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1.
Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus 总被引:50,自引:29,他引:21
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We have used Chinese hamster ovary (CHO) cells and a murine lymphoma cell line to study the recycling of the 215-kD and the 46-kD mannose 6-phosphate receptors to various regions of the Golgi to determine the site where the receptors first encounter newly synthesized lysosomal enzymes. For assessing return to the trans-most Golgi compartments containing sialyltransferase (trans-cisternae and trans-Golgi network), the oligosaccharides of receptor molecules on the cell surface were labeled with [3H]galactose at 4 degrees C. Upon warming to 37 degrees C, the [3H]galactose residues on both receptors were substituted with sialic acid with a t1/2 approximately 3 hrs. Other glycoproteins acquired sialic acid at least 8-10 times slower. Return of the receptors to the trans-Golgi cisternae containing galactosyltransferase could not be detected. Return to the cis/middle Golgi cisternae containing alpha-mannosidase I was measured by adding deoxymannojirimycin, a mannosidase I inhibitor, during the initial posttranslational passage of [3H]mannose-labeled glycoproteins through the Golgi, thereby preserving oligosaccharides which would be substrates for alpha-mannosidase I. After removal of the inhibitor, return to the early Golgi with subsequent passage through the Golgi complex was measured by determining the conversion of the oligosaccharides from high mannose to complex-type units. This conversion was very slow for the receptors and other glycoproteins (t1/2 approximately 20 h). Exposure of the receptors and other glycoproteins to the dMM-sensitive alpha-mannosidase without movement through the Golgi apparatus was determined by measuring the loss of mannose residues from these proteins. This loss was also slow. These results indicate that both Man-6-P receptors routinely return to the Golgi compartment which contains sialyltransferase and recycle through other regions of the Golgi region less frequently. We infer that the trans-Golgi network is the major site for lysosomal enzyme sorting in CHO and murine lymphoma cells. 相似文献
2.
A restricted set of apical proteins recycle through the trans-Golgi network in MDCK cells. 总被引:4,自引:1,他引:3
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Sorting of newly synthesized proteins destined for the apical plasma membrane takes place in the trans-Golgi network (TGN) in MDCK cells. This process is most likely receptor mediated and requires components that recycle between both compartments. We have developed an assay to detect apical proteins that recycle through the sialyltransferase-containing TGN. Cell surface glycoproteins were exogalactosylated apically using a mutant cell line derived from MDCK, MDCKII-RCAr. The mutant exhibits impaired galactosylation of glycoconjugates and thereby allows maximal incorporation of exogenously added galactose in the presence of galactosyltransferase. Upon reculture at 37 degrees C, a time-dependent increase of sialylated apical surface glycoproteins was observed by lectin binding as well as by the sialic acid-specific NaIO4/NaB[3H]4 labeling technique. This indicates that some galactosylated surface molecules had returned to the TGN. Recycling through the TGN was blocked, if exogalactosylated cells were incubated at 20 degrees C. Two-dimensional gel electrophoresis identified three apical proteins which recycle through the TGN, suggesting that this pathway is selective for a subset of the apical surface proteins. 相似文献
3.
Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network 总被引:6,自引:0,他引:6
By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans-Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post-endosomal, tubular reticulum that appears to be separate from the trans-most Golgi saccule. 相似文献
4.
The ability of particular cell surface glycoproteins to recycle and become
exposed to individual Golgi enzymes has been demonstrated. This study was
designed to determine whether endocytic trafficking includes significant
reentry into the overall oligosaccharide processing pathway. The Lec1
mutant of Chinese hamster ovary (CHO) cells lack N -
acetylglucosaminyltransferase I (GlcNAc-TI) activity resulting in surface
expression of incompletely processed Man5GlcNAc2 N -linked
oligosaccharides. An oligosaccharide tracer was created by exoglycosylation
of cell surface glycoproteins with purified porcine GlcNAc-TI and
UDP-[3H]GlcNAc. Upon reculturing, all cell surface glycoproteins that
acquired [3H]GlcNAc were acted upon by intracellular mannosidase II, the
next enzyme in the Golgi processing pathway of complex N -linked
oligosaccharides (t1/2= 3-4 h). That all radiolabeled cell surface
glycoproteins were included in this endocytic pathway indicates a common
intracellular compartment into which endocytosed cell surface glycoproteins
return. Significantly, no evidence was found for continued oligosaccharide
processing consistent with transit through the latter cisternae of the
Golgi apparatus. These data indicate that, although recycling plasma
membrane glycoproteins can be reexposed to individual Golgi-derived
enzymes, significant reentry into the overall contiguous processing pathway
is not evident.
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5.
Selective reentry of recycling cell surface glycoproteins to the biosynthetic pathway in human hepatocarcinoma HepG2 cells 总被引:9,自引:1,他引:8
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《The Journal of cell biology》1995,130(3):537-551
Return of cell surface glycoproteins to compartments of the secretory pathway has been examined in HepG2 cells comparing return to the trans- Golgi network (TGN), the trans/medial- and cis-Golgi. Transport to these sites was studied by example of the transferrin receptor (TfR) and the serine peptidase dipeptidylpeptidase IV (DPPIV) after labeling these proteins with the N-hydroxysulfosuccinimide ester of biotin on the cell surface. This experimental design allowed to distinguish between glycoproteins that return to these biosynthetic compartments from the cell surface and newly synthesized glycoproteins that pass these compartments during biosynthesis en route to the surface. Reentry to the TGN was measured in that surface glycoproteins were desialylated with neuraminidase and were monitored for resialylation during recycling. Return to the trans-Golgi was traced measuring the transfer of [3H]fucose residues to recycling surface proteins by fucosyltransferases. To study return to the cis-Golgi, surface proteins were metabolically labeled in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM). As a result surface proteins retained N-glycans of the oligomannosidic type. Return to the site of mannosidase I in the medial/cis-Golgi was measured monitoring conversion of these glycans to those of the complex type after washout of dMM. Our data demonstrate that DPPIV does return from the cell surface not only to the TGN, but also to the trans-Golgi thus linking the endocytic to the secretory pathway. In contrast, no reentry to sites of mannosidase I could be detected indicating that the early secretory pathway is not or is only at insignificant rates accessible to recycling DPPIV. In contrast to DPPIV, TfR was very efficiently sorted from endosomes to the cell surface and did not return to the TGN or to other biosynthetic compartments in detectable amounts, indicating that individual surface proteins are subject to different sorting mechanisms or sorting efficiencies during recycling. 相似文献
6.
R A van den Bosch A P du Maine H J Geuze A van der Ende G J Strous 《The EMBO journal》1988,7(11):3345-3351
Intracellular movement of cell surface 5'-nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5'-nucleotidase. Incubation of 125I-surface-labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5'-nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5'-nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5'-nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5'-nucleotidase is not inhibited by primaquine. 相似文献
7.
《The Journal of cell biology》1987,105(6):2655-2664
The trans-Golgi has been recognized as having a key role in terminal glycosylation and sorting of proteins. Here we show that tyrosine sulfation, a frequent modification of secretory proteins, occurs specifically in the trans-Golgi. The heavy chain of immunoglobulin M (IgM) produced by hybridoma cells was found to contain tyrosine sulfate. This finding allowed the comparison of the state of sulfation of the heavy chain with the state of processing of its N-linked oligosaccharides. First, the pre-trans-Golgi forms of the IgM heavy chain, which lacked galactose and sialic acid, were unsulfated, whereas the trans-Golgi form, identified by the presence of galactose and sialic acid, and the secreted form of the IgM heavy chain were sulfated. Second, the earliest form of the heavy chain detectable by sulfate labeling, as well as the heavy chain sulfated in a cell-free system in the absence of vesicle transport, already contained galactose and sialic acid. Third, sulfate-labeled IgM moved to the cell surface with kinetics identical to those of galactose-labeled IgM. Lastly, IgM labeled with sulfate at 20 degrees C was not transported to the cell surface at 20 degrees C but reached the cell surface at 37 degrees C. The data suggest that within the trans-Golgi, tyrosine sulfation of IgM occurred at least in part after terminal glycosylation and therefore appeared to be the last modification of this constitutively secreted protein before its exit from this compartment. Furthermore, the results establish the covalent modification of amino acid side chains as a novel function of the trans-Golgi. 相似文献
8.
Phosphorylation of the cation-independent mannose 6-phosphate receptor is closely associated with its exit from the trans-Golgi network 总被引:3,自引:0,他引:3
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《The Journal of cell biology》1993,120(1):67-75
We have previously shown that two serine residues present in two conserved regions of the bovine cation-independent mannose 6-phosphate receptor (CI-MPR) cytoplasmic domain are phosphorylated in vivo (residues 2421 and 2492 of the full length bovine CI-MPR precursor). In this study, we have used CHO cells to investigate the phosphorylation state of these two serines along the different steps of the CI-MPR exocytic and endocytic recycling pathways. Transport and phosphorylation of the CI-MPR in the biosynthetic pathway were examined using deoxymannojirimycin (dMM), a specific inhibitor of the cis-Golgi processing enzyme alpha-mannosidase I which leads to the accumulation of N-linked high mannose oligosaccharides on glycoproteins. Upon removal of dMM, normal processing to complex-type oligosaccharides (galactosylation and then sialylation) occurs on the newly synthesized glycoproteins, including the CI-MPR which could then be purified and analyzed on lectin affinity columns. Phosphorylation of the newly synthesized CI-MPR was concomitant with the sialylation of its oligosaccharides and appeared as a major albeit transient modification. Phosphorylation of the cell surface CI-MPR was examined during its endocytosis as well as its return to the Golgi using antibody tagging and exogalactosylation. The cell surface CI-MPR was not phosphorylated when it entered clathrin-coated pits or when it moved to the early and late endosomes. In contrast, the surface CI-MPR was phosphorylated when it had been resialylated upon its return to the trans-Golgi network. Subcellular fractionation experiments showed that the phosphorylated CI- MPR and the corresponding kinase were found in clathrin-coated vesicles. Collectively, these results indicate that phosphorylation of the two serines in the CI-MPR cytoplasmic domain is associated with a single step of transport of its recycling pathways and occurs when this receptor is in the trans-Golgi network and/or has left this compartment via clathrin-coated vesicles. 相似文献
9.
Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride. 总被引:39,自引:0,他引:39
Low concentrations of sodium metaperiodate induce specific oxidative cleavage of sialic acids between carbon 7 and carbon 8 or carbon 8 and carbon 9. The aldehydes formed can easily be reduced with NaB3H4 to tritiated 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid or 5-acetamido-3,5-dideoxy-L-arabino-2-octulosonic acid. At 0 degrees, the periodate anion penetrates the cell plasma membrane very slowly and only externally exposed sialic acids are oxidized. This was shown by (a) limited labeling of the sialoglycoproteins in a preparation of inside-out erythrocyte vesicles; (b) trapping 14C-labeled fetuin within resealed erythrocyte ghosts; fetuin was then poorly labeled, whereas the erythrocyte sialoglycoproteins were highly labeled; (c) comparison of labeled glycoproteins of mouse lymphoid cells before and after treatment with neuraminidase. This simple method of specifically introducing a radioactive label into cell surface sialic acids is useful in the study of cell surface sialic acid-containing glycoproteins. 相似文献
10.
Compartmentation of the Golgi complex: brefeldin-A distinguishes trans- Golgi cisternae from the trans-Golgi network 总被引:29,自引:17,他引:12
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The Golgi complex is composed of at least four distinct compartments, termed the cis-, medial, and trans-Golgi cisternae and the trans-Golgi network (TGN). It has recently been reported that the organization of the Golgi complex is disrupted in cells treated with the fungal metabolite, brefeldin-A. Under these conditions, it was shown that resident enzymes of the cis-, medial, and trans-Golgi return to the ER. We report here that 300-kD mannose 6-phosphate receptors, when pulse-labeled within the ER of brefeldin-A-treated cells, acquired numerous N-linked galactose residues with a half time of approximately 2 h, as measured by their ability to bind to RCA-I lectin affinity columns. In contrast, Limax flavus lectin chromatography revealed that less than 10% of these receptors acquired sialic acid after 8 h in brefeldin-A. Two lines of evidence suggested that proteins within and beyond the TGN did not return to the ER in the presence of brefeldin-A. First, the majority of 300-kD mannose 6-phosphate receptors present in the TGN and endosomes did not return to the ER after up to 6 h in brefeldin-A, as determined by their failure to contact galactosyltransferase that had relocated there. Moreover, although mannose 6-phosphate receptors did not acquire sialic acid when present in the ER of brefeldin-A-treated cells, they were readily sialylated when labeled at the cell surface and transported to the TGN. These experiments indicate that galactosyltransferase, a trans-Golgi enzyme, returns to the endoplasmic reticulum in the presence of brefeldin-A, while the bulk of sialyltransferase, a resident of the TGN, does not. Our findings support the proposal that the TGN is a distinct, fourth compartment of the Golgi apparatus that is insensitive to brefeldin-A. 相似文献
11.
Lysosomal acid phosphatase is transported to lysosomes via the cell surface. 总被引:31,自引:9,他引:22
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Lysosomal acid phosphatase (LAP) is transported as a transmembrane protein to dense lysosomes. The pathway of LAP to lysosomes includes the passage through the plasma membrane. LAP is transported from the trans-Golgi to the cell surface with a half-time of less than 10 min. Cell surface LAP is rapidly internalized. Most of the internalized LAP is transported back to the cell surface. On average, each LAP molecule cycles greater than 15 times between the cell surface and the endosomes before it is transferred to dense lysosomes. At equilibrium approximately 4 times more LAP precursor is present in endosomes than at the cell surface. Exposing cells to reduced temperature or weak bases such as NH4Cl, chloroquine and primaquine decreases the steady-state concentration of LAP at the cell surface. The recycling pathway is operative at greater than or equal to 20 degrees C and does not include passage of the Golgi/trans-Golgi network. LAP is transferred with a half-time of 5-6 h from the plasma membrane/endosome pool to dense lysosomes, from where it does not recycle to the endosome/plasma membrane pool at a measurable rate. 相似文献
12.
We have previously engineered transgenic insect cell lines to express mammalian glycosyltransferases and showed that these cells can sialylate N-glycoproteins, despite the fact that they have little intracellular sialic acid and no detectable CMP-sialic acid. In the accompanying study, we presented evidence that these cell lines can salvage sialic acids for de novo glycoprotein sialylation from extracellular sialoglycoproteins, such as fetuin, found in fetal bovine serum. This finding led us to create a new transgenic insect cell line designed to synthesize its own sialic acid and CMP-sialic acid. SfSWT-1 cells, which encode five mammalian glycosyltransferases, were transformed with two additional mammalian genes that encode sialic acid synthase and CMP-sialic acid synthetase. The resulting cell line expressed all seven mammalian genes, produced CMP-sialic acid, and sialylated a recombinant glycoprotein when cultured in a serum-free growth medium supplemented with N-acetylmannosamine. Thus the addition of mammalian genes encoding two enzymes involved in CMP-sialic acid biosynthesis yielded a new transgenic insect cell line, SfSWT-3, that can sialylate recombinant glycoproteins in the absence of fetal bovine serum. This new cell line will be widely useful as an improved host for baculovirus-mediated recombinant glycoprotein production. 相似文献
13.
The inherent promiscuity of the polysialic acid (PSA) biosynthetic pathway has been exploited by the use of exogenous unnatural sialic acid precursor molecules to introduce unnatural modifications into cellular PSA, and has found applications in nervous system development and tumor vaccine studies. The sialic acid precursor molecules N-propionyl- and N-butanoyl-mannosamine (ManPr, ManBu) have been variably reported to affect PSA biosynthesis ranging from complete inhibition to de novo production of modified PSA, thus illustrating the need for further investigation into their effects. In this study, we have used a monoclonal antibody (mAb) 13D9, specific to both N-propionyl-PSA and N-butanoyl-PSA (NPrPSA and NBuPSA), together with flow cytometry, to study precursor-treated tumor cells and NT2 neurons at different stages of their maturation. We report that both ManPr and ManBu sialic acid precursors are metabolized and the resultant unnatural sialic acids are incorporated into de novo surface sialylglycoconjugates in murine and human tumor cells and, for the first time, in human NT2 neurons. Furthermore, neither precursor treatment deleteriously affected endogenous PSA expression; however, with NT2 cells, PSA levels were naturally downregulated as a function of their maturation into polarized neurons independent of sialic acid precursor treatment. 相似文献
14.
O K Haffar A K Vallerga S A Marenda H J Witchel G L Firestone 《Molecular and cellular biology》1987,7(4):1508-1517
15.
Corinna Benz Markus Engstler Stefan Hillmer 《International journal for parasitology》2010,40(5):629-634
Bloodstream-form Trypanosoma brucei have two 14-3-3 proteins, which are required for parasite multiplication. We here describe the effects of 14-3-3 depletion on vesicular transport of variant surface glycoprotein (VSG). 14-3-3 depletion had no detectable effect on de novo synthesis and trafficking of VSG to the cell surface, or on VSG endocytosis. Despite strong inhibition of cell division, the flagellar pocket was not enlarged and the ultrastructure of internal organelles appeared normal. The Rab11-positive recycling endosome compartment was, however, fivefold smaller than normal, and the rate of return of recycling VSG to the surface was correspondingly reduced. Down-regulating 14-3-3 also prevented enlargement of the flagellar pocket by clathrin depletion. These results suggest that there is a remarkably specific requirement for 14-3-3 in normal functioning of the Rab11-positive recycling endosome compartment. 相似文献
16.
Sendai virus assembly: M protein binds to viral glycoproteins in transit through the secretory pathway. 总被引:10,自引:7,他引:3
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We have examined the relative ability of Sendai virus M (matrix) protein to associate with membranes containing viral glycoproteins at three distinct stages of the exocytic pathway prior to cell surface appearance. By the use of selective low-temperature incubations or the ionophore monensin, the transport of newly synthesized viral glycoproteins was restricted to either the pre-Golgi intermediate compartment (by incubation at 15 degrees C), the medial Golgi (in the presence of monensin), or the trans-Golgi network (by incubation at 20 degrees C). All three of these treatments resulted in a marked accumulation of the M protein on perinuclear Golgi-like membranes which in each case directly reflected the distribution of the viral F protein. Subsequent redistribution of the F protein to the plasma membrane by removal of the low-temperature (20 degrees C) block resulted in a concomitant redistribution of the M protein, thus implying association of the two components during intracellular transit. The extent of M protein-glycoprotein association was further examined by cell fractionation studies performed under each of the three restrictive conditions. Following equilibrium sedimentation of membranes derived from monensin-treated cells, approximately 40% of the recovered M protein was found to cofractionate with membranes containing the viral glycoproteins. Also, by flotation analyses, a comparable subpopulation of M protein was found to be membrane associated whether viral glycoproteins were restricted to the trans-Golgi network, the medial Golgi, or the pre-Golgi intermediate compartment. Additionally, transient expression of M protein alone from cloned cDNA showed that neither membrane association nor Golgi localization occurs in the absence of Sendai virus glycoproteins. 相似文献
17.
Membrane traffic in animal cells: cellular glycoproteins return to the site of Golgi mannosidase I 总被引:17,自引:11,他引:6
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The recycling of cellular glycoproteins to the site of Golgi mannosidase I, an enzyme of asparagine-linked oligosaccharide synthesis, was studied in K562 human erythroleukemia cells. Cells were metabolically labeled in the presence of deoxymannojirimycin, a reversible inhibitor of Golgi mannosidase I. This generates glycoproteins with immature oligosaccharides in their normal locations. Transport to the mannosidase I compartment was then assessed by testing for the conversion of oligosaccharides into mature forms during reculture without deoxymannojirimycin. Transferrin receptor (TfR) was acted on by mannosidase I during reculture, suggesting that it returned to the region of the Golgi complex where this enzyme resides. The slow rate of this transport (t1/2 greater than 6 h) implies that it is probably different than TfR movement during transferrin internalization (t1/2 = 10-20 min) and TfR transport to the sialyltransferase compartment in the Golgi complex (t1/2 = 2-3 h) (Snider, M. D., and O. C. Rogers, 1985, J. Cell Biol., 100:826-834). The total cell glycoprotein pool was also transported to the mannosidase I compartment with a half-time of 4 h. Because this transport is 5-10 times faster than the rate of de novo glycoprotein synthesis in these cells, it is likely that most of the glycoprotein traffic through the Golgi complex is composed of recycling molecules. 相似文献
18.
Cohort movement of different ligands and receptors in the intracellular endocytic pathway of alveolar macrophages 总被引:11,自引:0,他引:11
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. 相似文献
19.
Innamorati G Le Gouill C Balamotis M Birnbaumer M 《The Journal of biological chemistry》2001,276(16):13096-13103
The C terminus of the human V2 vasopressin receptor contains multiple phosphorylation sites including a cluster of amino acids that when phosphorylated prevents the return of the internalized receptor to the cell surface. To identify the step where the recycling process was interrupted, the trafficking of the V2 receptor was compared with that of the recycling V1a receptor after exposure to ligand. Initially, both receptors internalized in small peripheral endosomes, but a physical separation of their endocytic pathways was subsequently detected. The V1a receptor remained evenly distributed throughout the cytosol, whereas the V2 receptor accumulated in a large aggregation of vesicles in the proximity of the nucleus where it colocalized with the transferrin receptor and Rab11, a small GTP-binding protein that is concentrated in the perinuclear recycling compartment; only marginal colocalization of Rab11 with the V1a receptor was observed. Thus, the V2 receptor was sequestered in the perinuclear recycling compartment. Targeting to the perinuclear recycling compartment was determined by the receptor subtype and not by the inability to recycle, since the mutation S363A in the phosphorylation-dependent retention signal generated a V2 receptor that was recycled via the same compartment. The perinuclear recycling compartment was enriched in beta-arrestin after internalization of either wild type V2 receptor or its recycling mutant, indicating that long term interaction between the receptors and arrestin was not responsible for the intracellular retention. Thus, the fully phosphorylated retention domain overrides the natural tendency of the V2 receptor to recycle and, by preventing its exit from the perinuclear recycling compartment, interrupts its transit via the "long cycle." The data suggest that the inactivation of the domain, possibly by dephosphorylation, triggers the return of the receptor from the perinuclear compartment to the plasma membrane. 相似文献
20.
Summary Histochemical studies performed on specimens of intestine from 12 to 37-week human foetuses showed that the epithelial glycoproteins of the goblet cells of the small intestine are non-sulphated sialoglycoproteins containing neutral sugar (hexose, 6-deoxy hexose or N-acetyl hexosamine residues with Periodic acid-Schiff (PAS) reactive vicinal diols), sialic acids without O-acyl substituents, smaller and variable quantities of sialic acids with O-acyl substituents at positions C8 or C9 (or with two or three side chain substituents) and O-acyl sugars (neutral sugars with an ester substituent blocking PAS reactivity). In the lower small intestine glycoproteins containing 8 (or 9)-O-acyl sialic acids are first observed in goblet cells at the tips of the villi. As the foetus matures their quantity increases and they are found in goblet cells located along the length of the villi. Smaller quantities of O-acyl sialic acids and traces of O-acyl sugars occur in the goblet cells of the upper small intestine. The colonic goblet cells contain sulphosialoglycoproteins of two types. The first type, found in the majority of specimens, contains O-sulphate ester, neutral sugar, O-acyl sugars and 8 (or 9)-O-acyl sialic acids. The second type contains O-sulphate ester, neutral sugars, and sialic acids which are either without side chain O-acyl substituents or are a mixture of such acids and 8 (or 9)-O-acyl sialic acids; O-acyl sugars are reduced or absent. The degree of sulphation of the foetal colonic goblet cell epithelial glycoproteins differs with the region of the colon, the level of the crypt and the gestational age of the foetus in a manner consistent with that described by Lev & Orlic (1974). The detection of O-acyl sugars in foetal intestinal glycoproteins adds to the known examples of such sugars and strengthens the suggestion that they are a normal constituent of colonic epithelial glycoproteins.Part of this work was presented at the 32nd meeting of the Canadian Federation of Biological Sciences, Calgary, Alberta, June 1989 (abstract # 336). 相似文献