, but has a substantial effect for "immobile" receptors, D much less than 1 X 10(-11) cm2/s. We present numerical examples to show that it may be possible to distinguish between these models if one can monitor slowly diffusing receptors (D less than 1 X 10(-11) cm2/s) on cells whose coated pits have relatively short lifetimes (less than or equal to 1 min). Finally, we show that for the low-density lipoprotein (LDL) receptor on human fibroblasts (D = 4.5 X 10(-11) cm2/s), the predicted and observed values of K+ and phi are in close agreement. Therefore, even for slowly diffusing LDL receptor, unaided diffusion as the transport mechanism of receptors to coated pits is consistent with measured rates of LDL internalization.  相似文献   

13.
We have examined, by analyzing thin (15-20 nm) serial sections, whether coated pits involved in adsorptive pinocytosis in cultured fibroblasts give rise to free coated vesicles or represent permanently surface-associated structures from the neck of which uncoated receptosomes pinch off and carry ligand into the cell. Human skin fibroblasts and mouse L-929 fibroblasts were incubated with cationized ferritin (CF), a ligand known to bind to coated pit regions, at 37 degrees C before fixation. In thin sections, CF was found in coated vesicular profiles within the cytoplasm. Serial sections revealed that whereas many of these coated profiles communicated with the cell surface, thus representing pits, about 10% in L-cells and 36% in skin fibroblasts were actually free coated vesicles. Moreover, evidence for uncoated vesicular structures (receptosomes) budding off from the coated pits was not obtained. We therefore conclude that coated pits do pinch off from the plasma membrane to form free, coated vesicles (pinosomes).  相似文献   

14.
Electron microscopy and serial sections were used to examine the shape of clathrin-coated pits in sinusoidal endothelial cells of rat livers. Livers were perfused at 4 degrees C with either concanavalin A-horseradish peroxidase (conA-HRP), or HRP alone, followed by warm-up to 37 degrees C and fixation with glutaraldehyde. Alternatively, the livers were perfused with HRP at 37 degrees C, followed by fixation. All tissue was preserved using a membrane contrast enhancement technique (R-OTO) consisting of sequential osmium-ferrocyanide, thiocarbohydrazide, and osmium-ferrocyanide treatment. Peroxidase reaction product was used to identify structures participating in endocytosis. One hundred and ninety-three clathrin-coated structures were examined. Sixty-six were from livers perfused with conA-HRP at 4 degrees C, 63 were from livers perfused with only HRP at 4 degrees C, and 64 were from livers perfused with HRP at 37 degrees C. These coated structures were morphologically classified into three categories: (a) flat pits; (b) cup-shaped pits; (c) pits with a narrow neck. No isolated coated vesicles were found. In cells perfused at 4 degrees C followed by warming to 37 degrees C, the percentage of coated pits found connected to the cell surface by narrow necks was 31%, using conA-HRP, and 27% using HRP alone. In cells perfused continuously at 37 degrees C, the percentage of coated pits with narrow neck connections was 21% using HRP alone. These results suggest that the formation of coated pits connected to the surface by narrow necks is not an artifact of cell type, of experimental protocol or of incubation with a lectin.  相似文献   

15.
The entry of fowl plague virus, and avian influenza A virus, into Madin- Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 degrees C, viruses bound to the cell surface but were not internalized. Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37 degrees C, part of the virus became resistant to removal b neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium. The neuraminidase- resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0 degrees C and, after warming at 37 degrees C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles. Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures. At physiological pH, no fusion of the virus with the plasma membrane was observed. When prebound virus was incubated at a pH of 5.5 or below for 1 min at 37 degrees C, fusion was, however, detected by ferritin immunolabeling. t low multiplicity, 90% of the prebound virus became neuraminidase- resistant and was presumably fused after only 30 s at low pH. These experiments suggest that fowl plague virus enters MDCK cells by endocytosis in coated pits and coated vesicles and is transported to the lysosome where the low pH initiates a fusion reaction ultimately resulting in the transfer of the genome into the cytoplasm. The entry pathway of fowl plague virus thus resembles tht earlier described for Semliki Forest virus.  相似文献   

16.
Reconstitution of clathrin-coated pit budding from plasma membranes   总被引:16,自引:12,他引:4       下载免费PDF全文
Receptor-mediated endocytosis begins with the binding of ligand to receptors in clathrin-coated pits followed by the budding of the pits away from the membrane. We have successfully reconstituted this sequence in vitro. Highly purified plasma membranes labeled with gold were obtained by incubating cells in the presence of anti-LDL receptor IgG-gold at 4 degrees C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4 degrees C and then gently sonicating them to remove everything except the adherent membrane. Initially the gold label was clustered over flat, clathrin-coated pits. After these membranes were warmed to 37 degrees C for 5-10 min in the presence of buffer that contained cytosol extract, Ca2+, and ATP, the coated pits rounded up and budded from the membrane, leaving behind a membrane that was devoid of LDL gold. Simultaneous with the loss of the ligand, the clathrin triskelion and the AP-2 subunits of the coated pit were also lost. These results suggest that the budding of a coated pit to form a coated vesicle occurs in two steps: (a) the spontaneous rounding of the flat lattice into a highly invaginated coated pit at 37 degrees C; (b) the ATP, 150 microM Ca2+, and cytosolic factors(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.  相似文献   

17.
《The Journal of cell biology》1995,129(5):1241-1250
In polarized epithelial MDCK cells, all known endogenous endocytic receptors are found on the basolateral domain. The influenza virus hemagglutinin (HA) which is normally sorted to the apical plasma membrane, can be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomplete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the apical and basolateral surfaces of MDCK cells. The rates of internalization of HA-Y543 and HA-Y543,R546 from the basolateral surface of polarized MDCK cells resembled those in nonpolarized cells, whereas their rates of internalization from the apical cell surface were fivefold slower. However, HA-Y543,R546 was internalized approximately threefold faster than HA-Y543 at both membrane domains, indicating that apical endocytic pits in polarized MDCK cells retained the ability to discriminate between different internalization signals. Slower internalization from the apical surface could not be explained by a limiting number of coated pits: apical membrane contained 0.7 as many coated pits per cell cross-section as did basolateral membranes. 10-14% of HA-Y543 at the apical surface of polarized MDCK cells was found in coated pits, a percentage not significantly different from that observed in apical coated pits of nonpolarized MDCK cells, where internalization was fivefold faster. Thus, there was no lack of binding sites for HA-Y543 in apical coated pits of polarized cells. However, at the apical surface many more shallow pits, and fewer deep, mature pits, were observed than were seen at the basolateral. These results suggest that the slower internalization at the apical surface is due to slower maturation of coated pits, and not to a difference in recognition of internalization signals.  相似文献   

18.
We previously reported that in 3T3-L1 adipocytes 125I-insulin associates preferentially with microvilli and coated pits at low temperatures and early times of incubation. At higher temperatures it is internalized through a series of membrane limited intracellular compartments. In the present study, we used a high resolution probe, cationic ferritin (CF), to track adsorptive endocytosis in the 3T3-L1 adipocyte. We find that CF initially associates with coated pits at 2 min of incubation at 37 degrees C. With further incubation at 37 degrees C CF is internalized and after 2 to 10 min of incubation is predominantly localized to coated and non-coated clear vesicles. Approximately 50% of the apparent coated vesicles seen near the plasma membrane on single thin sections are shown by serial sectioning to be true vesicles (i.e., without a surface connection). At later time points CF is localized predominantly to lysosomal structures and, to a much smaller extent, Golgi-related structures. The remarkable similarity between 125I-insulin and CF with respect to post-binding processing suggests that while the membrane receptor confers the initial specificity, post-binding events are common for different types of ligands after they bind to cell surfaces and are subject to adsorptive endocytosis.  相似文献   

19.
Recent experiments suggest that low density lipoprotein (LDL) receptors on human fibroblasts are not inserted into the plasma membrane uniformly, as earlier experiments indicated, but are inserted into specialized regions, called plaques, where coated pits form. If the consequent reduction in the time required for LDL receptors to diffuse to coated pits were significant, this could alter conclusions drawn from previous calculations based on the assumption that LDL receptors are inserted uniformly. In particular, the conclusion could be wrong that diffusion of LDL receptors to coated pits is the rate limiting step in the interaction of cell surface LDL receptors with coated pits. Here we calculate the extent of the reduction in mean travel time of an LDL receptor to a coated pit, as a function of the plaque radius. We find that only if LDL receptor insertion is limited to a very small portion of the plasma membrane near coated pit sites is there a substantial decrease in the average time it would take an LDL receptor to diffuse to a coated pit. In order for preferential insertion of LDL receptors into plaques to cut the mean receptor travel time in half, plaques would have to take up no more than 10% of the cell surface area; to reduce the travel time by a factor of 10 plaques would have to cover only 2% of the cell surface, approximately twice the area covered by coated pits at 37°C.  相似文献   

20.
At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin- resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.  相似文献   

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