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1.
Grewal T Heeren J Mewawala D Schnitgerhans T Wendt D Salomon G Enrich C Beisiegel U Jäckle S 《The Journal of biological chemistry》2000,275(43):33806-33813
Annexins are calcium-binding proteins with a wide distribution in most polarized and nonpolarized cells that participate in a variety of membrane-membrane interactions. At the cell surface, annexin VI is thought to remodel the spectrin cytoskeleton to facilitate budding of coated pits. However, annexin VI is also found in late endocytic compartments in a number of cell types, indicating an additional important role at later stages of the endocytic pathway. Therefore overexpression of annexin VI in Chinese hamster ovary cells was used to investigate its possible role in endocytosis and intracellular trafficking of low density lipoprotein (LDL) and transferrin. While overexpression of annexin VI alone did not alter endocytosis and degradation of LDL, coexpression of annexin VI and LDL receptor resulted in an increase in LDL uptake with a concomitant increase of its degradation. Whereas annexin VI showed a wide intracellular distribution in resting Chinese hamster ovary cells, it was mainly found in the endocytic compartment and remained associated with LDL-containing vesicles even at later stages of the endocytic pathway. Thus, data presented in this study suggest that after stimulating endocytosis at the cell surface, annexin VI remains bound to endocytic vesicles to regulate entry of ligands into the prelysosomal compartment. 相似文献
2.
Osmotic swelling of endocytic compartments induced by internalized sucrose is restricted to mature lysosomes in cultured mammalian cells. 总被引:3,自引:1,他引:2
To determine which endocytic compartments are sensitive to sucrose-induced osmotic swelling, CHO and Vero cells were cultured for 1-3 days in media containing 0.03 to 0.05 M sucrose. (Sucrose is internalized but not digested by these cells.) To immunolocalize late endocytic compartments, cells were fixed with formaldehyde and labeled with antibodies against the 215-kDa mannose 6-phosphate receptor (prelysosomal compartment) and LAMP-1 and -2 (mature lysosomes). Early endosomes were labeled by a 2-min uptake of lucifer yellow, mature lysosomes by greater than or equal to 16-h uptake of lucifer yellow followed by a 2-h chase. The data showed that sucrose induced swelling of mature lysosomes only (mannose 6-phosphate receptor negative, LAMP-1 and LAMP-2 positive); early endosomes and the prelysosomal compartment were not affected by the presence of sucrose, i.e., osmotically swollen. Accumulation of lucifer yellow in the swollen compartment was insensitive to cycloheximide. These results suggest, by inference, that the complement of membrane transport proteins that regulate the osmotic properties of endocytic organelles must be discontinuously distributed along the endocytic pathway. 相似文献
3.
Pons M Ihrke G Koch S Biermer M Pol A Grewal T Jäckle S Enrich C 《Experimental cell research》2000,257(1):33-47
Annexin VI is an abundant calcium- and phospholipid-binding protein whose intracellular distribution and function are still controversial. Using a highly specific antibody, we have studied the distribution of annexin VI in NRK fibroblasts and the polarized hepatic cell line WIF-B by confocal microscopy. In NRK cells, annexin VI was almost exclusively found associated with endocytic compartments, which were defined by their ability to receive fluid-phase marker internalized from the cell surface. However, extensive colocalization of annexin VI and the endocytic marker was only observed after about 45 min, indicating that annexin VI was primarily in late endocytic compartments or (pre)lysosomes. Consistent with this, annexin VI was predominantly seen on structures that contained the lysosomal protein lgp120, although not on dense core lysosomes by electron microscopy. Two major populations of annexin VI-containing structures were present in polarized WIF-B hepatocytes. One correlated to lgp120-positive (pre)lysosomes and was still observed after treatment with brefeldin A (BFA), while the other appeared to be partially associated with Golgi membranes and was BFA-sensitive. The striking association with prelysosomal compartments in NRK and WIF-B cells suggests that annexin VI could play a role in fusion events in the late endocytic pathway, possibly by acting as a tether between membranes. 相似文献
4.
Epidermal growth factor-mediated caveolin recruitment to early endosomes and MAPK activation. Role of cholesterol and actin cytoskeleton 总被引:1,自引:0,他引:1
The endocytic compartment of eukaryotic cells is a complex intracellular structure involved in sorting, processing, and degradation of a great variety of internalized molecules. Recently, the uptake through caveolae has emerged as an alternative internalization pathway, which seems to be directly related with some signal transduction pathways. However, the mechanisms, molecules, and structures regulating the transport of caveolin from the cell surface into the endocytic compartment are largely unknown. In this study, normal quiescent fibroblasts (normal rat kidney (NRK)) were used to demonstrate that epidermal growth factor causes partial redistribution of caveolin from the cell surface into a cellubrevin early endocytic compartment. Treatment of NRK cells with cytochalasin D or latrunculin A inhibits this pathway and the concomitant activation of Mek and mitotic-activated protein (MAP) kinase; however, if cells were pre-treated with filipin, cytochalasin D does not inhibit the phosphorylation of MAP kinase induced by epidermal growth factor. From these results we conclude that in NRK cells the intact actin cytoskeleton is necessary for the EGF-mediated transport of caveolin from the cell surface into the early endocytic compartment and the activation of MAP kinase pathway. 相似文献
5.
A role for EHD4 in the regulation of early endosomal transport 总被引:1,自引:0,他引:1
All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)- and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway. 相似文献
6.
EHD1 regulates cholesterol homeostasis and lipid droplet storage 总被引:1,自引:0,他引:1
Naslavsky N Rahajeng J Rapaport D Horowitz M Caplan S 《Biochemical and biophysical research communications》2007,357(3):792-799
Endocytic transport is critical for the subcellular distribution of free cholesterol and the endocytic recycling compartment (ERC) is an important organelle that stores cholesterol and regulates its trafficking. The C-terminal EHD protein, EHD1, controls receptor recycling through the ERC and affects free cholesterol distribution in the cell. We utilized embryonic fibroblasts from EHD1 knockout mice (Ehd1(-/-)MEF) and SiRNA in normal MEF cells to assess the role of EHD1 in intracellular transport of cholesterol. Surprisingly, Ehd1(-/-)MEFs displayed reduced levels of esterified and free cholesterol, which returned to normal level upon re-introduction of wild-type, but not dysfunctional EHD1. Moreover, triglyceride and cholesterol storage organelles known as 'lipid droplets' were smaller in size in cells lacking EHD1, indicating that less esterified cholesterol and triglycerides were being stored. Decreased cellular cholesterol and reduced lipid droplet size in Ehd1(-/-)MEFs correlated with ineffectual cholesterol uptake via LDL receptor, suggesting involvement of EHD1 in LDL receptor internalization. 相似文献
7.
Pagler TA Golsabahi S Doringer M Rhode S Schütz GJ Pavelka M Wadsack C Gauster M Lohninger A Laggner H Strobl W Stangl H 《The Journal of biological chemistry》2006,281(50):38159-38171
Plasma high density lipoprotein (HDL) is inversely associated with the development of atherosclerosis. HDL exerts its atheroprotective role through involvement in reverse cholesterol transport in which HDL is loaded with cholesterol at the periphery and transports its lipid load back to the liver for disposal. In this pathway, HDL is not completely dismantled but only transfers its lipids to the cell. Here we present evidence that a Chinese hamster ovarian cell line (CHO7) adapted to grow in lipoprotein-deficient media degrades HDL and concomitantly internalizes HDL-derived cholesterol. Delivery of HDL cholesterol to the cell was demonstrated by a down-regulation of cholesterol biosynthesis, an increase in total cellular cholesterol content and by stimulation of cholesterol esterification after HDL treatment. This HDL degradation pathway is distinct from the low density lipoprotein (LDL) receptor pathway but also degrades LDL. 25-Hydroxycholesterol, a potent inhibitor of the LDL receptor pathway, down-regulated LDL degradation in CHO7 cells only in part and did not down-regulate HDL degradation. Dextran sulfate released HDL bound to the cell surface of CHO7 cells, and heparin treatment released protein(s) contributing to HDL degradation. The involvement of heparan sulfate proteoglycans and lipases in this HDL degradation was further tested by two inhibitors genistein and tetrahydrolipstatin. Both blocked HDL degradation significantly. Thus, we demonstrate that CHO7 cells degrade HDL and LDL to supply themselves with cholesterol via a novel degradation pathway. Interestingly, HDL degradation with similar properties was also observed in a human placental cell line. 相似文献
8.
de Diego I Schwartz F Siegfried H Dauterstedt P Heeren J Beisiegel U Enrich C Grewal T 《The Journal of biological chemistry》2002,277(35):32187-32194
Annexins are Ca(2+)- and phospholipid-binding proteins that are widely expressed in mammalian tissues and that bind to different cellular membranes. In recent years its role in membrane traffic has emerged as one of its predominant functions, but the regulation of its intracellular distribution still remains unclear. We demonstrated that annexin 6 translocates to the late endocytic compartment in low density lipoprotein-loaded CHO cells. This prompted us to investigate whether cholesterol, one of the major constituents of low density lipoprotein, could influence the membrane binding affinity and intracellular distribution of annexin 6. Treatment of crude membranes or early and late endosomal fractions with digitonin, a cholesterol-sequestering agent, displayed a strong reduction in the binding affinity of a novel EDTA-resistant and cholesterol-sensitive pool of annexin 6 proteins. In addition, U18666A-induced accumulation of cholesterol in the late endosomal compartment resulted in a significant increase of annexin 6 in these vesicles in vivo. This translocation/recruitment correlates with an increased membrane binding affinity of GST-annexin 6 to late endosomes of U18666A-treated cells in vitro. In conclusion, the present study shows that changes in the intracellular distribution and concentration of cholesterol in different subcellular compartments participate in the reorganization of intracellular pools of Ca(2+)-dependent and -independent annexin 6. 相似文献
9.
ARF6 Targets Recycling Vesicles to the Plasma Membrane: Insights from an Ultrastructural Investigation 总被引:13,自引:0,他引:13
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Crislyn D'Souza-Schorey Elly van Donselaar Victor W. Hsu Chunzhi Yang Philip D. Stahl Peter J. Peters 《The Journal of cell biology》1998,140(3):603-616
We have shown previously that the ADP- ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane. 相似文献
10.
Endocytosed cation-independent mannose 6-phosphate receptor traffics via the endocytic recycling compartment en route to the trans-Golgi network and a subpopulation of late endosomes
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Although the distribution of the cation-independent mannose 6-phosphate receptor (CI-MPR) has been well studied, its intracellular itinerary and trafficking kinetics remain uncertain. In this report, we describe the endocytic trafficking and steady-state localization of a chimeric form of the CI-MPR containing the ecto-domain of the bovine CI-MPR and the murine transmembrane and cytoplasmic domains expressed in a CHO cell line. Detailed confocal microscopy analysis revealed that internalized chimeric CI-MPR overlaps almost completely with the endogenous CI-MPR but only partially with individual markers for the trans-Golgi network or other endosomal compartments. After endocytosis, the chimeric receptor first enters sorting endosomes, and it then accumulates in the endocytic recycling compartment. A large fraction of the receptors return to the plasma membrane, but some are delivered to the trans-Golgi network and/or late endosomes. Over the course of an hour, the endocytosed receptors achieve their steady-state distribution. Importantly, the receptor does not start to colocalize with late endosomal markers until after it has passed through the endocytic recycling compartment. In CHO cells, only a small fraction of the receptor is ever detected in endosomes bearing substrates destined for lysosomes (kinetically defined late endosomes). These data demonstrate that CI-MPR takes a complex route that involves multiple sorting steps in both early and late endosomes. 相似文献
11.
The intracellular transport and degradation of in vivo endocytosed 125I-tyramine cellobiose-labelled low density lipoprotein (125I-TC-LDL) in rat liver cells were studied by means of subcellular fractionation in Nycodenz, sucrose and Percoll density gradients, as well as by means of analytical differential centrifugation. Initially, labelled LDL was located in endocytic vesicles of low densities. Subsequently, acid-soluble and acid-precipitable radioactivities were found in organelles with buoyant densities distinctly lower than that of the main peaks of the lysosomal marker enzymes acid phosphatase and N-acetyl-beta-glucosaminidase. These prelysosomal organelles may represent multivesicular bodies (MVBs). Finally, 6 h after injection and onwards, the acid-soluble radioactivity cosegregated completely with the two lysosomal marker enzymes, suggesting that the degradation products were in secondary lysosomes. The rate of intracellular processing of LDL was very slow compared to that of asialoglycoproteins, suggesting that LDL followed a unique intracellular pathway, that may be specific for this type of ligand. 相似文献
12.
Protein kinaseCdelta-calmodulin crosstalk regulates epidermal growth factor receptor exit from early endosomes
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Lladó A Tebar F Calvo M Moretó J Sorkin A Enrich C 《Molecular biology of the cell》2004,15(11):4877-4891
We have recently shown that calmodulin antagonist W13 interferes with the trafficking of the epidermal growth factor receptor (EGFR) and regulates the mitogen-activated protein kinase (MAPK) signaling pathway. In the present study, we demonstrate that in cells in which calmodulin is inhibited, protein kinase C (PKC) inhibitors rapidly restore EGFR and transferrin trafficking through the recycling compartment, although onward transport to the degradative pathway remains arrested. Analysis of PKC isoforms reveals that inhibition of PKCδ with rottlerin or its down-modulation by using small interfering RNA is specifically responsible for the release of the W13 blockage of EGFR trafficking from early endosomes. The use of the inhibitor Gö 6976, specific for conventional PKCs (α, β, and γ), or expression of dominant-negative forms of PKCλ, ζ, or ε did not restore the effects of W13. Furthermore, in cells treated with W13 and rottlerin, we observed a recovery of brefeldin A tubulation, as well as transport of dextran-fluorescein isothiocyanate toward the late endocytic compartment. These results demonstrate a specific interplay between calmodulin and PKCδ in the regulation of the morphology of and trafficking from the early endocytic compartment. 相似文献
13.
Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment 总被引:15,自引:0,他引:15
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《The Journal of cell biology》1995,129(6):1509-1522
Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross- linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf. 相似文献
14.
Identification of a membrane glycoprotein found primarily in the prelysosomal endosome compartment 总被引:5,自引:5,他引:0
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Cells contain an intracellular compartment that serves as both the "prelysosomal" delivery site for newly synthesized lysosomal enzymes by the mannose 6-phosphate (Man6P) receptor and as a station along the endocytic pathway to lysosomes. We have obtained mAbs to a approximately 57-kD membrane glycoprotein, (called here plgp57), found predominantly in this prelysosomal endosome compartment. This conclusion is supported by the following results: (a) plgp57 was primarily found in a population of late endosomes that were located just distal to the 20 degrees C block site in the endocytic pathway to lysosomes (approximately 83% of the prelysosomes were positive for plgp57 but less than 5% of the early endosomes had detectable amounts of this marker); (b) plgp57 and the cation-independent (CI) Man6P receptor were located in many of the same intracellular vesicles; (c) plgp57 was found in the membranes of an acidic compartment; (d) immunoelectron microscopy showed that plgp57 was located in characteristic multilamellar- and multivesicular-type vacuoles believed to be prelysosomal endosomes; and (e) cell fractionation studies demonstrated that plgp57 was predominantly found in low density organelles which comigrated with late endosomes and CI Man6P receptors, and only approximately 10-15% of the antigen was found in high density fractions containing the majority of secondary lysosomes. These results indicate that plgp57 is a novel marker for a unique prelysosomal endosome compartment that is the site of confluence of the endocytic and biosynthetic pathways to lysosomes. 相似文献
15.
The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is degraded in the vacuole when glucose is added to glucose-starved cells. Before it is delivered to the vacuole, however, FBPase is imported into intermediate carriers called Vid (vacuole import and degradation) vesicles. Here, using biochemical and genetic approaches, we identified a requirement for SEC28 in FBPase degradation. SEC28 encodes the epsilon-COP subunit of COPI (coat protein complex I) coatomer proteins. When SEC28 and other coatomer genes were mutated, FBPase degradation was defective and FBPase association with Vid vesicles was impaired. Coatomer proteins were identified as components of Vid vesicles, and they formed a protein complex with a Vid vesicle-specific protein, Vid24p. Furthermore, Vid24p association with Vid vesicles was impaired when coatomer genes were mutated. Kinetic studies indicated that Sec28p traffics to multiple locations. Sec28p was in Vid vesicles, endocytic compartments, and the vacuolar membrane in various mutants that block the FBPase degradation pathway. Sec28p was also found in vesicles adjacent to the vacuolar membrane in the ret2-1 coatomer mutant. We propose that Sec28p resides in Vid vesicles, and these vesicles converge with the endocytic pathway. After fusion, Sec28p is distributed on the vacuolar membrane, where it concentrates on vesicles that pinch off from this organelle. FBPase also utilizes the endocytic pathway for transport to the vacuole, as demonstrated by its presence in endocytic compartments in the Deltavph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway. 相似文献
16.
The End2 mutation in CHO cells slows the exit of transferrin receptors from the recycling compartment but bulk membrane recycling is unaffected 总被引:10,自引:5,他引:5
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《The Journal of cell biology》1993,122(6):1231-1241
We have characterized a new CHO cell line (12-4) derived from a parental line, TRVb-1, that expresses the human transferrin receptor. This mutant belongs to the end2 complementation group of endocytosis mutants. Like other end2 mutants, the endosomes in 12-4 cells show a partial acidification defect. These cells internalize LDL and transferrin at 70% of the rate of parental cells and externalize transferrin at 55% of the parental rate (Johnson, L. S., J. F. Presley, J. C. Park, and T. E. McGraw. J. Cell Physiol. 1993). In this report, we have used fluorescence microscopy to determine which step in receptor trafficking is affected in the mutants. Transferrin is sorted from LDL and is delivered to a peri-centriolar recycling compartment at rates similar to parental cells. However, the rate constant for exit of transferrin from the recycling compartment in mutant cells is 0.025 min- 1 vs 0.062 min-1 in the parental line. We also measured the trafficking of a bulk membrane marker, 6-[N-[7-nitrobenzo-2-oxa-1,3-diazol-4-yl]- amino]hexanoyl- sphingosylphosphorylcholine (C6-NBD-SM) that labels the exofacial side of the plasma membrane. C6-NBD-SM enters the same recycling compartment as transferrin, and it exits the recycling compartment at a rate of 0.060-0.065 min-1 in both parental and 12-4 cells. We conclude that bulk membrane flow in the recycling pathway of 12-4 cells is normal, but exit of transferrin from the recycling compartment is slowed due to retention in this compartment. Thus, in the mutant cell line the recycling compartment carries out a sorting function, retaining transferrin over bulk membrane. 相似文献
17.
Disruptions in Golgi structure and membrane traffic in a conditional lethal mammalian cell mutant are corrected by epsilon-COP 总被引:9,自引:3,他引:6
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《The Journal of cell biology》1994,125(6):1213-1224
The CHO cell temperature-sensitive mutant ldlF exhibits two defects in membrane traffic at the nonpermissive temperature (39.5 degrees C): rapid degradation of LDL receptors, possibly caused by endocytic missorting, and disruption of ER-through-Golgi transport. Here, we show that at 39.5 degrees C, the Golgi in ldlF cells dissociated into vesicles and tubules. This dissociation was inhibited by AlF4-, suggesting trimeric G proteins are involved in the dissociation mechanism. This resembled the effects of brefeldin A on wild-type cells. We isolated a hamster cDNA that specifically corrected the ts defects of ldlF cells, but not those of other similar ts mutants (ldlE, ldlG, ldlH, and End4). Its predicted protein sequence is conserved in humans, rice, Arabidopsis, and Caenorhabditis elegans, and is virtually identical to that of bovine epsilon-COP, a component of the coatomer complex implicated in membrane transport. This provides the first genetic evidence that coatomers in animal cells can play a role both in maintaining Golgi structure and in mediating ER-through-Golgi transport, and can influence normal endocytic recycling of LDL receptors. Thus, along with biochemical and yeast genetics methods, mammalian somatic cell mutants can provide powerful tools for the elucidation of the mechanisms underlying intracellular membrane traffic. 相似文献
18.
Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells 总被引:17,自引:7,他引:10
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We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome. 相似文献
19.
The endosomal compartment is a major sorting station controlling the balance between endocytic recycling and lysosomal degradation, and its homeostasis is emerging as a central factor in various neurodegenerative diseases such as Alzheimer's and Parkinson's. Membrane trafficking is generally coordinated by the recognition of specific signals in transmembrane protein cargos by different transport machineries. A number of different protein trafficking complexes are essential for sequence-specific recognition and retrieval of endosomal cargos, recycling them to other compartments and acting to counter-balance the default endosomal sorting complex required for transport–mediated degradation pathway. In this review, we provide a summary of the key endosomal transport machineries, and the molecular mechanisms by which different cargo sequences are specifically recognised. 相似文献
20.
Mesa R Magadán J Barbieri A López C Stahl PD Mayorga LS 《Experimental cell research》2005,304(2):339-353
The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN). 相似文献