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Turnover of brush-border glycoproteins in human intestinal absorptive cells: Do lysosomes have a regulatory function?
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MINI-REVIEW TURNOVER OF BRUSH-BORDER GLYCOPROTEINS IN HUMAN INTESTINAL ABSORPTIVE CELLS: DO LYSOSOMES HAVE A REGULATORY FUNCTION? John Laboratories University
Blok2, of
Jack
A.M.
Leo
Fransen',
for Electron Microscopy' Leiden, Rijnsburgerweg The Netherlands
A.
Ginsell
and Cell 10, 2333
Biology2, AA Leiden
Introduction The absorptive cells lining the small intestine are columnar cells whose surface membranes show a striking polarization. At the luminal side the plasma membrane is folded into finger-like extrusions, the microvilli, which are covered with a prominent cell coat. Separated from this brush-border domain by the junctional complexes, is the basolateral membrane, which is strongly interdigitated with that of neighbouring epithelial cells (Fig. 1) The cell coat of the luminal plasma membrane contains disaccharidases and peptidases), which the enzymes (e.g., in addition to the pancreatic proteases are involved in the degradation of intraluminal food constituents, thus making the end-products of these processes available for uptake in the intestine. These enzymes, which are glycoproteins, are synthesized by the absorptive cells themselves (Bennett and Leblond, 1970; Ginsel et al., 1979). Since the lumen of the small intestine can be assumed to be a hostile environment for the brush-border glycoproteins due to the presence of pancreatic proteases and bile salts (Alpers and Tedesco, 1975), the absorptive cells maintain a continuous synthesis of these macroprobably to compensate for the extracellular molecules, loss of brush-border enzymes. In addition to the extracellular turnover of cell-coat material, an intracellular catabolic mechanism has also been mentioned (Daems et al. 1973; Seetharam et al., 1976; Ginsel et al., 1979). Autoradiographical data (Ginsel et al., 1979; Blok et al., 1981a,b) as well as a recent immunocytochemical study (Fransen et al., 1984) have indeed shown that part of the newly synthesized brush-border constituents are transferred to the lysosomes present in the apical cytoplasm of the absorptive cells. In this mini-review, data concerning the above described phenomena will be summarized and special attention will be given to the role of lysosomes in intracellular catabolism of brush-border enzymes. In the first 030!3-1651/84/120993-22l$O3.Q0/0
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part the biosynthesis of some brush-border enzymes will be considered briefly. The second part will focus on the intracellular transport route of brush-border glycoproteins to the microvilli and the cell organelles involved in this process. A possible involvement of the basolateral membrane in the transport process will also be discussed. The third part gives an overview of the lysosoma1 catabolism of brush-border constituents and speculates on the physiological meaning of such a process. Synthesis of brush-border enzymes It is generally accepted that the peptidases and disaccharidases of the microvillar membrane are synthesized in a way that is essentially similar to the synthesis of other macromolecules destined for secretion (Lennarz, 1983). Thus, the protein backbone is synthesized by the ribosomes of the rough endoplasmic reticulum (RER), linking the various amino acids to yield the translation product. Post-translational modification of the protein core in the RER by the addition of various sugar residues, especially mannose, yield the intermediate products which are known as high-mannose glycoproteins. After transfer to the Golgi apparatus these glycoproteins are trimmed and converted to complex glycoproteins by the addition of other sugars such as N-acetyl glucosamine, galactose, fucose and sialic acid (Bennett, 1970; Bennett et al., 1974; Ginsel et al., 1979; Bennett et al., 1981). An example is the study by Danielsen et al. (1983a) who have used a cell-free microsomal translation system with 35S-methionine as radioactive marker to investigate the synthesis of the brush-border enzyme aminopeptidase A (see also Wacker et al., 1981, for sucrase-isomaltase). Using immunoprecipitation and SDS-gel electrophoresis they found in pulse-chase experiments that the enzyme was synthesized as a primary translational peptide with an M, of 115,000. At later time intervals this product was processed to one of an II, of 140,000, which was sensitive to the action of endo-glycosidase H. This indicates the conversion to a high-mannose glycosylated intermediate, since complex glycoproteins are resistent to treatment by this enzyme. After transfer of the brush--. border enzymes to the Golgi apparatus they are converted cell of the small intestine after silver protein.-Fig. 1. Absorptive ate staining. A fine granular staining is present on the microvilli (mv), dense bodies (edb), a multivesicular body (mvb) and the Golgi web (tw) positive (arrowheads) as apparatus (Ga). In the terminal well as negative (small arrows) apical vesicles and tubules can be observed. Glycogen is present dispersed in the cytoplasm. N, nucleus; m, mitochondrion: x 12,800.
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to the complex mature form., as shown by studies on the synthesis of the peptidases aminopeptidase A and N and dipeptidyl peptidase IV and the disaccharidases sucraseisomaltase and maltase-glycoamylase (Danielsen, 1982; Danielsen et al., 1981, 1983; Erickson et al., 1983; Hauri et al., 1980, 1982; for reviews, see Hauri, 1983 and Sjastrom et al., 1983). Although in general the synthesis of peptidases and disaccharidases is similar, one major difference can be observed. The peptidases are syn-thesized as monomers, whereas the disaccharidases are assembled as dimers. The latter (sucrase-isomaltase, mal-tase-glycoamylase, lactase-phlorizin hydrolase) are converted to monomers by the action of the pancreatic proteolytic enzyme elastase (Hauri et al., 1979). The intracellular transport route of brush-border glycoproteins The conversion of brush-border glycoproteins from the high-mannose to the complex glycosylated form, as described above, takes place in the Golgi apparatus of the absorptive cells. Electron-microscopical autoradiography has shown that the in vivo or in vitro presence of tritiated sugars such as galactose, fucose and sialic acid, which are either terminally or subterminally located in the glycoproteins, results in a significant labeling of Golgi stacks within a short time (Figs. 2 and 4; Bennett, 1970; Bennett et al., 1974; Ginsel et al., 1979; Bennett et al., 1981). To identify the intracellular transport route of the brush-border glycoproteins, Ginsel et al. ,(1979) performed EM-autoradiographical experiments in which human intestinal explants were labeled in tissue culture for 10 min with 3H-fucose and chased for various periods up to 24 hr. It was found that after a 10 min pulse, 40% of the silver grains were located over the Golgi apparatus of the cells. This label decreased after chasing, but a peak of radiolabel appeared after a 35 min chase period in vesicular and tubular structures prominently present in the apex of the absorptive cells (Fig. the same time the number of silver 2) - At approximately grains located on the microvilli started to increase (Figs. 2 and 5). Similar results had been found in vivo in the rat (Bennett and Leblond, 1970; Bennett et al., 1974, 1981). These findings led to the hypothesis that the brush-border glycoproteins are completed in the Golgi apparatus and packaged there in small vesicular and tubular structures, by which they are transported directly to the microvillar membrane (Ginsel et al., 1979; Blok et al., 1981b; see also Michaels and Leblond, 1976). Insertion of the brush-border glycoproteins into the micro-. villar membrane was thought to take place by fusion of the vesicles and tubules with this membrane (exocytosis). At about the same time, Quaroni et al. (1979a,b), and
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Fig. 2. Distribution, in percentages, of the silver grains over the Golgi apparatus, apical vesicles and tubules, microvilli and glycocalyx. The absorptive cells were labeled for 10 min with 3H-fucose and chased for various periods indicated on the abscissa. The data indicate a transport of brush-border glycoproteins via the apical vesicles and tubules to the microvilli and later to the glycocalyx.
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Fig. 3. Distribution, in percentages, of the silver grains over the Golgi apparatus, apical vesicles and tubules, and multivesicular and dense bodies. The cells were labeled and chased as described in Fig. 2. The data suggest that brush-border glycoproteins are also transported from the Golgi apparatus and apical vesicles and tubules to the multivesicular bodies and later to the dense bodies. Fig. 4. EM-autoradiograph Sf the Golgi zone of an absorptive cell after a 10 min pulse with H-fucose. Silver grains are located over Golgi cisternae (arrowheads) as well as vesicles (arrows). x 24,600. Fig. 5. EM-autoradiograph of the apical cytoplasm of an absorptive cell after a 3 hr chase period. Silver grains are located over the microvilli (mv), a dense body (edb) and an apical vesicle (arrowhead). x 21,000.
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Hauri et al. (1979) presented biochemical data which sug-gested that there might also be an indirect transport route of brush-border glycoproteins, i.e., via the later-al plasma membrane to the microvilli. In these studies Quaroni et al. (1979a,b) injected rats with tritiated fucose and after various time intervals isolated membrane fractions of the absorptive cells, consisting of a Golgi, a basolateral, and a brush-border fraction, and determined the amount of radiolabel present in them. Hauri et al. (1979) performed similar experiments, but in addition immunoprecipitated sucrase-isomaltase, one of the most abundant enzymes of the disaccharidases present in the intestine, from the above-mentioned membrane fractions. They found that the radioactivity was initially present in the Golgi fraction and,at later time-points (60 min), in the brush-border membrane fraction. However, a peak of radioactive labeled glycoproteins was also found after 30-60 min in the basolateral membrane fraction. On these grounds it was postulated that at least part of the brush-border glycoproteins are transported via the lateral membrane to the apical membrane. How can the discrepancies between the autoradiographical and biochemical data be explained? Autoradiography as well as the biochemical techniques used-by Quaroni et al. (1979a,b) suffer from the lack of specific microvillar enzyme labeling, since all gl coconjugates pro3 H-fucose duced by the cells that incorporate are detected. The experiments performed by Hauri et al. (19791, had the benefit of a specific immunoprecipitahowever, tion step of a well-known brush-border enzyme. However, even this biochemical approach is hampered by the problem of obtaining pure fractions without cross-contamination of the various membranes present in the different It is not unlikely that the basodomains in the cell. lateral membrane fraction was contaminated by (trans?) Golgi membranes or intracellular transport vesicles, resulting in an apparent peak-labeling of this organelle. Although EM-autoradiography also showed silver grains over the lateral membrane (Cinsel, unpublished obser1981b) in the above-menvations; see also Blok et al., tioned pulse-chase experiments, the amount was at most only 5% of the total number and in all Sikelihood reflects synthesis of enzymes (e.g. Na+/K -ATPase) normally present in this domain. Our recent immunocytochemical studies (Fransen et al., 1984) performed in close collaboration with Hauri's group and with the use of cryosections and the protein A-gold technique (Geuze et al., 1981) have shown that one of the monoclonal antibodies directed against sucrase-isomaltase (HBB 2/614/88) labeled the structures that according to our hypothesis are involved in the intracellular transport, i.e., gold particles were present, randomly
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distributed over the Golgi apparatus, and over apical vesicles and tubules. Lysosomes were also labeled, an observation which will be discussed in detail below. However, in contrast to the numerous gold particles present over the microvillar membrane, no labeling was detectable over the basolateral membrane. Since this technique is more specific with respect to localization than those used in the autoradiographical and biochemical studies referred to above, transport of brush-border glyin our coproteins via the lateral plasma membrane is opinion, of minor importance, if it occurs at all. In sum, brush-border glycoproteins are completed in the Golgi apparatus and packaged in small vesicular and tubular structures. These apical vesicles and tubules transfer the glycoproteins to the apex of the cells where they can contribute to the apical membrane by an exocytic mechanism. In all likelihood, the lateral membrane is by-passed in this process. Effects of drugs on the transport of brush-border glycoproteins To investigate the transport route of brush-border glycoproteins and the cell organelles involved in it, several investigators have attempted to manipulate the process by using drugs such as monensin and colchicine. The effects of these derivates on the transport process will be discussed in detail here. Monensin It has been shown in a large number of cell types that the carboxylic ionophore monensin leads to diminution of the intracellular transport of secretory products (for reviews, see Tartakoff, 1982, 1983). At the ultrastructural level the presence of monensin results in severe swelling of the Golgi apparatus. Besides the latter effect, which was seen in vivo in the rat (Ellinger and Pavelka, 1984) as well as in vitro in explants originating from man (Fransen, Ginsel, Blok, unpublished observations) and the pig (Danielsen et al., 1983) it was found in intestinal absorptive cells that the drug interferes with the formation of complex-glycosylated brush-border glycoproteins (Danielsen et al., 1983). Together with these effects the transport of brushborder glycoproteins to the apical membrane of the cells is severely inhibited. Autoradiographical studies at the electronmicroscopical level showed that after culture of human ex lants for 60 min in the presence of 1 ~JM monensin and TH-fucose the number of silver grains present over the Golgi apbaratus increased from 3.5% in control cultures to 45.5% of the total number (Fransen, Ginsel, Blok; unpublished observations). Concomittant with this effect the number of silver grains present over the 1.
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microvilli decreased by approximately 50% in the presence of monensin. These results indicate that monensin is a potent inhibitor of the terminal process of the synthesis of brush-border glycoproteins. These macromolecules accumulated in the Golgi stacks (Fransen, Ginsel, Blok; unpublished observations). Colchicine Several studies on the inhibition of cellular secretion of macromolecules induced by colchicine have shown that the drug interferes with intracellular transport (for a review see Dustin, 1978). In the case of intestinal absorptive cells it has been shown (Reaven and Reaven, 1977; Pavelka and Gangl, 1983) that the presence of colchicine results in a decrease of the number of microtubules present in the cytoplasm of these cells. In addition, the drug induces a redistribution of apical vesicles and tubules, which are involved in the transport process of brush-border glycoproteins. Electron microscopy has shown that the presence of colchicine in cultures of human explants leads to a decrease in the number of these transport vesicles in the terminal web zone of whereas they were more abundant in the absorptive cells, the Golgi area (Ginsel et al., 1975; Buschmann, 1983; Michaels, 1983). Two other morphological changes were induced by the drug: Golgi stacks were displaced from their normal perior supranuclear position, showing a random distribution more apically in the absorptive cells (Reaven and Reaven, 1977; Blok and Ginsel, unpublished observations), and morphological structures developed at the basolateral membrane reminiscent of apical microvilli (Pavelka and Gangl, 1983; Pavelka et al., 1983). With respect to the intracellular transport of brushborder glycoproteins, autoradiographical (Blok et al., Ellinger et al., 1983) and biochemical data 1981b; (Quaroni et al., 197933; Blok et al., 1981b) have shown that in vivo in the rat and in vitro in human intestinal explants colchicine leads to a 50% reduction of the 3H-fucose-labeled macromolecules in the microvillar These observations indicate that microtubules membrane. are involved in the vectorial transport of brush-border glycoproteins from the Golgi apparatus to the microvillar membrane. The drug, however, also induced an increased transfer of radiolabeled material to the lateral plasma membrane (Quaroni et al., 197933; Blok et al., 1981b; Ellinger et which suggests that interference with the al., 1983), normal transport route to the microvillar membrane leads to the transfer of this material to the basolateral membrane. This process might even result, as already mentioned, in the formation of microvillar structures in this domain in the rat (Pavelka and Gangl, 1983; Pavelka 2.
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et al., 1983). However, this phenomenon was not observed in cultured human intestinal explants (Blok and Ginsel, unpublished observations). In our opinion, this is an artificially, drug-induced route, as indicated by our recent immunocytochemical observations (Fransen et al., 1984). With antibodies against sucrase-isomaltase, no labeling of the basolateral membrane was found. An increased transfer of radiolabeled material to the lysosomes of the absorptive cells in the presence of colchicine was also observed in the above-mentioned experiments on cultured human explants (Blok et al., 1981b). According to our hypothesis on the role of lysosomes in the regulation of intracellular transport of brush-border glycoproteins, which will be discussed in detail below, this might reflect an increased activity of the crinophagic mechanism, by which secretory vesicles fuse directly with lysosomes, ascribable to the impairment of the intracellular transport to the apical membrane. Lysosomes and intracellular degradation of cell-coat qlycoproteins In the apical cytoplasm of the absorptive cells two types of lysosomes can be distinguished at the ultrastructural level: multivesicular and dense bodies (Fig. The former organelles are spherical bodies with a 1). mean diameter of 0.35 urn and an electron-dense matrix with numerous vesicles. The dense bodies show a more homogeneous dense matrix and have a mean diameter of 0.53 urn (Ginsel et al., 1973; Blok et al., 1981a). After silver-proteinate staining, which detects the presence of glycoconjugates (Thiery, 1967), it was found that both types of lysosome showed an affinity for this stain similar to that of the glycoproteins present in the brushborder membrane (Fig. 1; Ginsel et al., 1973; Blok et al., 1982a). This was found in absorptive cells of normal subjects as well as in those of patients with various lysosomal storage diseases in which lysosomal function is impaired due to the absence of one or more hydrolases (Ginsel et al., 1973, 1978). Silver-staining material was also observed in the apical vesicles and tubules involved in intracellular transport and in the Golgi apparatus, with the intensity increasing from the cis to the trans side of the organelle. EM-autoradiographical pulse-chase experiments in cultured human intestinal explants showed that radioactive labeled material appeared at approximately the same time in the lysosomes and over the microvilli of the absorptive cells (Ginsel et al., 1979; Figs. 2 and 3). An increased amount of radiolabel, up to 25% of the total amount, was found in lysosomes of absorptive cells of patients suffering from a lysosomal storage disease (Ginsel et al., 1979). From these and earlier observations
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Fig. 6. EM-autoradiograph of the apical part of an absorptive cell cultured for 24 hr in the presence of 10V4 M chloroquine; the first 20 hr in the presence of 3H-fucose followed by a 4 hr chase. The dense bodies (edb) are significantly enlarged and show and increase in the amount of silver grains over their matrix (compare with Fig. grains over the microvilli is similar to 5). The number of silver that shown in Fig. 5. mvb, multivesicular body; x 16,800.
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Fig. 7. Ultrathin frozen section of an absorptive cell labeled an antibody against sucrase-isomaltase followed by a protein colloidal gold conjugate. Gold particles are present over the villi (mv) and a dense body (edb). Note the absence of label the lateral membrane (arrowheads). x 28,000.
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it was postulated that brush-border glycoproteins enter the lysosomes and that these organelles have a crinophagic function (i.e. fusion of transport vesicles with lysosomes) in the intracellular degradation of the macromolecules (Daems et al., 1973; Ginsel et al., 1973, 1979). Bennett and Leblond (1971), however, performed similar EM-autoradiographical studies in the rat and concluded that the radiolabel found in the lysosomes was due to de-novo synthesis of lysosomal enzymes. Both brush-border and lysosomal enzymes consist of glycoproteins and can be detected with the cytochemical and EM-autoradiographical procedures employed. For fur-therevaluation of the nature of the glycoproteins present in the lysosomes of the absorptive cells, Blok et al. (1981a, 1982a) performed experiments in which human intestinal explants were cultured in the presence of chloroquine. This antimalarial drug has been shown to inhibit intralysosomal degradation of various
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macromolecules such as proteins and mucopolysaccharides (Lee and Schofield, 1973; Mego and Chung, 1979; Seglen et al., 19791, most probably by raising the pH in these cell organelles and thus repressing the activity of the lysosomal enzymes with their acid pH optima (Ohkuma and EM-autoradiographic results showed that Poole, 1978). after 24 hr the presence of chloroquine resulted in an accumulation of silver grains over the lysosomes amounting to 17% of the total compared with 2.5% in the controls (Fig. 6, compare with Fig. 5; Blok et al., 1981a). However, the amount of the lysosomal enzymes B-glucuronidase and acid phosphatase as determined biochemically, was not influenced by the presence of the drug. however, From these data it was concluded (Blok et al., 1981a; 1981) that the radiolabel found see also Bennett et al., in the lysosomes in the autoradiographical experiments represents brush-border glycoproteins entering these organelles rather than that of lysosomal enzymes. This conclusion is supported by recent EM-immunocytochemical data obtained by Fransen et al. (1984), who used cryosections and a highly specific monoclonal antibody against sucrase-isomaltase and found that at least this microvillar membrane enzyme is present in the lysosomes (Fig. 7). Further immunocytochemical studies using antibodies against maltase-glycoamylase, lactase-phlorizin hydrolase, dipeptidyl peptidase IV, and aminopeptidase N are performed by our group at the moment to establish whether entry of brush-border enzymes into lysosomes is a general phenomenon. How do brush-border glycoproteins enter the lysosomes? In general, two routes are distinguished by which membrane-bound macromolecules can enter the lysosomes: endocytosis and crinophagy. The latter process was first described by Smith and Farquhar (1966) in the anterior pituitary gland, where secretory vesicles fuse with lysosomes. In 1973 it was postulated that brush-border glycoproteins enter the lysosomes of human intestinal absorptive cells by a similar mechanism (Daems et al., 1973; Ginsel et al., 1973). The presence of such a mechanism was suggested by the frequent morphological observation of fusion of apical vesicles and tubules, with lysosomes, and the similarity of silver-proteinate staining of apical vesicles and tubules, lysosomes, and the brush-border glycoproteins in the microvillar membrane of the absorptive cells. Additional indications for the existence of this mechanism were provided by an autoradiographical study which showed that radiolabeled brush-border glycoproteins arrived simultaneously at the microvilli and in the the latter being one of the types multivesicular bodies,
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Fig. 8. Apical part of a cell cultured for 2 hr in the presence of horseradish peroxidase. Multivesicular (mvb) and dense bodies (edb) show peroxidase reaction product. A large number of positively (arrows) and negatively (arrowheads) stained apical vesicles and tubules are present. x 18,600. of lysosome present in the apical cytoplasm (Ginsel et al., 1979). At a later stage the radiolabel appeared in the dense lysosome-like bodies. However, since endocytosis is a very rapid process, uptake of brush-border glycoproteins by this process in these studies could certainly not be completely excluded. Indeed, the intestine is known to be capable of endocytosing external macromolecular substances (for reviews see Walker and Isselbacher, 1974; Walker, 1981). Human intestinal absorptive cells are able to take up horseradish peroxidase (HRP) by an endocytotic mechanism (Blok et al., 1981c). This marker, which does not adhere to the brush-border glycoproteins and thus represents a fluidphase endocytotic pathway (Silverstein et al., 1977), was transported via apical vesicles and tubules to the lysosomes, first to the multivesicular bodies and later to the dense bodies (Fig. 8; Blok et al., 1981c). Similar experiments in which ferritin was used to visualize endocytosis showed, however, that this marker, which has a distinct affinity for the brush-border
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Fig. 9. Schematic representation of the postulated turnover of brush-border glycoproteins in human small-intestinal absorptive cells. The glycoproteins are completed in the Golgi apparatus Cl), transported to the apex of the cell via apical vesicles and tubules (2), and via exocytosis to cell coat contribute present at the base of the microvilli to the (31, whence it is transported tip of the microvilli (41, and finally released into the lumen (5). Part of the brush-border glycoproteins are transported directly to the multivesicular bodies by crinophagy (6) and then to the dense bodies (71, where they are degraded by the acid hydrolases, the breakdown products diffusing through the lysosomal membrane into the cytoendocytosis does plasm (8). Fluid-phase occur, but the brush-border glycoproteins are selectively excluded from this process.
glycoproteins, was not taken up by the absorptive cells (Blok et al., 1981c). These results indicate that although endocytosis of externally present macromolecules occurs, brush-border membrane glycoproteins are very probably excluded from this uptake mechanism. The nature of the interaction of the ferritin particles with the brush-border glycoproteins is unknown, however, and the possibility must be considered that prior to endocytosis of the latter, dissociation of the ferritin molecules occurs. An attempt was therefore made to inhibit fluidphase endocytosis of HRP by culturing human explants in the presence of cytochalasin D, a drug which interferes with the microfilament system (Blok et al., 198233). Morphometrical analysis of the number of (endocytotic) vesicles containing HRP present in the apical cytoplasm, showed that the drug inhibited endocytotic uptake of HRP by at least 60%. Autoradiographically, however, an equal amount of radioactive labeled material was found in the lysosomes of both control and cytochalasin -D-treated cultures, despite the inhibition of the endocytotic uptake of HRP. These results indicate that endocytosis of brush-border glycoproteins is a process which, if present, is of minor importance in the delivery of brush-border glycoproteins to the lysosomes (Fig. 9). It seems more likely
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that the brush-border glycoproteins enter the lysosornes one by which the apical vesby a crinophagic mechanism, icles and tubules fuse with the lysosomes to dispose of the excessively produced brush-border glycoproteins, as will be discussed here in more detail. What
is the functional role of the crinophagic mechanism? It is of interest to speculate on the role of the crinophagic mechanism in the physiology of the absorptive cells. It is possible that the synthesis of brushborder glycoproteins is influenced by the environmental conditions to which these macromolecules may be exposed in vivo. After food consumption, the increase of pancreatic proteases and bile salts in the lumen of the intestine might lead to enhanced degradation of brushborder enzymes (see Alpers and Tedesco, 1975). To maintain the functioning of the microvillar enzymes in the brush border, the absorptive cells would have to respond by increased exocytosis of brush-border enzymes. This might be realized by increased synthesis of these macromolecules. However, transcriptional and translational processes might be too time consuming to compensate for those deleterious effects with sufficient rapidity. Therefore it is conceivable that the absorptive cells maintain a relatively high steady-state synthesis of brush-border glycoproteins to ensure normal functioning of the brush-border dependent degradation of food constituents. When the level if harmful factors (e.g., pancreatic proteases and bile salts) decreases, crinophagy might function as a regulatory mechanism, brushborder glycoproteins being recycled by lysosomal degradation (Fig. 9). It has to be kept in mind, however, that only 5% of the synthesized material is degraded in this way. A crinophagic regulatory mechanism might permit the absorptive cells to maintain their normal digesting functions despite changes in the environmental conditions. Acknowledgements The authors are indebted to Prof. W.Th. Daems and Prof. J.P. Scherft for their critical reading of the manuscript. They also wish to thank Mrs. G.C.A.M. Spigtvan den Bercken and L.D.C. Verschragen for excellent technical assistance. References Alpers, D.H. and Tedesco, of pancreatic proteases brush border proteins. 401, 28-40. Bennett, G. (1970) Migration apparatus to cell coat
F.J. (1975) The possible role in the turnover of intestinal Biochimica et Biophysics Acta in
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duodenal epithelium. Journal of Cell Biology 45, 668673. Bennett, G. and Leblond, C.P. (1970) Formation of cell coat material for the whole surface of columnar cells in the rat small intestine, as visualized by radioautoradiography with L-fucose3H. Journal of Cell Biology 46, 409-417. Bennett, G. and Leblond, C.P. (1971) Passage of fucose3H label from the Golgi apparatus into dense and multivesicular bodies in the duodenal columnar cells and hepatocytes of the rat. Journal of Cell Biology 51, 875-881. Bennett, G., Leblond, C.P. and Haddad, A. (1974) Migration of glycoprotein from the Golgi apparatus to the surface of various cell types as shown by radioautography after labeled fucose injection into rats. Journal of Cell Biology 60, 258-284. Bennett, G., Kan, F.W.K. and O'Shaughnessy, D. (1981) The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of t3H)-N-acetylmannosamine. II Observations in tissues other than liver. Journal of Cell Biology 88, 16-28. Blok, J., Mulder-Stapel, A.A., Ginsel, L.A. and Daems, W.Th. (1981a). The effect of chloroguine on lysosomal function and cell-coat glycoprotein transport in the absorptive cells of cultured human small-intestinal tissue. Cell and Tissue Research 218, 227-251. Blok, J., Ginsel, L.A., Mulder-Stapel, A.A., Onderwater, J.J.M. and Daems, W.Th. (1981b). The effec of colchicine on the intracellular transport of f H-fucoselabelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue. Cell and Tissue Research 215, 1-12. Blok, J., Mulder-Stapel, A.A., Ginsel, L.A. and Daems, W.Th. (1981c). Endocytosis in absorptive cells of cultured human small-intestinal tissue: horseradish peroxidase, lactoperoxidase and ferritin as markers. Cell and Tissue Research 216, 1-13. Blok, J., Mulder-Stapel, A.A., Daems, W.Th. and Ginsel, L.A. (1982a) The effect of chloroguine on the intralysosomal degradation of cell-coat glycoproteins in the absorptive cells of cultured human small-intestinal tissue as shown by silver proteinate staining. Histochemistry 73, 429-438. Blok, J., Scheven, B.A.A., Mulder-Stapel, A.A., Ginsel, L.A. and Daems, W.Th. (1982b). Endocytosis in absorptive cells of cultured human small-intestinal tissue: Effect of cytochalasin B and D. Cell and Tissue Research 113-126. --222, (1983) Morphometry of the small-intestinal Buschmann, R.J.
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International
Reports,
Vol. 8, No. 12, December
1984
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rat and the effects of colenterocytes of the fasted Cell and Tissue Research --231, 289-299. chicine. Daems, W.Th., van Gemund, J.J., Vio, P.M.A., Willighagen, R.G.J. and den Tandt, W.R. (1973). The use of intestinal suction biopsy material for the study of lysosomal storage diseases. In: Hers, H.G., van Hoof, H. (eds) Lysosomal storage diseases, pp. 575-598. Academic Press, New York. Danielsen, E.M., SjZjstrBm, H. and Nor-en, 0. (1981) Biosynthesis of intestinal microvillas proteins. Putative precursor forms of microvillus aminopeptidase and sucrase-isomaltase isolated from Ca2+-precipitated enterocyte membranes. Federation of the European Biochemical Societies Letters 127, 129-132. Danielsen, E.M. (1982). Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes. Biochemical Journal 204, 161165. Danielsen, E.M., SjijstrZjm, H. and Noren, 0. (1983a) Biosynthesis of intestinal microvillar proteins. Pulsechase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV. Biochemical Journal 210, 389-393. -.Danielsen, E.M., Cowell, G.K. and Poulsen, S.S. (198333) Biosynthesis of intestinal microvillar proteins. Role of the Golgi complex and microtubules. Biochemical Journal 216, 37-42. Dustin, P. (1978) Microtubules, Springer Verlag, Berlin, Heidelberg. Ellinger, A. and Pavelka, M. (1984) Effect of monensin on the Golgi apparatus of absorptive cells in the small intestine of the rat. Cell and Tissue Research 235, 187-194. Ellinger, A., Pavelka, M. and Gangl, A. (1983) Effect of colchicine on rat small intestinal absorptive cells. II. Distribution of label after incorporation of 3Hfucose into plasma membrane glycoproteins. Journal of Ultrastructure Research 85, 260-271. Erickson, Y.S. (1983) PurifiTation and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV. Biochimica et Biophysica Acta 756, 258-265. Fransen, J.A.M., Hauri, H.P., Sterchi, E., Ginsel, L.A. and Blok, J. (1984) Irmnuno-electronmicroscopical localization of microvillus membrane disaccharidases in the human small-intestinal epithelium with monoclonal antibodies. Submitted for publication. Geuze, H-J., Slot, J.W., van der Ley, P.A. and Scheffer, R.C.T. (1981) Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen sections. Journal of Cell Biology --89, 653-665.
Cell Biology International
1012
Reports, Vol. 8, No. 12, December 1984
Ginsel, L.A., Daems, W.Th., Emeis, J.J., Vio, P.M.A. and van Gemund, J.J. (1973) Fine structure and silverstaining patterns of lysosome-like bodies in absorptive cells of the small intestine in normal children and children with a lysosomal storage disease. Virchows Archiv Abteilung B Zellpathologie 13, 119144. Ginsel, L.A., Debets, W.F. and Daems, W.Th. (1975) The effect of colchicine on the distribution of apical vesicles and tubules in absorptive cells of cultured human intestine. In: Borqers, M., de Brabander, M. (eds) Microtubules and microtubule inhibitors, pp. 187-198, North-Holland Publication Company, Amsterdam. Ginsel, L.A., Cambier, P.H. and Daems, W.Th. (1978) Fucosidosis and I-cell disease: a fine structural and silver-staining study of abnormal inclusion bodies in small-intestinal cells. Virchows Archiv B: Cell Pathology 27, 99-117. Ginsel, L.A., Onderwater, J.J.M. and Daems, W.Th. (1979) Transport of radiolabelled glycoprotein to cell-surface and lysosome-like bodies of absorptive cells of cultured small-intestinal tissue from normal subjects and patients with a lysosomal storage disease. Virchows Archiv B: Cell Pathology 30, 245-273. Hauri, H.P. (1983) Biosynthesis and transport of plasma membrane glycoproteins in the rat intestinal epithelial cell: studies with sucrase-isomaltase. In: Brush Ciba Foundation Symposium 95, pp. border membranes, 132-149, Pitman, London. Hauri, H.P., Quaroni, A. and Isselbacher, K.J. (1979) Biogenesis of intestinal plasma membrane: posttranslational route and cleavage of sucrase-isomaltase. Proceedings of the National Academy of Sciences of the USA 3, 5183-5186. Hauri, H.P., Quaroni, A. and Isselbacher, K.J. (1980) Monoclonal antibodies to sucrase-isomaltase: probes for the study of postnatal development and biogenesis of the intestinal microvillus membrane. Proceedings of the National Academy of Sciences of the USA 77, 6629-6633. Hauri, H.P., Wacker, H., Rickli, E.E., Bigler-Meier, B., Quaroni, A. and Semenza, G. (1982) Biosynthesis of Purification and NH2-terminal sucrase-isomaltase. amino acid sequence of the rat sucrase-isomaltase precursor (pro-sucrase-isomaltase) from fetal intesJournal of Biological Chemistry tinal transplants. 257,
4522-4528.
role of intracellular Lennarz, W. (1983) Overview: brane systems in glycosylation of proteins. Fleischer, S., Fleischer, B. (eds) Methods ology, vol. Academic Press, --98, pp. 91-97,
memIn: in EnzymNew York.
Cell Biology
International
Reports,
Vol. 8, No. 12, December
1984
1013
and Schofield, B. (1973) Inactivation of lysoS-0. somal function by chloroquine. Biochemical PharmacoloCJY 22, 3109-3114. (1979) Effects of some antiMego, J.L. and Chung, C.H. malarials and related substances on intralysosomal Biochemical Pharmacology 28, 465-470. proteolysis. -_Michaels, J.E. (1983) The effects of colchicine on the distribution of glycoprotein-containing vesicles in epithelial cells of the murine colon. Cell and Tissue Research 228, 323-335. Michaels, J.E. and Leblond, C.P. (1976) Transport of glycoprotein from Golgi apparatus to cell surface as shown by radioautoby means of carrier vesicles, raphy of mouse colonic epithelium after injection of jH-fucose . Journal de Microscopic et Biologie Cellulaire 25, 243-248. Ohkuma, S. and Poole, B. (1978) Fluorescent probe measurement of the intralysosomal pH in living cells and the pertubation of pH by various agents. Proceedings of the National Academy of Sciences of the USA 75, 3327-3331. M. and Gangl, A. (1983) Effect of colchicine on Pavelka, the intestinal transport of endogenous lipid. Gastroenterology, 84, 544-555. Pavelka, M., Ellinger, A. and Gangl, A. (1983) Effects of colchicine on rat small intestinal absorptive cells. I. Formation of basolateral microvillar borders. Journal of Ultrastructural Research 85, 249-259. Quaroni, A., Kirsch, K. and Weiser, M.M. (1979a). Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of L-(1,5,6-3H)fucose labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo. Biochemical Journal 182, 203-212. --Quaroni, A., Kirsch, K. and Weiser, M.M. (1979b) Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Effec of colchicine on the ref H)-fucose distribution of L-(1,5,6labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes. Biochemical Journal 182, 213-221. Reaven, E.P. and Reaven, E.M. (1977) Distribution and content of microtubules in relation to the transport of lipid. Journal of Cell Biology 75, 559-572. Seetharam, B., Grimme, N., Coodwin, C. and Alpers, D.H. (1976) Differential sensitivity of intestinal brush border enzymes to pancreatic and lysosomal enzymes. Life Sciences 18, 89-96. Seglen, P.O., Grinde, B. and Solheim, A.E. (1979) Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. European Journal of Biochemistry 2, 215-225. L&Z,
Cell Biology International
1014
Reports, Vol. 8, No. 12, December 1984
Silverstein, S.C., Steinman, R.M. and Cohn, Endocytosis. Annual Review of Biochemistry
Z.A. (1977) 46, 669-
722.
Sj6strBm, H., Nor&, O., Danielsen, E.M. and Skovbjerg, H. (1983) Structure of microvillar enzymes in different phases of their life cycles. In: Brush border membranes, Ciba Foundation Symposium 95, pp. 50-72, Pitman, London. M.G. (1966) Lysosome function Smith, R.E. and Farquhar, in the regulation of the secretory process in cells of the anterior pituitary gland. Journal of Cell Biology 31, 319-347. Tartakoff, ATM. (1982) Simplifying the Golgi complex. Trends in Biochemical Sciences 1, 174-176. of the structure and Tartakoff, A.M. (1983) Pertubation function of the Golgi complex by monovalent carboxylic ionophores. In: Fleischer, S., Fleischer, B. (eds) Methods in enzymology, vol. 98, pp. 47-59, Academic Press, New York. des polysaccharides Thiery, J.P. (1967) Mise en Evidence sur coupes fine en microscopic electronique. Journal de Microscopic 5, 987-1018. Wacker, H., Jaussi, R., Sonderegger, P., Dokow, M., Ghersa, P., Hauri, H.P., Christen, P. and Semenza, G. (1981) Cell-free synthesis of the one chain precursor of a major intrinsic protein complex of the smallintestinal brush-border membrane (pro-sucrase-isomaltase). Federation of the European Biochemical Societies Letters 136, 329-332. uptake in the gut: immunoWalker, W.A. (1981) Antigen logic implications. Immunology Today 2, 30-34. Walker, W.A. and Isselbacher, K.J. (1974) Uptake and transport of macromolecules by the intestine. Possible role in clinical disorders. Gastroenterology 67, 531-550.
Received:
10th September 1984.
Accepted:
24th October 1984.
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MINI-REVIEW TURNOVER OF BRUSH-BORDER GLYCOPROTEINS IN HUMAN INTESTINAL ABSORPTIVE CELLS: DO LYSOSOMES HAVE A REGULATORY FUNCTION? John Laboratories University
Blok2, of
Jack
A.M.
Leo
Fransen',
for Electron Microscopy' Leiden, Rijnsburgerweg The Netherlands
A.
Ginsell
and Cell 10, 2333
Biology2, AA Leiden
Introduction The absorptive cells lining the small intestine are columnar cells whose surface membranes show a striking polarization. At the luminal side the plasma membrane is folded into finger-like extrusions, the microvilli, which are covered with a prominent cell coat. Separated from this brush-border domain by the junctional complexes, is the basolateral membrane, which is strongly interdigitated with that of neighbouring epithelial cells (Fig. 1) The cell coat of the luminal plasma membrane contains disaccharidases and peptidases), which the enzymes (e.g., in addition to the pancreatic proteases are involved in the degradation of intraluminal food constituents, thus making the end-products of these processes available for uptake in the intestine. These enzymes, which are glycoproteins, are synthesized by the absorptive cells themselves (Bennett and Leblond, 1970; Ginsel et al., 1979). Since the lumen of the small intestine can be assumed to be a hostile environment for the brush-border glycoproteins due to the presence of pancreatic proteases and bile salts (Alpers and Tedesco, 1975), the absorptive cells maintain a continuous synthesis of these macroprobably to compensate for the extracellular molecules, loss of brush-border enzymes. In addition to the extracellular turnover of cell-coat material, an intracellular catabolic mechanism has also been mentioned (Daems et al. 1973; Seetharam et al., 1976; Ginsel et al., 1979). Autoradiographical data (Ginsel et al., 1979; Blok et al., 1981a,b) as well as a recent immunocytochemical study (Fransen et al., 1984) have indeed shown that part of the newly synthesized brush-border constituents are transferred to the lysosomes present in the apical cytoplasm of the absorptive cells. In this mini-review, data concerning the above described phenomena will be summarized and special attention will be given to the role of lysosomes in intracellular catabolism of brush-border enzymes. In the first 030!3-1651/84/120993-22l$O3.Q0/0
0
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part the biosynthesis of some brush-border enzymes will be considered briefly. The second part will focus on the intracellular transport route of brush-border glycoproteins to the microvilli and the cell organelles involved in this process. A possible involvement of the basolateral membrane in the transport process will also be discussed. The third part gives an overview of the lysosoma1 catabolism of brush-border constituents and speculates on the physiological meaning of such a process. Synthesis of brush-border enzymes It is generally accepted that the peptidases and disaccharidases of the microvillar membrane are synthesized in a way that is essentially similar to the synthesis of other macromolecules destined for secretion (Lennarz, 1983). Thus, the protein backbone is synthesized by the ribosomes of the rough endoplasmic reticulum (RER), linking the various amino acids to yield the translation product. Post-translational modification of the protein core in the RER by the addition of various sugar residues, especially mannose, yield the intermediate products which are known as high-mannose glycoproteins. After transfer to the Golgi apparatus these glycoproteins are trimmed and converted to complex glycoproteins by the addition of other sugars such as N-acetyl glucosamine, galactose, fucose and sialic acid (Bennett, 1970; Bennett et al., 1974; Ginsel et al., 1979; Bennett et al., 1981). An example is the study by Danielsen et al. (1983a) who have used a cell-free microsomal translation system with 35S-methionine as radioactive marker to investigate the synthesis of the brush-border enzyme aminopeptidase A (see also Wacker et al., 1981, for sucrase-isomaltase). Using immunoprecipitation and SDS-gel electrophoresis they found in pulse-chase experiments that the enzyme was synthesized as a primary translational peptide with an M, of 115,000. At later time intervals this product was processed to one of an II, of 140,000, which was sensitive to the action of endo-glycosidase H. This indicates the conversion to a high-mannose glycosylated intermediate, since complex glycoproteins are resistent to treatment by this enzyme. After transfer of the brush--. border enzymes to the Golgi apparatus they are converted cell of the small intestine after silver protein.-Fig. 1. Absorptive ate staining. A fine granular staining is present on the microvilli (mv), dense bodies (edb), a multivesicular body (mvb) and the Golgi web (tw) positive (arrowheads) as apparatus (Ga). In the terminal well as negative (small arrows) apical vesicles and tubules can be observed. Glycogen is present dispersed in the cytoplasm. N, nucleus; m, mitochondrion: x 12,800.
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to the complex mature form., as shown by studies on the synthesis of the peptidases aminopeptidase A and N and dipeptidyl peptidase IV and the disaccharidases sucraseisomaltase and maltase-glycoamylase (Danielsen, 1982; Danielsen et al., 1981, 1983; Erickson et al., 1983; Hauri et al., 1980, 1982; for reviews, see Hauri, 1983 and Sjastrom et al., 1983). Although in general the synthesis of peptidases and disaccharidases is similar, one major difference can be observed. The peptidases are syn-thesized as monomers, whereas the disaccharidases are assembled as dimers. The latter (sucrase-isomaltase, mal-tase-glycoamylase, lactase-phlorizin hydrolase) are converted to monomers by the action of the pancreatic proteolytic enzyme elastase (Hauri et al., 1979). The intracellular transport route of brush-border glycoproteins The conversion of brush-border glycoproteins from the high-mannose to the complex glycosylated form, as described above, takes place in the Golgi apparatus of the absorptive cells. Electron-microscopical autoradiography has shown that the in vivo or in vitro presence of tritiated sugars such as galactose, fucose and sialic acid, which are either terminally or subterminally located in the glycoproteins, results in a significant labeling of Golgi stacks within a short time (Figs. 2 and 4; Bennett, 1970; Bennett et al., 1974; Ginsel et al., 1979; Bennett et al., 1981). To identify the intracellular transport route of the brush-border glycoproteins, Ginsel et al. ,(1979) performed EM-autoradiographical experiments in which human intestinal explants were labeled in tissue culture for 10 min with 3H-fucose and chased for various periods up to 24 hr. It was found that after a 10 min pulse, 40% of the silver grains were located over the Golgi apparatus of the cells. This label decreased after chasing, but a peak of radiolabel appeared after a 35 min chase period in vesicular and tubular structures prominently present in the apex of the absorptive cells (Fig. the same time the number of silver 2) - At approximately grains located on the microvilli started to increase (Figs. 2 and 5). Similar results had been found in vivo in the rat (Bennett and Leblond, 1970; Bennett et al., 1974, 1981). These findings led to the hypothesis that the brush-border glycoproteins are completed in the Golgi apparatus and packaged there in small vesicular and tubular structures, by which they are transported directly to the microvillar membrane (Ginsel et al., 1979; Blok et al., 1981b; see also Michaels and Leblond, 1976). Insertion of the brush-border glycoproteins into the micro-. villar membrane was thought to take place by fusion of the vesicles and tubules with this membrane (exocytosis). At about the same time, Quaroni et al. (1979a,b), and
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golgi apparatus 0
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02
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Fig. 2. Distribution, in percentages, of the silver grains over the Golgi apparatus, apical vesicles and tubules, microvilli and glycocalyx. The absorptive cells were labeled for 10 min with 3H-fucose and chased for various periods indicated on the abscissa. The data indicate a transport of brush-border glycoproteins via the apical vesicles and tubules to the microvilli and later to the glycocalyx.
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40 ,”
60
golgi apparatus
.0-T\
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Fig. 3. Distribution, in percentages, of the silver grains over the Golgi apparatus, apical vesicles and tubules, and multivesicular and dense bodies. The cells were labeled and chased as described in Fig. 2. The data suggest that brush-border glycoproteins are also transported from the Golgi apparatus and apical vesicles and tubules to the multivesicular bodies and later to the dense bodies. Fig. 4. EM-autoradiograph Sf the Golgi zone of an absorptive cell after a 10 min pulse with H-fucose. Silver grains are located over Golgi cisternae (arrowheads) as well as vesicles (arrows). x 24,600. Fig. 5. EM-autoradiograph of the apical cytoplasm of an absorptive cell after a 3 hr chase period. Silver grains are located over the microvilli (mv), a dense body (edb) and an apical vesicle (arrowhead). x 21,000.
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Hauri et al. (1979) presented biochemical data which sug-gested that there might also be an indirect transport route of brush-border glycoproteins, i.e., via the later-al plasma membrane to the microvilli. In these studies Quaroni et al. (1979a,b) injected rats with tritiated fucose and after various time intervals isolated membrane fractions of the absorptive cells, consisting of a Golgi, a basolateral, and a brush-border fraction, and determined the amount of radiolabel present in them. Hauri et al. (1979) performed similar experiments, but in addition immunoprecipitated sucrase-isomaltase, one of the most abundant enzymes of the disaccharidases present in the intestine, from the above-mentioned membrane fractions. They found that the radioactivity was initially present in the Golgi fraction and,at later time-points (60 min), in the brush-border membrane fraction. However, a peak of radioactive labeled glycoproteins was also found after 30-60 min in the basolateral membrane fraction. On these grounds it was postulated that at least part of the brush-border glycoproteins are transported via the lateral membrane to the apical membrane. How can the discrepancies between the autoradiographical and biochemical data be explained? Autoradiography as well as the biochemical techniques used-by Quaroni et al. (1979a,b) suffer from the lack of specific microvillar enzyme labeling, since all gl coconjugates pro3 H-fucose duced by the cells that incorporate are detected. The experiments performed by Hauri et al. (19791, had the benefit of a specific immunoprecipitahowever, tion step of a well-known brush-border enzyme. However, even this biochemical approach is hampered by the problem of obtaining pure fractions without cross-contamination of the various membranes present in the different It is not unlikely that the basodomains in the cell. lateral membrane fraction was contaminated by (trans?) Golgi membranes or intracellular transport vesicles, resulting in an apparent peak-labeling of this organelle. Although EM-autoradiography also showed silver grains over the lateral membrane (Cinsel, unpublished obser1981b) in the above-menvations; see also Blok et al., tioned pulse-chase experiments, the amount was at most only 5% of the total number and in all Sikelihood reflects synthesis of enzymes (e.g. Na+/K -ATPase) normally present in this domain. Our recent immunocytochemical studies (Fransen et al., 1984) performed in close collaboration with Hauri's group and with the use of cryosections and the protein A-gold technique (Geuze et al., 1981) have shown that one of the monoclonal antibodies directed against sucrase-isomaltase (HBB 2/614/88) labeled the structures that according to our hypothesis are involved in the intracellular transport, i.e., gold particles were present, randomly
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distributed over the Golgi apparatus, and over apical vesicles and tubules. Lysosomes were also labeled, an observation which will be discussed in detail below. However, in contrast to the numerous gold particles present over the microvillar membrane, no labeling was detectable over the basolateral membrane. Since this technique is more specific with respect to localization than those used in the autoradiographical and biochemical studies referred to above, transport of brush-border glyin our coproteins via the lateral plasma membrane is opinion, of minor importance, if it occurs at all. In sum, brush-border glycoproteins are completed in the Golgi apparatus and packaged in small vesicular and tubular structures. These apical vesicles and tubules transfer the glycoproteins to the apex of the cells where they can contribute to the apical membrane by an exocytic mechanism. In all likelihood, the lateral membrane is by-passed in this process. Effects of drugs on the transport of brush-border glycoproteins To investigate the transport route of brush-border glycoproteins and the cell organelles involved in it, several investigators have attempted to manipulate the process by using drugs such as monensin and colchicine. The effects of these derivates on the transport process will be discussed in detail here. Monensin It has been shown in a large number of cell types that the carboxylic ionophore monensin leads to diminution of the intracellular transport of secretory products (for reviews, see Tartakoff, 1982, 1983). At the ultrastructural level the presence of monensin results in severe swelling of the Golgi apparatus. Besides the latter effect, which was seen in vivo in the rat (Ellinger and Pavelka, 1984) as well as in vitro in explants originating from man (Fransen, Ginsel, Blok, unpublished observations) and the pig (Danielsen et al., 1983) it was found in intestinal absorptive cells that the drug interferes with the formation of complex-glycosylated brush-border glycoproteins (Danielsen et al., 1983). Together with these effects the transport of brushborder glycoproteins to the apical membrane of the cells is severely inhibited. Autoradiographical studies at the electronmicroscopical level showed that after culture of human ex lants for 60 min in the presence of 1 ~JM monensin and TH-fucose the number of silver grains present over the Golgi apbaratus increased from 3.5% in control cultures to 45.5% of the total number (Fransen, Ginsel, Blok; unpublished observations). Concomittant with this effect the number of silver grains present over the 1.
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microvilli decreased by approximately 50% in the presence of monensin. These results indicate that monensin is a potent inhibitor of the terminal process of the synthesis of brush-border glycoproteins. These macromolecules accumulated in the Golgi stacks (Fransen, Ginsel, Blok; unpublished observations). Colchicine Several studies on the inhibition of cellular secretion of macromolecules induced by colchicine have shown that the drug interferes with intracellular transport (for a review see Dustin, 1978). In the case of intestinal absorptive cells it has been shown (Reaven and Reaven, 1977; Pavelka and Gangl, 1983) that the presence of colchicine results in a decrease of the number of microtubules present in the cytoplasm of these cells. In addition, the drug induces a redistribution of apical vesicles and tubules, which are involved in the transport process of brush-border glycoproteins. Electron microscopy has shown that the presence of colchicine in cultures of human explants leads to a decrease in the number of these transport vesicles in the terminal web zone of whereas they were more abundant in the absorptive cells, the Golgi area (Ginsel et al., 1975; Buschmann, 1983; Michaels, 1983). Two other morphological changes were induced by the drug: Golgi stacks were displaced from their normal perior supranuclear position, showing a random distribution more apically in the absorptive cells (Reaven and Reaven, 1977; Blok and Ginsel, unpublished observations), and morphological structures developed at the basolateral membrane reminiscent of apical microvilli (Pavelka and Gangl, 1983; Pavelka et al., 1983). With respect to the intracellular transport of brushborder glycoproteins, autoradiographical (Blok et al., Ellinger et al., 1983) and biochemical data 1981b; (Quaroni et al., 197933; Blok et al., 1981b) have shown that in vivo in the rat and in vitro in human intestinal explants colchicine leads to a 50% reduction of the 3H-fucose-labeled macromolecules in the microvillar These observations indicate that microtubules membrane. are involved in the vectorial transport of brush-border glycoproteins from the Golgi apparatus to the microvillar membrane. The drug, however, also induced an increased transfer of radiolabeled material to the lateral plasma membrane (Quaroni et al., 197933; Blok et al., 1981b; Ellinger et which suggests that interference with the al., 1983), normal transport route to the microvillar membrane leads to the transfer of this material to the basolateral membrane. This process might even result, as already mentioned, in the formation of microvillar structures in this domain in the rat (Pavelka and Gangl, 1983; Pavelka 2.
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et al., 1983). However, this phenomenon was not observed in cultured human intestinal explants (Blok and Ginsel, unpublished observations). In our opinion, this is an artificially, drug-induced route, as indicated by our recent immunocytochemical observations (Fransen et al., 1984). With antibodies against sucrase-isomaltase, no labeling of the basolateral membrane was found. An increased transfer of radiolabeled material to the lysosomes of the absorptive cells in the presence of colchicine was also observed in the above-mentioned experiments on cultured human explants (Blok et al., 1981b). According to our hypothesis on the role of lysosomes in the regulation of intracellular transport of brush-border glycoproteins, which will be discussed in detail below, this might reflect an increased activity of the crinophagic mechanism, by which secretory vesicles fuse directly with lysosomes, ascribable to the impairment of the intracellular transport to the apical membrane. Lysosomes and intracellular degradation of cell-coat qlycoproteins In the apical cytoplasm of the absorptive cells two types of lysosomes can be distinguished at the ultrastructural level: multivesicular and dense bodies (Fig. The former organelles are spherical bodies with a 1). mean diameter of 0.35 urn and an electron-dense matrix with numerous vesicles. The dense bodies show a more homogeneous dense matrix and have a mean diameter of 0.53 urn (Ginsel et al., 1973; Blok et al., 1981a). After silver-proteinate staining, which detects the presence of glycoconjugates (Thiery, 1967), it was found that both types of lysosome showed an affinity for this stain similar to that of the glycoproteins present in the brushborder membrane (Fig. 1; Ginsel et al., 1973; Blok et al., 1982a). This was found in absorptive cells of normal subjects as well as in those of patients with various lysosomal storage diseases in which lysosomal function is impaired due to the absence of one or more hydrolases (Ginsel et al., 1973, 1978). Silver-staining material was also observed in the apical vesicles and tubules involved in intracellular transport and in the Golgi apparatus, with the intensity increasing from the cis to the trans side of the organelle. EM-autoradiographical pulse-chase experiments in cultured human intestinal explants showed that radioactive labeled material appeared at approximately the same time in the lysosomes and over the microvilli of the absorptive cells (Ginsel et al., 1979; Figs. 2 and 3). An increased amount of radiolabel, up to 25% of the total amount, was found in lysosomes of absorptive cells of patients suffering from a lysosomal storage disease (Ginsel et al., 1979). From these and earlier observations
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Fig. 6. EM-autoradiograph of the apical part of an absorptive cell cultured for 24 hr in the presence of 10V4 M chloroquine; the first 20 hr in the presence of 3H-fucose followed by a 4 hr chase. The dense bodies (edb) are significantly enlarged and show and increase in the amount of silver grains over their matrix (compare with Fig. grains over the microvilli is similar to 5). The number of silver that shown in Fig. 5. mvb, multivesicular body; x 16,800.
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Fig. 7. Ultrathin frozen section of an absorptive cell labeled an antibody against sucrase-isomaltase followed by a protein colloidal gold conjugate. Gold particles are present over the villi (mv) and a dense body (edb). Note the absence of label the lateral membrane (arrowheads). x 28,000.
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it was postulated that brush-border glycoproteins enter the lysosomes and that these organelles have a crinophagic function (i.e. fusion of transport vesicles with lysosomes) in the intracellular degradation of the macromolecules (Daems et al., 1973; Ginsel et al., 1973, 1979). Bennett and Leblond (1971), however, performed similar EM-autoradiographical studies in the rat and concluded that the radiolabel found in the lysosomes was due to de-novo synthesis of lysosomal enzymes. Both brush-border and lysosomal enzymes consist of glycoproteins and can be detected with the cytochemical and EM-autoradiographical procedures employed. For fur-therevaluation of the nature of the glycoproteins present in the lysosomes of the absorptive cells, Blok et al. (1981a, 1982a) performed experiments in which human intestinal explants were cultured in the presence of chloroquine. This antimalarial drug has been shown to inhibit intralysosomal degradation of various
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macromolecules such as proteins and mucopolysaccharides (Lee and Schofield, 1973; Mego and Chung, 1979; Seglen et al., 19791, most probably by raising the pH in these cell organelles and thus repressing the activity of the lysosomal enzymes with their acid pH optima (Ohkuma and EM-autoradiographic results showed that Poole, 1978). after 24 hr the presence of chloroquine resulted in an accumulation of silver grains over the lysosomes amounting to 17% of the total compared with 2.5% in the controls (Fig. 6, compare with Fig. 5; Blok et al., 1981a). However, the amount of the lysosomal enzymes B-glucuronidase and acid phosphatase as determined biochemically, was not influenced by the presence of the drug. however, From these data it was concluded (Blok et al., 1981a; 1981) that the radiolabel found see also Bennett et al., in the lysosomes in the autoradiographical experiments represents brush-border glycoproteins entering these organelles rather than that of lysosomal enzymes. This conclusion is supported by recent EM-immunocytochemical data obtained by Fransen et al. (1984), who used cryosections and a highly specific monoclonal antibody against sucrase-isomaltase and found that at least this microvillar membrane enzyme is present in the lysosomes (Fig. 7). Further immunocytochemical studies using antibodies against maltase-glycoamylase, lactase-phlorizin hydrolase, dipeptidyl peptidase IV, and aminopeptidase N are performed by our group at the moment to establish whether entry of brush-border enzymes into lysosomes is a general phenomenon. How do brush-border glycoproteins enter the lysosomes? In general, two routes are distinguished by which membrane-bound macromolecules can enter the lysosomes: endocytosis and crinophagy. The latter process was first described by Smith and Farquhar (1966) in the anterior pituitary gland, where secretory vesicles fuse with lysosomes. In 1973 it was postulated that brush-border glycoproteins enter the lysosomes of human intestinal absorptive cells by a similar mechanism (Daems et al., 1973; Ginsel et al., 1973). The presence of such a mechanism was suggested by the frequent morphological observation of fusion of apical vesicles and tubules, with lysosomes, and the similarity of silver-proteinate staining of apical vesicles and tubules, lysosomes, and the brush-border glycoproteins in the microvillar membrane of the absorptive cells. Additional indications for the existence of this mechanism were provided by an autoradiographical study which showed that radiolabeled brush-border glycoproteins arrived simultaneously at the microvilli and in the the latter being one of the types multivesicular bodies,
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Fig. 8. Apical part of a cell cultured for 2 hr in the presence of horseradish peroxidase. Multivesicular (mvb) and dense bodies (edb) show peroxidase reaction product. A large number of positively (arrows) and negatively (arrowheads) stained apical vesicles and tubules are present. x 18,600. of lysosome present in the apical cytoplasm (Ginsel et al., 1979). At a later stage the radiolabel appeared in the dense lysosome-like bodies. However, since endocytosis is a very rapid process, uptake of brush-border glycoproteins by this process in these studies could certainly not be completely excluded. Indeed, the intestine is known to be capable of endocytosing external macromolecular substances (for reviews see Walker and Isselbacher, 1974; Walker, 1981). Human intestinal absorptive cells are able to take up horseradish peroxidase (HRP) by an endocytotic mechanism (Blok et al., 1981c). This marker, which does not adhere to the brush-border glycoproteins and thus represents a fluidphase endocytotic pathway (Silverstein et al., 1977), was transported via apical vesicles and tubules to the lysosomes, first to the multivesicular bodies and later to the dense bodies (Fig. 8; Blok et al., 1981c). Similar experiments in which ferritin was used to visualize endocytosis showed, however, that this marker, which has a distinct affinity for the brush-border
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Fig. 9. Schematic representation of the postulated turnover of brush-border glycoproteins in human small-intestinal absorptive cells. The glycoproteins are completed in the Golgi apparatus Cl), transported to the apex of the cell via apical vesicles and tubules (2), and via exocytosis to cell coat contribute present at the base of the microvilli to the (31, whence it is transported tip of the microvilli (41, and finally released into the lumen (5). Part of the brush-border glycoproteins are transported directly to the multivesicular bodies by crinophagy (6) and then to the dense bodies (71, where they are degraded by the acid hydrolases, the breakdown products diffusing through the lysosomal membrane into the cytoendocytosis does plasm (8). Fluid-phase occur, but the brush-border glycoproteins are selectively excluded from this process.
glycoproteins, was not taken up by the absorptive cells (Blok et al., 1981c). These results indicate that although endocytosis of externally present macromolecules occurs, brush-border membrane glycoproteins are very probably excluded from this uptake mechanism. The nature of the interaction of the ferritin particles with the brush-border glycoproteins is unknown, however, and the possibility must be considered that prior to endocytosis of the latter, dissociation of the ferritin molecules occurs. An attempt was therefore made to inhibit fluidphase endocytosis of HRP by culturing human explants in the presence of cytochalasin D, a drug which interferes with the microfilament system (Blok et al., 198233). Morphometrical analysis of the number of (endocytotic) vesicles containing HRP present in the apical cytoplasm, showed that the drug inhibited endocytotic uptake of HRP by at least 60%. Autoradiographically, however, an equal amount of radioactive labeled material was found in the lysosomes of both control and cytochalasin -D-treated cultures, despite the inhibition of the endocytotic uptake of HRP. These results indicate that endocytosis of brush-border glycoproteins is a process which, if present, is of minor importance in the delivery of brush-border glycoproteins to the lysosomes (Fig. 9). It seems more likely
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that the brush-border glycoproteins enter the lysosornes one by which the apical vesby a crinophagic mechanism, icles and tubules fuse with the lysosomes to dispose of the excessively produced brush-border glycoproteins, as will be discussed here in more detail. What
is the functional role of the crinophagic mechanism? It is of interest to speculate on the role of the crinophagic mechanism in the physiology of the absorptive cells. It is possible that the synthesis of brushborder glycoproteins is influenced by the environmental conditions to which these macromolecules may be exposed in vivo. After food consumption, the increase of pancreatic proteases and bile salts in the lumen of the intestine might lead to enhanced degradation of brushborder enzymes (see Alpers and Tedesco, 1975). To maintain the functioning of the microvillar enzymes in the brush border, the absorptive cells would have to respond by increased exocytosis of brush-border enzymes. This might be realized by increased synthesis of these macromolecules. However, transcriptional and translational processes might be too time consuming to compensate for those deleterious effects with sufficient rapidity. Therefore it is conceivable that the absorptive cells maintain a relatively high steady-state synthesis of brush-border glycoproteins to ensure normal functioning of the brush-border dependent degradation of food constituents. When the level if harmful factors (e.g., pancreatic proteases and bile salts) decreases, crinophagy might function as a regulatory mechanism, brushborder glycoproteins being recycled by lysosomal degradation (Fig. 9). It has to be kept in mind, however, that only 5% of the synthesized material is degraded in this way. A crinophagic regulatory mechanism might permit the absorptive cells to maintain their normal digesting functions despite changes in the environmental conditions. Acknowledgements The authors are indebted to Prof. W.Th. Daems and Prof. J.P. Scherft for their critical reading of the manuscript. They also wish to thank Mrs. G.C.A.M. Spigtvan den Bercken and L.D.C. Verschragen for excellent technical assistance. References Alpers, D.H. and Tedesco, of pancreatic proteases brush border proteins. 401, 28-40. Bennett, G. (1970) Migration apparatus to cell coat
F.J. (1975) The possible role in the turnover of intestinal Biochimica et Biophysics Acta in
of glycoprotein the columnar
from cells
of
Golgi the
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Cell Biology
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Reports,
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duodenal epithelium. Journal of Cell Biology 45, 668673. Bennett, G. and Leblond, C.P. (1970) Formation of cell coat material for the whole surface of columnar cells in the rat small intestine, as visualized by radioautoradiography with L-fucose3H. Journal of Cell Biology 46, 409-417. Bennett, G. and Leblond, C.P. (1971) Passage of fucose3H label from the Golgi apparatus into dense and multivesicular bodies in the duodenal columnar cells and hepatocytes of the rat. Journal of Cell Biology 51, 875-881. Bennett, G., Leblond, C.P. and Haddad, A. (1974) Migration of glycoprotein from the Golgi apparatus to the surface of various cell types as shown by radioautography after labeled fucose injection into rats. Journal of Cell Biology 60, 258-284. Bennett, G., Kan, F.W.K. and O'Shaughnessy, D. (1981) The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of t3H)-N-acetylmannosamine. II Observations in tissues other than liver. Journal of Cell Biology 88, 16-28. Blok, J., Mulder-Stapel, A.A., Ginsel, L.A. and Daems, W.Th. (1981a). The effect of chloroguine on lysosomal function and cell-coat glycoprotein transport in the absorptive cells of cultured human small-intestinal tissue. Cell and Tissue Research 218, 227-251. Blok, J., Ginsel, L.A., Mulder-Stapel, A.A., Onderwater, J.J.M. and Daems, W.Th. (1981b). The effec of colchicine on the intracellular transport of f H-fucoselabelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue. Cell and Tissue Research 215, 1-12. Blok, J., Mulder-Stapel, A.A., Ginsel, L.A. and Daems, W.Th. (1981c). Endocytosis in absorptive cells of cultured human small-intestinal tissue: horseradish peroxidase, lactoperoxidase and ferritin as markers. Cell and Tissue Research 216, 1-13. Blok, J., Mulder-Stapel, A.A., Daems, W.Th. and Ginsel, L.A. (1982a) The effect of chloroguine on the intralysosomal degradation of cell-coat glycoproteins in the absorptive cells of cultured human small-intestinal tissue as shown by silver proteinate staining. Histochemistry 73, 429-438. Blok, J., Scheven, B.A.A., Mulder-Stapel, A.A., Ginsel, L.A. and Daems, W.Th. (1982b). Endocytosis in absorptive cells of cultured human small-intestinal tissue: Effect of cytochalasin B and D. Cell and Tissue Research 113-126. --222, (1983) Morphometry of the small-intestinal Buschmann, R.J.
Cell Biology
International
Reports,
Vol. 8, No. 12, December
1984
1011
rat and the effects of colenterocytes of the fasted Cell and Tissue Research --231, 289-299. chicine. Daems, W.Th., van Gemund, J.J., Vio, P.M.A., Willighagen, R.G.J. and den Tandt, W.R. (1973). The use of intestinal suction biopsy material for the study of lysosomal storage diseases. In: Hers, H.G., van Hoof, H. (eds) Lysosomal storage diseases, pp. 575-598. Academic Press, New York. Danielsen, E.M., SjZjstrBm, H. and Nor-en, 0. (1981) Biosynthesis of intestinal microvillas proteins. Putative precursor forms of microvillus aminopeptidase and sucrase-isomaltase isolated from Ca2+-precipitated enterocyte membranes. Federation of the European Biochemical Societies Letters 127, 129-132. Danielsen, E.M. (1982). Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes. Biochemical Journal 204, 161165. Danielsen, E.M., SjijstrZjm, H. and Noren, 0. (1983a) Biosynthesis of intestinal microvillar proteins. Pulsechase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV. Biochemical Journal 210, 389-393. -.Danielsen, E.M., Cowell, G.K. and Poulsen, S.S. (198333) Biosynthesis of intestinal microvillar proteins. Role of the Golgi complex and microtubules. Biochemical Journal 216, 37-42. Dustin, P. (1978) Microtubules, Springer Verlag, Berlin, Heidelberg. Ellinger, A. and Pavelka, M. (1984) Effect of monensin on the Golgi apparatus of absorptive cells in the small intestine of the rat. Cell and Tissue Research 235, 187-194. Ellinger, A., Pavelka, M. and Gangl, A. (1983) Effect of colchicine on rat small intestinal absorptive cells. II. Distribution of label after incorporation of 3Hfucose into plasma membrane glycoproteins. Journal of Ultrastructure Research 85, 260-271. Erickson, Y.S. (1983) PurifiTation and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV. Biochimica et Biophysica Acta 756, 258-265. Fransen, J.A.M., Hauri, H.P., Sterchi, E., Ginsel, L.A. and Blok, J. (1984) Irmnuno-electronmicroscopical localization of microvillus membrane disaccharidases in the human small-intestinal epithelium with monoclonal antibodies. Submitted for publication. Geuze, H-J., Slot, J.W., van der Ley, P.A. and Scheffer, R.C.T. (1981) Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen sections. Journal of Cell Biology --89, 653-665.
Cell Biology International
1012
Reports, Vol. 8, No. 12, December 1984
Ginsel, L.A., Daems, W.Th., Emeis, J.J., Vio, P.M.A. and van Gemund, J.J. (1973) Fine structure and silverstaining patterns of lysosome-like bodies in absorptive cells of the small intestine in normal children and children with a lysosomal storage disease. Virchows Archiv Abteilung B Zellpathologie 13, 119144. Ginsel, L.A., Debets, W.F. and Daems, W.Th. (1975) The effect of colchicine on the distribution of apical vesicles and tubules in absorptive cells of cultured human intestine. In: Borqers, M., de Brabander, M. (eds) Microtubules and microtubule inhibitors, pp. 187-198, North-Holland Publication Company, Amsterdam. Ginsel, L.A., Cambier, P.H. and Daems, W.Th. (1978) Fucosidosis and I-cell disease: a fine structural and silver-staining study of abnormal inclusion bodies in small-intestinal cells. Virchows Archiv B: Cell Pathology 27, 99-117. Ginsel, L.A., Onderwater, J.J.M. and Daems, W.Th. (1979) Transport of radiolabelled glycoprotein to cell-surface and lysosome-like bodies of absorptive cells of cultured small-intestinal tissue from normal subjects and patients with a lysosomal storage disease. Virchows Archiv B: Cell Pathology 30, 245-273. Hauri, H.P. (1983) Biosynthesis and transport of plasma membrane glycoproteins in the rat intestinal epithelial cell: studies with sucrase-isomaltase. In: Brush Ciba Foundation Symposium 95, pp. border membranes, 132-149, Pitman, London. Hauri, H.P., Quaroni, A. and Isselbacher, K.J. (1979) Biogenesis of intestinal plasma membrane: posttranslational route and cleavage of sucrase-isomaltase. Proceedings of the National Academy of Sciences of the USA 3, 5183-5186. Hauri, H.P., Quaroni, A. and Isselbacher, K.J. (1980) Monoclonal antibodies to sucrase-isomaltase: probes for the study of postnatal development and biogenesis of the intestinal microvillus membrane. Proceedings of the National Academy of Sciences of the USA 77, 6629-6633. Hauri, H.P., Wacker, H., Rickli, E.E., Bigler-Meier, B., Quaroni, A. and Semenza, G. (1982) Biosynthesis of Purification and NH2-terminal sucrase-isomaltase. amino acid sequence of the rat sucrase-isomaltase precursor (pro-sucrase-isomaltase) from fetal intesJournal of Biological Chemistry tinal transplants. 257,
4522-4528.
role of intracellular Lennarz, W. (1983) Overview: brane systems in glycosylation of proteins. Fleischer, S., Fleischer, B. (eds) Methods ology, vol. Academic Press, --98, pp. 91-97,
memIn: in EnzymNew York.
Cell Biology
International
Reports,
Vol. 8, No. 12, December
1984
1013
and Schofield, B. (1973) Inactivation of lysoS-0. somal function by chloroquine. Biochemical PharmacoloCJY 22, 3109-3114. (1979) Effects of some antiMego, J.L. and Chung, C.H. malarials and related substances on intralysosomal Biochemical Pharmacology 28, 465-470. proteolysis. -_Michaels, J.E. (1983) The effects of colchicine on the distribution of glycoprotein-containing vesicles in epithelial cells of the murine colon. Cell and Tissue Research 228, 323-335. Michaels, J.E. and Leblond, C.P. (1976) Transport of glycoprotein from Golgi apparatus to cell surface as shown by radioautoby means of carrier vesicles, raphy of mouse colonic epithelium after injection of jH-fucose . Journal de Microscopic et Biologie Cellulaire 25, 243-248. Ohkuma, S. and Poole, B. (1978) Fluorescent probe measurement of the intralysosomal pH in living cells and the pertubation of pH by various agents. Proceedings of the National Academy of Sciences of the USA 75, 3327-3331. M. and Gangl, A. (1983) Effect of colchicine on Pavelka, the intestinal transport of endogenous lipid. Gastroenterology, 84, 544-555. Pavelka, M., Ellinger, A. and Gangl, A. (1983) Effects of colchicine on rat small intestinal absorptive cells. I. Formation of basolateral microvillar borders. Journal of Ultrastructural Research 85, 249-259. Quaroni, A., Kirsch, K. and Weiser, M.M. (1979a). Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of L-(1,5,6-3H)fucose labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo. Biochemical Journal 182, 203-212. --Quaroni, A., Kirsch, K. and Weiser, M.M. (1979b) Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Effec of colchicine on the ref H)-fucose distribution of L-(1,5,6labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes. Biochemical Journal 182, 213-221. Reaven, E.P. and Reaven, E.M. (1977) Distribution and content of microtubules in relation to the transport of lipid. Journal of Cell Biology 75, 559-572. Seetharam, B., Grimme, N., Coodwin, C. and Alpers, D.H. (1976) Differential sensitivity of intestinal brush border enzymes to pancreatic and lysosomal enzymes. Life Sciences 18, 89-96. Seglen, P.O., Grinde, B. and Solheim, A.E. (1979) Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. European Journal of Biochemistry 2, 215-225. L&Z,
Cell Biology International
1014
Reports, Vol. 8, No. 12, December 1984
Silverstein, S.C., Steinman, R.M. and Cohn, Endocytosis. Annual Review of Biochemistry
Z.A. (1977) 46, 669-
722.
Sj6strBm, H., Nor&, O., Danielsen, E.M. and Skovbjerg, H. (1983) Structure of microvillar enzymes in different phases of their life cycles. In: Brush border membranes, Ciba Foundation Symposium 95, pp. 50-72, Pitman, London. M.G. (1966) Lysosome function Smith, R.E. and Farquhar, in the regulation of the secretory process in cells of the anterior pituitary gland. Journal of Cell Biology 31, 319-347. Tartakoff, ATM. (1982) Simplifying the Golgi complex. Trends in Biochemical Sciences 1, 174-176. of the structure and Tartakoff, A.M. (1983) Pertubation function of the Golgi complex by monovalent carboxylic ionophores. In: Fleischer, S., Fleischer, B. (eds) Methods in enzymology, vol. 98, pp. 47-59, Academic Press, New York. des polysaccharides Thiery, J.P. (1967) Mise en Evidence sur coupes fine en microscopic electronique. Journal de Microscopic 5, 987-1018. Wacker, H., Jaussi, R., Sonderegger, P., Dokow, M., Ghersa, P., Hauri, H.P., Christen, P. and Semenza, G. (1981) Cell-free synthesis of the one chain precursor of a major intrinsic protein complex of the smallintestinal brush-border membrane (pro-sucrase-isomaltase). Federation of the European Biochemical Societies Letters 136, 329-332. uptake in the gut: immunoWalker, W.A. (1981) Antigen logic implications. Immunology Today 2, 30-34. Walker, W.A. and Isselbacher, K.J. (1974) Uptake and transport of macromolecules by the intestine. Possible role in clinical disorders. Gastroenterology 67, 531-550.
Received:
10th September 1984.
Accepted:
24th October 1984.