Dissection of the asynchronous transport of intestinal microvillar hydrolases to the cell surface

Novel subcellular fractionation procedures and pulse-chase techniques were used to study the intracellular transport of the microvillar membrane hydrolases sucrase-isomaltase and dipeptidylpeptidase IV in the differentiated colon adenocarcinoma cell line Caco-2. The overall rate of transport to the cell surface was two fold faster for dipeptidylpeptidase IV than for sucrase-isomaltase, while no significant differences were observed in transport rates from the site of complex glycosylation to the brush border. The delayed arrival of sucrase-isomaltase in the compartment where complex glycosylation occurs was only in part due to exit from the endoplasmic reticulum. A major slow-down could be ascribed to maturation in and transit of this enzyme through the Golgi apparatus. These results suggest that the observed asynchronism is due to more than one rate-limiting step along the rough endoplasmic reticulum to trans-Golgi pathway.     

Asynchronous transport to the cell surface of intestinal brush border hydrolases is not due to differential trimming of N-linked oligosaccharides

Intestinal brush border enzyme glycoproteins are transported to the microvillar membrane at different rates in the differentiated intestinal cell line Caco-2. This asynchronism is due to at least two rate-limiting events, a pre- and an intra-Golgi step (Stieger B., Matter, K., Baur, B., Bucher, K., H?chli, M., and Hauri, H.P. (1988) J. Cell Biol. 106, 1853-1861). A possible cause for the asynchronous protein transport might be differential trimming of N-linked oligosaccharide side chains. The effects of two trimming inhibitors on the intracellular transport of sucrase-isomaltase, a slowly migrating hydrolase, and dipeptidylpeptidase IV, a rapidly migrating hydrolase, are described. 1-Deoxymannojirimycin, an inhibitor of Golgi alpha-mannosidase I, had no influence on the rate of appearance of these hydrolases in the brush border membrane as assessed by subcellular fractionation. In the presence of N-methyl-1-deoxynojirimycin, an inhibitor of glucosidase I, 30-40% of the newly synthesized molecules appeared at the cell surface, and half-time for appearance of this pool was identical to that found in control cells. The reduced maximal transport to the cell surface observed with N-methyl-1-deoxynojirimycin may suggest that proper glycosylation is necessary for an efficient transport from the Golgi apparatus to the microvillar membrane. Inhibition of glucosidase I does not prevent the acquisition of endoglycosidase H resistance. Furthermore, evidence is presented that the processing in the presence of N-methyl-1-deoxynojirimycin leads to glycosylated endoglycosidase H-resistant glycoproteins.     

Intracellular localization and endocytosis of brush border enzymes in the enterocyte-like cell line Caco-2.

Immunoelectron microscopy was used to localize the brush border hydrolases sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPPIV) in the human colon carcinoma cell line Caco-2. Both enzymes were detected at the microvillar membrane, in small vesicles and multivesicular bodies (MVBs), and in lysosomal bodies. In addition, DPPIV was found in the Golgi apparatus, a variety of apical vesicles and tubules, and at the basolateral membrane. To investigate whether the hydrolases present in the lysosomal bodies were endocytosed from the apical membrane, endocytic compartments were marked with the endocytic tracer cationized ferritin (CF). After internalization from the apical membrane through coated pits, CF was first recovered in apical vesicles and tubules, and larger electronlucent vesicles (early endosomes), and later accumulated in MVBs (late endosomes) and lysosomal bodies. DPPIV was localized in a subpopulation of both early and late endocytic vesicles, which contained CF after 3 and 15 min of uptake, respectively. Also, internalization of the specific antibody against DPPIV and gold labeling on cryosections showed endocytosed DPPIV in both early and late endosomes. However, unlike CF, no accumulation of DPPIV was seen in MVBs or lysosomal bodies after longer chase times. The results indicate that in Caco-2 cells the majority of brush border hydrolases present in lysosomal bodies are not endocytosed from the brush border membrane. Furthermore, the labeling patterns obtained, suggest that late endosomes may be involved in the recycling of endocytosed DPPIV to the microvilli.     

Intracellular transport and conformational maturation of intestinal brush border hydrolases.

Brush border hydrolases of the differentiated intestinal cell line Caco-2 are transported to the microvillar membrane at different rates. This asynchronism is due to at least two rate-limiting events, a pre- and an intra-Golgi step. The retardation of sucrase-isomaltase, a slowly migrating hydrolase, versus dipeptidylpeptidase IV, a rapidly transported enzyme, is neither due to differential trimming of N-linked carbohydrates nor due to oligomerization. In this study, the conformational maturation of biosynthetically labeled sucrase-isomaltase and dipeptidylpeptidase IV was probed by conformation-specific antibodies and proteases. These assays enabled us to correlate the conformational maturation of the two enzymes with their rates of transport. Furthermore, two naturally occurring mutants of sucrase-isomaltase with impaired intracellular transport displayed an immature conformation. It is proposed that differential kinetics of folding might be the underlying cause for both the pre- and the intra-Golgi steps of asynchronous intracellular transport. Furthermore, a proper tertiary structure might be a prerequisite for sucrase-isomaltase to leave the Golgi apparatus.     

Endocytosis, recycling, and lysosomal delivery of brush border hydrolases in cultured human intestinal epithelial cells (Caco-2)

Lysosomes of intestinal epithelial cells in vivo and in culture display strong immunoreactivity with monoclonal antibodies against various brush border enzymes as visualized by immunoelectron microscopy. Novel subcellular fractionation procedures were developed to study, by the pulse-chase technique and by internalization assays, the pathway along which two microvillar hydrolases, sucrase-isomaltase and dipeptidylpeptidase IV, are transported to lysosomes in the differentiated colon adenocarcinoma cell line Caco-2. 7-9% of metabolically labeled sucrase-isomaltase of dipeptidylpeptidase IV were present in lysosomes after 7-8 h of chase as intact complex-glycosylated molecules. Appearance of these enzymes in lysosomes was biphasic. Endocytosis studies with radioiodinated antienzyme monoclonal antibodies (monovalent antigen-binding fragments) and by means of cell surface iodination revealed only slow transport of the enzymes to lysosomes at a low level. However, both enzymes were internalized with different efficiencies and recycled to the cell surface via endosomes. These results suggest that in Caco-2 cells a significant amount of newly synthesized sucrase-isomaltase and dipeptidylpeptidase IV is directly imported into lysosomes bypassing the brush border membrane.     

Development of the small intestine in the hypophysectomized rat. II. Influence of cortisone, thyroxine, growth hormone, and prolactin.

The intestinal deficiencies caused by hypophysectomy of rats at 6 days of age can be repaired to varying degrees by thyroxine or cortisone but not by growth hormone or prolactin. Administration of daily doses of thyroxine alone from 19–22 days raises duodenal alkaline phosphatase activity to normal levels at 24 days; it has a strong effect on jejunal sucrase and maltase, although these activities remain below those of controls. Thyroxine causes a marked increase in rough endoplasmic reticulum and restores the Golgi complexes to their normal appearance. It also elicits an intensification of periodic acid-Schiff (PAS) stainability of the brush border. Cortisone acetate given from 19 to 22 days elevates sucrase and maltase to normal levels but does not fully restore phosphatase activity. Like thyroxine, cortisone causes intensification of PAS staining of the brush border and also increases rough endoplasmic reticulum. It seems to stimulate Golgi activity, but results in the appearance of a variety of abnormal forms. The defects in Golgi configuration, brush border carbohydrate content, and activity of glycoprotein enzymes that are bound to the brush border may all reflect impaired glycosylation in the hypophyseoprivic state; the results of thyroxine or cortisone administration suggest that both hormones may affect glycosylation but in different ways.     

Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells

A panel of monoclonal antibodies was produced against purified microvillus membranes of human small intestinal enterocytes. By means of these probes three disaccharidases (sucrase-isomaltase, lactase-phlorizin hydrolase, and maltase-glucoamylase) and four peptidases (aminopeptidase N, dipeptidylpeptidase IV, angiotension I-converting enzyme, and p-aminobenzoic acid peptide hydrolase) were successfully identified as individual entities by SDS PAGE and localized in the microvillus border of the enterocytes by immunofluorescence microscopy. The antibodies were used to study the expression of small intestinal hydrolases in the colonic adenocarcinoma cell line Caco 2. This cell line was found to express sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV, but not the other three enzymes. Pulse-chase studies with [35S]methionine and analysis by subunit-specific monoclonal antibodies revealed that sucrase-isomaltase was synthesized and persisted as a single-chain protein comprising both subunits. Similarly, lactase-phlorizin hydrolase was synthesized as a large precursor about twice the size of the lactase subunits found in the human intestine. Aminopeptidase N and dipeptidylpeptidase IV, known to be dimeric enzymes in most mammals, were synthesized as monomers. Transport from the rough endoplasmic reticulum to the trans-Golgi apparatus was considerably faster for the peptidases than for the disaccharidases, as probed by endoglycosidase H sensitivity. These results suggest that the major disaccharidases share a common biosynthetic mechanism that differs from that for peptidases. Furthermore, the data indicate that the transport of microvillus membrane proteins to and through the Golgi apparatus is a selective process that may be mediated by transport receptors.     

Flow cytometry, a very useful technique for the characterization of intestinal membrane vesicles

The plasma membrane of enterocytes comprises two structurally and functionally distinct domains. These are the apical brush border, containing digestive hydrolases and glycocalyx, and the basolateral domain, characterized by other specific markers. Using a fast and easy subcellular fractionation, we purified four membrane vesicle fractions from rabbit small intestinal mucosa: brush border, basolateral, rough endoplasmic reticulum and Golgi + smooth endoplasmic reticulum. Using flow cytometry, the fluorescence polarization of diphenylhexatriene was determined in brush border and in basolateral + Golgi + smooth endoplasmic reticulum membrane fractions in order to investigate changes in the membrane fluidity of both fractions and to compare the results obtained with those of spectroscopic techniques. Moreover, it was possible with flow cytometry to detect and quantify basolateral and brush border markers by using polyclonal and monoclonal antibodies. The advantages of flow cytometry in the detection of brush border membrane markers found in small amounts in the basolateral domain are discussed. Finally, flow cytometry holds great promise for the analysis and sorting of subcellular fractions.     

Distribution of glycosyltransferases among Golgi apparatus subfractions from liver and hepatomas of the rat.

Glycosyltransferase activities of highly purified fractions of Golgi apparatus, plasma membrane and endoplasmic reticulum, all from the same homogenates, were analyzed and compared. Additionally, Golgi apparatus were unstacked and the individual cisternae separated into fractions enriched in cis, median and trans elements using the technique of preparative free-flow electrophoresis. Golgi apparatus from both liver and hepatomas were enriched in all glycosyltransferases compared to endoplasmic reticulum and plasma membranes. However, Golgi apparatus from hepatomas showed both elevated fucosyltransferase and galactosyltransferase activities but reduced sialyltransferase and dipeptidyl peptidase IV (DPP IV) activities compared to liver. Activity of N-acetylglucosaminyltransferase was approximately the same in both liver and hepatoma Golgi apparatus. With normal liver, sialyl- and galactosyltransferase activities and DPP IV showed a marked cis-to-trans gradient of activity. Fucosyltransferase was concentrated in two regions of the electrophoretic separations, one corresponding to cis cisternae and one corresponding to trans cisternae. N-Acetylglucosaminyltransferase activity was more widely distributed but the endogenous acceptor activity was predominantly cis. With hepatoma Golgi apparatus, the pattern for DPP IV was similar to that for liver but those of sialyl- and galactosyltransferases differed markedly from liver. Instead of activity increasing cis to trans, the activities for sialyl- and galactosyltransferases decreased. For fucosyltransferases, activity dependent on exogenous acceptor was medial whereas with endogenous acceptor, two activity peaks, cis and trans, still were observed. For N-acetylglucosaminyltransferase the pattern for hepatoma was similar to that for liver. The results indicate alterations in the distribution of glycosyltransferase activities within the Golgi apparatus in hepatotumorigenesis that may reflect altered cell surface glycosylation patterns.     

Subcellular fractionation of Hymenolepis diminuta with special reference to the localization of marker enzymes

A method for subcellular fractionation of Hymenolepis diminuta using whole worm homogenization and differential centrifugation is presented. Different fractions obtained in this study were screened for the presence of enzymes that serve as markers for plasma membrane, brush border, mitochondria, Golgi complex, endoplasmic reticulum, peroxisomes, lysosomes and cytosol. The purity of fractions was also monitored by transmission electron microscopy. The purity of fractions, particularly the brush border membranes, are compared to those obtained by previous methods for H. diminuta or other tissues.     

Sites of sulfatation in the chondrocytes of the articular cartilage of the rabbit

35S sulfate uptake by the articular cartilage chondrocytes, from biopsies of rabbit, have been studied by high resolution autoradiography. The Golgi apparatus, rough endoplasmic reticulum, cytosol, cytoplasmic membrane and extracellular space were considered as cell compartments in the quantitative analysis of the autoradiograms. The results obtained show: 1) a high activity of radiosotope incorporation in the Golgi apparatus; 2) a fast rhythm of transfer of the substances labelled in the Golgi apparatus to the cell membrane; 3) significant labelling of the rough endoplasmic reticulum, throughout the experiment. It is concluded: 1) The grains observed in the rough endoplasmic reticulum show a significant radioisotope uptake on this level, and this evidence some sulfotransferase activity. 2) The high 35S sulfate uptake level which is observed in the Golgi apparatus demonstrates that the highest sulfotransferase enzyme activity is located in this cell area, thus showing that the "early" sulfation that began in the rough endoplasmic reticulum was completed by a "late" sulfation in the Golgi apparatus. It is here that complete chondromucoprotein building takes place before being excreted. 3) The high transfer level of the labelled substances from the Golgi apparatus shows that the sulfated product secretion for building the cartilage matrix takes place rapidly since a great label increase can be already observed at the beginning of the chase period in the outer surrounding area of the chondrocyte membrane.     

Intestinal uptake of dipeptides and beta-lactam antibiotics. I. The intestinal uptake system for dipeptides and beta-lactam antibiotics is not part of a brush border membrane peptidase

The uptake of beta-lactam antibiotics into small intestinal enterocytes occurs by the transport system for small peptides. The role of membrane-bound peptidases in the brush border membrane of enterocytes from rabbit and pig small intestine for the uptake of small peptides and beta-lactam antibiotics was investigated using brush border membrane vesicles. The enzymatic activity of aminopeptidase N was inhibited by beta-lactam antibiotics in a non-competitive manner whereas dipeptidylpeptidase IV was not affected. The peptidase inhibitor bestatin led to a strong competitive inhibition of aminopeptidase N whereas the uptake of cephalexin into brush border membrane vesicles was only slightly inhibited at high bestatin concentrations (greater than 1 mM). Modification of brush border membrane vesicles with the histidine-modifying reagent diethyl pyrocarbonate led to a strong irreversible inhibition of cephalexin uptake whereas the activity of aminopeptidase N remained unchanged. A modification of serine residues with diisopropyl fluorophosphate completely inactivated dipeptidylpeptidase IV whereas the transport activity for cephalexin and the enzymatic activity of aminopeptidase N were not influenced. With polyclonal antibodies raised against aminopeptidase N from pig renal microsomes the aminopeptidase N from solubilized brush border membranes from pig small intestine could be completely precipitated; the binding protein for beta-lactam antibiotics and oligopeptides of apparent Mr 127,000 identified by direct photoaffinity labeling with [3H]benzylpenicillin showed no crossreactivity with the aminopeptidase N anti serum and was not precipitated by the anti serum. These results clearly demonstrate that peptidases of the brush border membrane like aminopeptidase N and dipeptidylpeptidase IV are not directly involved in the intestinal uptake process for small peptides and beta-lactam antibiotics and are not a constituent of this transport system. This suggests that a membrane protein of Mr 127,000 is (a part of) the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of small intestinal enterocytes.     

Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport

Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.     

Uncoordinated,transient mosaic patterns of intestinal hydrolase expression in differentiating human enterocytes

The heterogenous expression of brush border membrane hydrolases by the human enterocyte-like Caco-2 cell line during morphological and functional differentiation in vitro was investigated at the cellular level. Indirect immunofluorescence revealed that the heretogenous (“mosaic”) expression of sucrase-isomaltase, lactase, aminopeptidase N, and alkaline phosphatase was, in fact, transient in nature. The labeling indexes for each hydrolase gradually increased during culture at postconfluence in order to reach a maximum (≥90%) after 30 days, concomitant with an upregulation of their respective protein expression levels. In contrast, dipeptidylpeptidase IV labeling remained relatively constant. Backscattered electron imaging analysis in midstage (12 days postconfluence) monolayers demonstrated a lack of correlation between brush border membrane development and expression of each enzyme studied. Moreover, double immunostaining revealed that none of the other four hydrolases correlated directly with sucrase-isomaltase expression. Finally, immunodetection for the proliferation-associated antigen Kl-67 revealed a transient mosaic pattern of proliferation which was inversely related to Caco-2 cell differentiation. These data indicate that enterocytic differentiation-related (as well as proliferation-related) gene expression in Caco-2 cells is regulated but uncoordinated at the cellular level, suggesting that an overall control mechanism is lacking. © 1996 Wiley-Liss, Inc.     

The structure and function of the Golgi apparatus: a hundred years of questions

Over the last century, the Golgi apparatus has attracted the attention of researchers world-wide. This highly variable and polymorphic organelle plays a central role in intracellular membrane traffic. Not only does it receive all the secretory material and membrane synthesized by the endoplasmic reticulum and modifies these products by glycosylation, but also packages them and sends them in vesicular carriers to their correct destinations. It is also capable of the synthesis of complex polysaccharides used for building cell walls, a feature unique for higher plants. Yet, the current models of Golgi function are based on those established for yeast and mammalian cells and may not be completely relevant to plants. This review is an attempt to summarize the current knowledge of the plant Golgi apparatus and, where possible, to discuss the applicability of the current models of Golgi function to the plant cell.     

Biosynthesis of intestinal microvillar proteins. Dimerization of aminopeptidase N and lactase-phlorizin hydrolase

The pig intestinal brush border enzymes aminopeptidase N (EC 3.4.11.2) and lactase-phlorizin hydrolase (EC 3.2.1.23-62) are present in the microvillar membrane as homodimers. Dimethyl adipimidate was used to cross-link the two [35S]methionine-labeled brush border enzymes from cultured mucosal explants. For aminopeptidase N, dimerization did not begin until 5-10 min after synthesis, and maximal dimerization by cross-linking of the transient form of the enzyme required 1 h, whereas the mature form of aminopeptidase N cross-linked with unchanged efficiency from 45 min to 3 h of labeling. Formation of dimers of this enzyme therefore occurs prior to the Golgi-associated processing, and the slow rate of dimerization may be the rate-limiting step in the transport from the endoplasmic reticulum to the Golgi complex. For lactase-phlorizin hydrolase, the posttranslational processing includes a proteolytic cleavage of its high molecular weight precursor. Since only the mature form and not the precursor of this enzyme could be cross-linked, formation of tightly associated dimers only takes place after transport out of the endoplasmic reticulum. Dimerization of the two brush border enzymes therefore seems to occur in different organelles of the enterocyte.     

Inhibition of Golgi Apparatus Glycosylation Causes Endoplasmic Reticulum Stress and Decreased Protein Synthesis

Nucleotide sugar transporters of the Golgi apparatus play an essential role in the glycosylation of proteins, lipids, and proteoglycans. Down-regulation of expression of the transporters for CMP-sialic acid, GDP-fucose, or both unexpectedly resulted in accumulation of glycoconjugates in the Golgi apparatus rather than in the plasma membrane. Pulse-chase experiments with radiolabeled sugars and amino acids showed decreased synthesis and secretion of both nonglycoproteins and glycoproteins. Further studies revealed that the above silencing induced endoplasmic reticulum stress and inhibited protein translation initiation. Together these results suggest that global inhibition of Golgi apparatus glycosylation may lead to important secondary metabolic changes, unrelated to glycosylation.     

Glycosylated nuclear lectin CBP70 also associated with endoplasmic reticulum and the Golgi apparatus: does the "classic pathway" of glycosylation also apply to nuclear glycoproteins?

The subcellular plurilocalization of some lectins (galectin-1, galectin-3, galectin-10, calreticulin, etc.) is an intriguing problem, implying different partners according to their localization, and involvement in a variety of cellular activities. For example, the well-known lectin, galectin-3, a lactose-binding protein, can act inside the nucleus in splicing events, and at the plasma membrane in adhesion, and it was demonstrated that galectin-3 interacts in the cytoplasm with Bcl-2, an antiapoptotic protein. Some years ago, our group isolated a nuclear lectin CBP70, capable of recognizing N-acetylglucosamine residues. This lectin, first isolated from the nucleus of HL60 cells, was also localized in the cytoplasm. It has been demonstrated that CBP70 is a glycosylated lectin, with different types of glycosylation, comparing cytoplasmic and nuclear forms. In this article, we have studied the localization of CBP70 in undifferentiated HL60 cells by electron microscopy, immunofluorescence analysis, and subcellular fractionation. The results obtained clearly demonstrated that CBP70 is a plurilocalized lectin that is found in the nucleus, at the endoplasmic reticulum, the Golgi apparatus, and mitochondria, but not at the plasma membrane. Because CBP70, a nuclear glycoprotein, was found to be associated also with the endoplasmic reticulum and the Golgi apparatus where the glycosylation take place, it raised the question: where does the glycosylation of nuclear proteins occur?     

Is the Golgi apparatus the obligatory final step for lipoprotein secretion by intestinal cells?

It is currently admitted that the synthesis and excretion of triglyceride-rich lipoproteins (chylomicrons and 'small chylomicrons') by intestinal epithelial cells involves the Golgi apparatus as an obligatory final step before exocytosis. The cells of the proximal intestine of the trout are an excellent model for investigating functional compartmentalization in the course of lipid absorption. Using this model, our data invalidate morphological data which were the basis for considering the Golgi apparatus as the mandatory final stage for their secretion. In particular, we show that triglyceride-rich particles can be transported directly from the endoplasmic reticulum to the intercellular space. Two pathways of intestinal lipoprotein excretion appear to coexist. One follows the classical export route, the second functions in a manner that bypasses the Golgi apparatus. The arguments used to affirm the requirement for the Golgi apparatus as a final step (glycosylation of apoprotein B, membrane vehicle for exocytosis) are discussed.     

Signal-anchor sequences are an essential factor for the Golgi-plasma membrane localization of type II membrane proteins

Despite studies of the mechanism underlying the intracellular localization of membrane proteins, the specific mechanisms by which each membrane protein localizes to the endoplasmic reticulum, Golgi apparatus, and plasma membrane in the secretory pathway are unclear. In this study, a discriminant analysis of endoplasmic reticulum, Golgi apparatus and plasma membrane-localized type II membrane proteins was performed using a position-specific scoring matrix derived from the amino acid propensity of the sequences around signal-anchors. The possibility that the sequence around the signal-anchor is a factor for identifying each localization group was evaluated. The discrimination accuracy between the Golgi apparatus and plasma membrane-localized type II membrane proteins was as high as 90%, indicating that, in addition to other factors, the sequence around signal-anchor is an essential component of the selection mechanism for the Golgi and plasma membrane localization. These results may improve the use of membrane proteins for drug delivery and therapeutic applications.