Biomedical subjects

B van Deurs

Publications and source records attributed to B van Deurs.

At least 37 records · Page 2Linked to original sources

PubMed1999

Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles.

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

Animals↗

PubMed1999

Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor.

We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosylphosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-beta-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles.

Adolescent↗

PubMed1998

Analysis of GLUT4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions.

The effects of insulin stimulation and muscle contractions on the subcellular distribution of GLUT4 in skeletal muscle have been studied on a preparation of single whole fibers from the rat soleus. The fibers were labeled for GLUT4 by a preembedding technique and observed as whole mounts by immunofluorescence microscopy, or after sectioning, by immunogold electron microscopy. The advantage of this preparation for cells of the size of muscle fibers is that it provides global views of the staining from one end of a fiber to the other and from one side to the other through the core of the fiber. In addition, the labeling efficiency is much higher than can be obtained with ultracryosections. In nonstimulated fibers, GLUT4 is excluded from the plasma membrane and T tubules. It is distributed throughout the muscle fibers with approximately 23% associated with large structures including multivesicular endosomes located in the TGN region, and 77% with small tubulovesicular structures. The two stimuli cause translocation of GLUT4 to both plasma membrane and T tubules. Quantitation of the immunogold electron microscopy shows that the effects of insulin and contraction are additive and that each stimulus recruits GLUT4 from both large and small depots. Immunofluorescence double labeling for GLUT4 and transferrin receptor (TfR) shows that the small depots can be further subdivided into TfR-positive and TfR-negative elements. Interestingly, we observe that colocalization of TfR and GLUT4 is increased by insulin and decreased by contractions. These results, supported by subcellular fractionation experiments, suggest that TfR-positive depots are only recruited by contractions. We do not find evidence for stimulation-induced unmasking of resident surface membrane GLUT4 transporters or for dilation of the T tubule system (Wang, W., P.A. Hansen, B.A. Marshall, J.O. Holloszy, and M. Mueckler. 1996. J. Cell Biol. 135:415-430).

Animals↗

PubMed1998

Transport of apically but not basolaterally internalized ricin to the Golgi apparatus is stimulated by 8-Br-cAMP in MDCK cells.

The plant toxin ricin has to be transported to the Golgi apparatus after endocytosis to exert its toxic effect. In this study we show that transport of apically endocytosed ricin to the Golgi apparatus is stimulated by 8-Br-cAMP in polarized MDCK cells. This stimulation is counteracted by the PKA inhibitor H-89. In contrast, there is no increase in the transport to the Golgi apparatus of ricin internalized from the basolateral membrane. These results suggest that protein kinase A selectively regulates endosome to Golgi transport in these cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Expression of mutant dynamin inhibits toxicity and transport of endocytosed ricin to the Golgi apparatus.

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dynG273D at the nonpermissive temperature or the dynK44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69-80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915-934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949-966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dynWT than in cells expressing dynK44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dynK44A. In contrast, ricin transport to lysosomes as measured by degradation of 125I-ricin was essentially unchanged in cells expressing dynK44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor.

Biological Transport↗

PubMed1998

Expression of caveolin-1 and polarized formation of invaginated caveolae in Caco-2 and MDCK II cells.

We have studied caveolin-1 expression and the frequency and distribution of typical invaginated caveolae as they are identified by electron microscopy in the polarized epithelial cell lines MDCK II and Caco-2. In wild-type MDCK II cells caveolin expression is high and more than 400 caveolae/mm filter were observed at the basolateral membrane. No caveolae were found at the apical surface. By contrast, wild-type Caco-2 cells do not express caveolin-1 and have extremely few, if any caveolae. Caco-2 cells were stably transfected with the gene for caveolin-1 in order to investigate if the formation of caveolae is polarized also in these cells. We have isolated Caco-2 clones expressing different levels of caveolin-1, where the level of expression varies from 10-100% of the endogenous level in MDCK II cells. Caveolin-1 expression in Caco-2 cells gives rise to a marked immunofluorescense labeling mainly at the lateral plasma membrane. By electron microscopy an increase from less than 4 caveolae/mm filter in wild-type Caco-2 cells to 21-76 caveolae/mm filter in Caco-2 clones transfected with caveolin-1 was revealed and these caveolae were exclusively localized to the basolateral membrane. Thus expression of heterologous caveolin-1 in Caco-2 cells leads to polarized formation of caveolae, but there is a lack of correlation between the amount of caveolin expressed in the cells and the number of caveolae, suggesting that factors in addition to caveolin are required for generation of caveolae.

Animals↗

PubMed1997

Intracellular transport and processing of protein toxins produced by enteric bacteria.

Bacterial toxins are associated with disease in humans and animals. Toxins can either be preformed in food or produced by bacteria in the intestine. There are two types of toxins: heat-labile protein toxins and heat stabile toxins. Heat labile toxins are produced by Bacillus cereus, Clostridium perfringens, Escherichia coli, and Vibrio cholerae, and heat-stabile enterotoxins consisting of relatively few amino acids are produced by Escherichia coli and acts by activation of guanylate cyclase. Similarly, heat-stabile entero-toxins are also produced by Staphylococcus aureus, a common cause of food poisoning in the United States, and Yersenia enterocolitica. Protein toxins produced by enteric bacteria can intoxicate intestinal cells and can also be taken up from the gut and reach other cells in the body. For example the Shiga-like toxins (vero-toxins) can intoxicate endothelial cells in the kidney and cause kidney failure. Intracellular transport and processing of a few of the protein toxins produced by enteric bacteria, namely Clostridium difficile toxin A and B, cholera toxin and the related heat-labile toxin produced by Escherichia coli, and Shiga toxin and Shiga-like toxins are presented.

Animals↗

PubMed1997

Galectin-4 and small intestinal brush border enzymes form clusters.

Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed that galectin-4 is indeed an intestinal brush border protein; we also localized galectin-4 throughout the cell, mainly associated with membraneous structures, including small vesicles, and to the rootlets of microvillar actin filaments. This was confirmed by subcellular fractionation, showing about half the amount of galectin-4 to be in the microvillar fraction, the rest being associated with insoluble intracellular structures. A direct association between the lectin and aminopeptidase N was evidenced by a colocalization along microvilli in double immunogold labeling and by the ability of an antibody to galectin-4 to coimmunoprecipitate aminopeptidase N and sucrase-isomaltase. Furthermore, galectin-4 was released from microvillar, right-side-out vesicles as well as from mucosal explants by a brief wash with 100 mM lactose, confirming its extracellular localization. Galectin-4 is therefore secreted by a nonclassical pathway, and the brush border enzymes represent a novel class of natural ligands for a member of the galectin family. Newly synthesized galectin-4 is rapidly "trapped" by association with intracellular structures prior to its apical secretion, but once externalized, association with brush border enzymes prevents it from being released from the enterocyte into the intestinal lumen.

Amino Acid Sequence↗

PubMed1996

Thapsigargin-induced transport of cholera toxin to the endoplasmic reticulum.

Cholera toxin is normally observed only in the Golgi apparatus and not in the endoplasmic reticulum (ER) although the enzymatically active A subunit of cholera toxin has a KDEL sequence. Here we demonstrate transport of horseradish peroxidase-labeled cholera toxin to the ER by electron microscopy in thapsigargin-treated A431 cells. Thapsigargin treatment strongly increased cholera toxin-induced cAMP production, and the formation of the catalytically active A1 fragment was somewhat increased. Binding of cholera toxin to the cell surface and transport of toxin to the Golgi apparatus were not changed in thapsigargin-treated cells, suggesting increased retrograde transport of cholera toxin from the Golgi apparatus to the ER. The data demonstrate that retrograde transport of cholera toxin can take place and that the transport is under regulation. The results are consistent with the idea that retrograde transport can be important for the action of cholera toxin.

Calcium↗

PubMed1996

Effect of calmodulin antagonists on endocytosis and intracellular transport of ricin in polarized MDCK cells.

The effect of calmodulin antagonists on endocytosis, transcytosis, recycling, and transport to the Golgi apparatus from both the apical and the basolateral plasma membrane of polarized Madin-Darby canine kidney cells has been investigated by using the plant toxin ricin as a membrane marker. The calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) stimulated apical endocytosis of ricin, whereas basolateral endocytosis was unaffected. A stimulation of the apical uptake of the fluid-phase marker horseradish peroxidase by calmodulin antagonists was also found both by biochemical and by ultrastructural studies. Furthermore, W-7 reduced the recycling of ricin to the apical plasma membrane, whereas the recycling to the basolateral plasma membrane was not changed. Transport of ricin to the Golgi apparatus was also selectively affected by the calmodulin antagonist W-7. After basolateral endocytosis of ricin, transport to the Golgi apparatus was reduced, whereas after apical endocytosis the fraction of endocytosed ricin transport to the Golgi apparatus was increased. Transcytosis of ricin from the basolateral to the apical pole was increased in the presence of calmodulin antagonists, whereas these compounds did not have any significant effect on the apical to basolateral transcytosis. Thus, the results obtained indicate that calmodulin is involved in regulation of apical endocytosis and recycling as well as in transcytosis of ricin from the basolateral plasma membrane. Furthermore, the data suggest that calmodulin plays a role in regulation of ricin transport to the Golgi apparatus.

Animals↗

PubMed1996

Importance of glycolipid synthesis for butyric acid-induced sensitization to shiga toxin and intracellular sorting of toxin in A431 cells.

The human epidermoid carcinoma cell line A431 becomes highly sensitive to Shiga toxin upon treatment with butyric acid. This strong sensitization (> 1000-fold) is accompanied by an increase in the fraction of cell-associated toxin transported to the Golgi apparatus and to the endoplasmic reticulum (ER). Furthermore, our previous work showed that the length of the fatty acyl chain of Gb3, the Shiga toxin receptor, also was changed (longer fatty acids). We have not investigated the importance of this change by testing whether glycolipid synthesis is required for the changed intracellular sorting and the toxin sensitivity. We demonstrate here that inhibition of glycosphingolipid synthesis by inhibition of N-acyltransferase with fumonisin B1, by inhibition of glucosylceramide synthetase by PDMP or PPMP, or by inhibition of serine palmitoyl transferase by beta-fluoroalanine, inhibited the butyric acid-induced change in sensitivity and the increase in the fraction of cell-associated Shiga toxin transported to the Golgi apparatus and the ER. The block in butyric acid-induced sensitization caused by beta-fluoroalanine could be abolished by simultaneous addition of sphinganine or sphingosine. Thus, the data suggest that the fatty acyl chain length of glycosphingolipids is important for intracellular sorting and translocation of Shiga toxin to the cytosol.

Acyltransferases↗

PubMed1996

Endocytosis, intracellular transport, and cytotoxic action of Shiga toxin and ricin.

Protein toxins such as ricin and Shiga toxin with intracellular targets have to be endocytosed and translocated to the cytosol to inhibit the protein synthesis and thereby kill the cell. Ricin is internalized by both clathrin-dependent and -independent endocytic mechanisms, whereas Shiga toxin seems to be taken up exclusively from clathrin-coated pits. After endocytosis, internalized membrane and content are delivered to endosomes, where sorting for further routing in the cell takes place. Toxins that remain membrane bound at low endosomal pH can be recycled to the cell surface or transcytosed in polarized epithelia. A large proportion of internalized toxin is transported to lysosomes for degradation. Most importantly, a fraction of the internalized ricin and Shiga toxin molecules is delivered to the trans-Golgi network (TGN). Shiga toxin can, in some very sensitive cells, be transported retrogradely through the Golgi cisterns all the way back to the endoplasmic reticulum (ER), and it is possible that also ricin is transported retrogradely to the ER. In this review, a cell biological overview of these intracellular transport steps is presented, and evidence is provided that the delivery to the TGN and the subsequent retrograde transport to the ER are required for optimal intoxication. Moreover, it is argued that knowledge of this transport is important for targeted drug delivery such as the application of immunotoxins in cancer therapy.

Bacterial Toxins↗

PubMed1996

Destabilization of plasma membrane structure by prevention of actin polymerization. Microtubule-dependent tubulation of the plasma membrane.

Electron microscopy of thick (0.2-1.0 micron) sections of cytochalasin D-treated cells fixed in the presence of Ruthenium red revealed an extensive, surface-connected tubular compartment in HEp-2 cells. The tubules measured 120-220 nm in diameter and at least up to 6 microns in length. Morphometric analysis showed that in control cells about 0.2% of the total plasma membrane area (defined as all Ruthenium red-labeled membrane) appeared as vesicular or tubular profiles beneath the cell surface. However, after 15-30 minutes of cytochalasin D incubation about 4% of the total plasma membrane area is tubulated, and after 60-105 minutes as much as about 15% of the total plasma membrane appears as tubules. Clathrin-coated pits and caveolae-like structures were occasionally associated with the tubular membrane. Moreover, immunogold labeling showed that the tubular membrane contained transferrin receptors at about the same density as the nontubulated plasma membrane. Examination of cells in which endosomes and lysosomes were labeled with horseradish peroxidase before or after exposure to cytochalasin D showed that these organelles remained spherical, and that no horseradish peroxidase was present in the tubules. Moreover, the surface to volume ratio remained constant with increasing time of cytochalasin D incubation. Accordingly, the surface-connected tubules were not derived from endocytic structures but were formed by invagination of the plasma membrane. The tubule formation is reversible. When microtubules are depolymerized by nocodazole or colchicine treatment before the cells are exposed to cytochalasine D, tubule formation is strongly inhibited. Hence, the cytochalasin D-induced plasma membrane tubulation depends on intact microtubules.

Actins↗

PubMed1996

Inhibition of the vacuolar H(+)-ATPase with bafilomycin reduces delivery of internalized molecules from mature multivesicular endosomes to lysosomes in HEp-2 cells.

In the human carcinoma cell line HEp-2, endosomes are multivesicular bodies (MVBs) which gradually mature and eventually fuse with other mature endosomes and/or with preexisting lysosomes. Selective inhibition of the vacuolar H(+)-ATPase with bafilomycin A1 (Baf) did not influence endocytic uptake and recycling of the protein toxin ricin. Moreover, quantification of immunogold labeling on ultracryosections revealed that the frequency by which MVBs containing internalized cationized gold (Cat.Au) also labeled for the cation-independent mannose-phosphate receptor (MPR) was the same with and without Baf, suggesting that formation and maturation of MVBs were unchanged in the presence of Baf. However, degradation of ricin was strongly reduced by bafilomycin, and this reduction was not only due to increased pH in lysosomes. Thus, quantitative lmmunogold labeling showed that the Baf-induced alkalinization reduced the transfer of internalized Cat.Au to lysosomes, as defined by their content of human lysosome-associated membrane protein 1 (h-lamp-1) and cathepsin D. Accordingly, although low vacuolar pH does not seem to be required for transport to MPR-containing endosomes, it seems to be important for a late fusion step along the endocytic pathway.

Anti-Bacterial Agents↗

PubMed1995

Furin-induced cleavage and activation of Shiga toxin.

Shiga toxin has a single A subunit non-covalently associated with a pentamer of B subunits. The toxin has a trypsin-sensitive region near the COOH-terminal end of the A-chain, and upon cleavage, two disulfide bonded fragments, A1 and A2, are generated. These fragments are also formed upon incubation with cells. The disulfide loop contains the sequence (Arg-X-X-Arg), which is a consensus motif for cleavage by the membrane-anchored protease furin. We found that a soluble form of furin cleaves intact A-chain producing A1 and A2 fragments, and furin also seems to be responsible for rapid cellular cleavage of Shiga toxin. LoVo cells, which normally do not produce functional furin, cleave intact A-chain very efficiently when transfected with furin (LoVo/fur), whereas a control cell (LoVo/neo) cleaves the toxin very slowly. To investigate the role of this cleavage for intoxication of cells, we studied the ability of unnicked and furin-nicked toxin to inhibit protein synthesis in LoVo/fur and LoVo/neo cells. LoVo/fur cells were intoxicated equally well with unnicked and nicked toxin, whereas in LoVo/neo cells nicked toxin was about 20 times more active than unnicked toxin. The results suggest that cleavage of Shiga toxin is important for intoxication of cells, and they indicate that furin can cleave and thereby activate Shiga toxin in cells.

Amino Acid Sequence↗

PubMed1995

Phorbol myristate acetate selectively stimulates apical endocytosis via protein kinase C in polarized MDCK cells.

We have examined the regulation of endocytosis in polarized Madin-Darby canine kidney (MDCK) cells. Using quantitative electron microscopy and biochemical measurements, we found that incubation with the tumor promoter phorbol 12-myristate 13-acetate (TPA) stimulated endocytosis of horseradish peroxidase (HRP) and ricin four- to fivefold at the apical side in MDCK cells, whereas the uptake at the basolateral membrane was unaffected. The use of several protein kinase inhibitors and TPA analogues indicated that the stimulation of apical endocytosis was mediated via protein kinase C independently of protein kinase A. This stimulation occurred even when the clathrin-dependent pathway was inhibited by acidification of the cytosol, suggesting that the TPA-stimulated uptake was associated with a clathrin-independent mechanism. Moreover, we found that TPA also stimulated recycling of ricin to the apical domain. Ultrastructural analysis of MDCK cells preincubated with TPA revealed that neither the morphology nor the size of the endosomes was altered compared to control cells. Using morphometry, no marked change in the apical plasma membrane area was detected after incubation with TPA. These data indicate that the TPA-stimulated endocytosis involved neither ruffling nor formation of macropinosomes in MDCK cells. Finally, we found that TPA also selectively stimulated apical endocytosis in the human colon adenocarcinoma cell line (Caco-2). The data suggest that protein kinase C is involved in a strong stimulation of apical endocytosis and might participate in the regulation of membrane trafficking between the apical plasma membrane and apical endosomes in polarized epithelial cells.

Animals↗

PubMed1995

Role of processing and intracellular transport for optimal toxicity of Shiga toxin and toxin mutants.

Cleavage of Shiga toxin A-fragment at a highly trypsin-sensitive site increases its enzymatic activity. To investigate the role of this cleavage site in intoxication of cells, we studied the routing, cleavage, and toxicity of mutant toxin where the trypsin-sensitive site had been eliminated. Ultrastructural analysis of toxin tagged with horseradish peroxidase demonstrated that wild-type and mutant toxins were transported from endosomes to the trans-Golgi network and further through the Golgi cisterns to the endoplasmic reticulum. Wild-type toxin was much more efficient than the mutants in provoking rapid intoxication, but after prolonged incubation time also mutants were highly toxic. The cells were able to cleave both wild-type Shiga toxin and the mutants, but the cellular location for cleavage appears to differ. Wild-type toxin was cleaved in the presence of brefeldin A, which disrupts the Golgi cisterns. This indicates that the cleavage occurs in the endosomes or in the trans-Golgi network. In contrast, the mutant Shiga-His (R248H/R251H) was not cleaved in the presence of brefeldin A, indicating that the cleavage can occur only after the toxin has left the trans-Golgi network. In vitro experiments showed that the cytosolic enzyme calpain is able to cleave Shiga-His, and results from in vivo experiments are consistent with the possibility that cleavage is carried out by calpain after the mutant A-fragment has reached the cytosol.

Amino Acid Sequence↗

PubMed1995

A transferrin-like GPI-linked iron-binding protein in detergent-insoluble noncaveolar microdomains at the apical surface of fetal intestinal epithelial cells.

A GPI-anchored 80-kD protein was found to be the major component of detergent-insoluble complexes, prepared from fetal porcine small intestine, constituting about 25% of the total amount of protein. An antibody was raised to the 80-kD protein, and by immunogold electron microscopy of ultracryosections of mucosal tissue, the protein was localized to the apical surface of the enterocytes, whereas it was absent from the basolateral plasma membrane. Interestingly, it was mainly found in patches of flat or invaginated apical membrane domains rather than at the surface of microvilli. Caveolae were not found in association with these labeled microdomains. In addition, the 80-kD protein was seen in apical endocytic vacuoles and in tubulo-vesicular structures, suggesting that the apical microdomains are involved in endocytosis of the 80-kD protein. By its NH2-terminal amino acid sequence, iron-binding capacity and partial immunological cross-reactivity with serum transferrin, the 80-kD protein was shown to belong to the transferrin family, and it is probably homologous to melanotransferrin, a human melanoma-associated antigen. The 80-kD iron-binding protein was fully detergent-soluble immediately after synthesis and only became insoluble after gaining resistance to endo H, supporting a mechanism for exocytic delivery to the apical cell surface by way of detergent-insoluble glycolipid "rafts" that fuse with the plasmalemma at restricted sites devoid of microvilli.

Age Factors↗