Cell surface display and intracellular trafficking of free glycosylphosphatidylinositols in mammalian cells

In addition to serving as membrane anchors for cell surface proteins, glycosylphosphatidylinositols (GPIs) can be found abundantly as free glycolipids in mammalian cells. In this study we analyze the subcellular distribution and intracellular transport of metabolically radiolabeled GPIs in three different cell lines. We use a variety of membrane isolation techniques (subcellular fractionation, plasma membrane vesiculation to isolate pure plasma membrane fractions, and enveloped viruses to sample cellular membranes) to provide direct evidence that free GPIs are not confined to their site of synthesis, the endoplasmic reticulum, but can redistribute to populate other subcellular organelles. Over short labeling periods (2.5 h), radiolabeled GPIs were found at similar concentration in all subcellular fractions with the exception of a mitochondria-enriched fraction where GPI concentration was low. Pulse-chase experiments over extended chase periods showed that although the total amount of cellular radiolabeled GPIs decreased, the plasma membrane complement of labeled GPIs increased. GPIs at the plasma membrane were found to populate primarily the exoplasmic leaflet as detected using periodate oxidation of the cell surface. Transport of GPIs to the cell surface was inhibited by Brefeldin A and blocked at 15 degrees C, suggesting that GPIs are transported to the plasma membrane via a vesicular mechanism. The rate of transport of radiolabeled GPIs to the cell surface was found to be comparable with the rate of secretion of newly synthesized soluble proteins destined for the extracellular space.     

Transport of cholesterol from the endoplasmic reticulum to the plasma membrane

We have studied the transport of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane in Chinese hamster ovary cells using a cell fractionation assay. We found that transport is dependent on metabolic energy, but that the maintenance of the high differential concentration of cholesterol in the plasma membrane is not an energy-requiring process. We have tested a variety of inhibitors for their effect on cholesterol transport and found that cytochalasin B, colchicine, monensin, cycloheximide, and NH4Cl did not have any effect. The cholesterol transport process shows a sharp temperature dependence; it ceases at 15 degrees C, whereas cholesterol synthesis continues. When synthesis occurs at 15 degrees C, the newly synthesized cholesterol accumulates in the endoplasmic reticulum and in a low density, lipid-rich vesicle fraction. These results suggest that cholesterol is transported via a vesicular system.     

Intracellular transport of the major glycoprotein of zymogen granule membranes in the rat pancreas. Demonstration of high turnover at the plasma membrane

The intracellular transport and destination of the major glycoprotein associated with zymogen granule membranes in the pancreas (GP-2) was established. In suspensions of isolated acinar cells from rat pancreas, pulse-chase experiments were performed. The incorporation of the first newly synthesized GP-2 molecules into zymogen granule membranes occurred at about 60 min after beginning of the pulse. We demonstrated by using two different methods that newly made GP-2 reaches the cell surface within the same time span. After 6-8 h chase considerable more newly synthesized GP-2 has reached the cell surface than would be expected on account of secreted newly synthesized zymogens. These observations strongly suggest that at least part of the GP-2 molecules bypass the mature zymogen granule compartment on their way to the plasma membrane. GP-2 is the only protein that appears in discernable quantity in the plasma membrane during 1-4 h after a pulse label. Nevertheless GP-2 comprises only a small percentage of externally 125I-iodinated plasma membrane proteins. We conclude that GP-2 has a high turnover rate at the plasma membrane level. Treatment of the acinar cells with the N-glycosylation inhibitor tunicamycin does not block the intracellular transport of GP-2.     

Lipid transport during mitosis. Alternative pathways for delivery of newly synthesized lipids to the cell surface

A number of cellular processes involving vesicle transport are inhibited during mitosis. In the present study we asked whether the transport of a newly synthesized glycerophospholipid and (some) sphingolipids from their site(s) of intracellular synthesis to the plasma membranes of Chinese hamster ovary cells was also inhibited at mitosis. (i) For phospholipids, we examined the movement of phosphatidylethanolamine (PE) following its de novo synthesis from [3H]ethanolamine (Sleight, R. G., and Pagano, R. E. (1983) J. Biol. Chem. 258, 9050-9058). Plasma membrane PE was distinguished from intracellular PE by its derivatization with the amino-reactive reagent, trinitrobenzenesulfonic acid, under nonpermeating conditions. Both the steady state amount of PE and the rate of appearance of newly synthesized PE at the cell surface were quantified. Transport of newly synthesized PE to the plasma membrane was not inhibited at mitosis but was found to be a rapid process similar to that previously reported for interphase cells. (ii) For sphingolipids, we examined the transport of fluorescent analogs of sphingomyelin and glucosylceramide (GlcCer) to the plasma membrane following their de novo synthesis from the fluorescent sphingolipid precursor, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]aminocaproyl D-erythro-sphingosine (Lipsky, N. G., and Pagano, R. E. (1985a) J. Cell Biol. 100, 27-34). Transport of fluorescent sphingomyelin and glucosylceramide to the plasma membrane was inhibited in mitotic cells but not in interphase or G1 cells. These results are discussed in terms of alternative mechanisms for delivery of the newly synthesized lipids to the outer leaflet of the plasma membrane bilayer.     

Brefeldin A arrests the intracellular transport of viral envelope proteins in primary cultured rat hepatocytes and HepG2 cells.

We have studied the effect of brefeldin A (BFA) on the intracellular transport of the envelope proteins of vesicular stomatitis virus (VSV) and sindbis virus in primary cultured rat hepatocytes. BFA (2.5 micrograms/ml) inhibited not only the secretion of plasma proteins into the medium, but also the assembly of both G protein of VSV and E1 and E2 proteins (envelope proteins) of sindbis virus into respective virions. Concomitantly, both the acquisition of endo-beta-N-acetylglucosaminidase H resistance by the G protein and the proteolytic conversion of PE2 to E2 were found to be inhibited in the BFA-treated cells, suggesting that the intracellular transport of the envelope proteins was arrested in the endoplasmic reticulum. Such inhibitory effects of the drug were variable depending upon the culture conditions of the hepatocytes. In the 1-day-cultured cells, even in the presence of the drug, newly synthesized envelope proteins were assembled into the virions after a 3 h chase period, at the same time as secretion of plasma proteins into the medium resumes. In contrast, in 4-day-cultured hepatocytes, BFA continuously blocked the entry of the envelope proteins into the virions and the release of plasma proteins into the medium for at least 5 h. BFA also completely inhibited the exocytotic pathway in HepG2 cells. These results indicate that the duration time of the effect of BFA is different from one cell to another and may change depending upon the culture conditions of the cells.     

Catabolism of newly synthesized decorin by explant cultures of bovine ligament.

The catabolism of newly synthesized decorin by explant cultures of bovine collateral ligament was investigated. The tissue was placed in explant culture for 6 days then incubated with radiolabeled sulfate for 6 h and replaced in culture for 5 days to allow for the loss of the radiolabeled large proteoglycan. The metabolic fate of the remaining radiolabeled decorin present in the matrix of the tissue over the next 9-day period was determined. It was shown that this pool of decorin was lost from ligament explant cultures either directly into the culture medium or taken up and degraded within the cells of the tissue. The intracellular degradation of the radiolabeled pool of decorin by ligament explant cultures was shown to result in the generation of [35S]sulfate. This process required metabolically active cells and involved the lysosomal system since sulfate generation was inhibited when cultures were maintained at 4 degrees C or in the presence of either 10 mM ammonium chloride or 0. 05 mM chloroquine. The inhibition of intracellular processing of decorin resulted in an increase in the rate of loss of this proteoglycan into the medium of the cultures. The inhibition of intracellular degradation of decorin was reversible on incubation of the explant cultures at 37 degrees C or removal of ammonium chloride from the culture medium. After removal of the ammonium chloride from the culture medium the rate of intracellular catabolism was greater than that observed in cultures maintained in medium alone, which suggested that there was an intracellular accumulation of native and/or partially degraded material within the cells.     

Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins

A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.     

Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.     

Translocation of nascent non-signal sequence protein in heated Escherichia coli

Exposure of Escherichia coli to heat resulted in 1) selective inhibition of protein synthesis, 2) synthesis of heat shock proteins, and 3) altered subcellular distribution of newly synthesized proteins. Either 5 min or 1 h at 48 degrees C increases outer membrane proteins of Coomassie Blue-stained gels. After 1 h, there was a loss of stained proteins from the soluble fraction. Much greater changes in the distribution of radiolabeled (newly synthesized) proteins were observed, with marked increases in the number of outer membrane protein species and a corresponding loss of soluble fraction proteins. Three major species of radiolabeled proteins from heat-treated cells remain in the soluble fraction; these proteins have apparent Mr 56,000, 69,200, and 79,400. Cells were labeled with L-[35S] methionine at either 37 or 48 degrees C and chased with non-radiolabeled methionine before a temperature shift to either 48 or 37 degrees C, respectively. Only proteins synthesized at elevated temperature participated in translocation. It is suggested that heat disordering of membrane lipids promotes interlipidic connections between the inner and outer membrane providing pathways for protein movement to the outer membrane and may be the mechanism whereby a cell quickly responds to environmental temperature stress. The response does not require but may trigger synthesis of mRNA.     

Temperature dependence of prolactin endocytosis and casein exocytosis in epithelial mammary cells

As previously reported in epithelial mammary cells of lactating rabbit, prolactin exerts a stimulatory effect on casein secretion. After binding to a membrane receptor, the complex hormone-receptor is internalized in mammary cells. Peptide hormone action involves the generation of second messengers. These second messengers can be emitted as soon as hormone is linked to the membrane receptor. However, it is not excluded that endocytosis and transfer of prolactin inside the cell take part in the emission of second messenger and related secretory response. In order to precise intracellular transport pathways in the lactating mammary cell, we have examined the effects of reduced temperature on the one hand on prolactin endocytosis, on the other hand on casein secretion and on the stimulating effect of prolactin on casein secretion. Endocytosed prolactin was cytochemically localized mainly on the plasma membrane at 4 degrees C. At 25 degrees C, the hormone accumulated, during 60 min, in endosomes and multivesicular bodies. At 37 degrees C, prolactin was detectable after 15 and 30 min inside the cells and disappeared after 60 min. Transport and exocytosis of secretory proteins were only partly inhibited at 25 degrees C as attested by autoradiography localization and biochemical assays of newly synthesized caseins. However, at 25 degrees C, prolactin was no more able to stimulate casein exocytosis. These results show that intracellular transport of prolactin and secretagogue effect of the hormone does not proceed at 25 degrees C. However, secretory mechanisms of the cell are always able to be stimulated by exogenous arachidonic acid at this temperature. Low temperature appears as a good means to study intracellular transport in the mammary cell.     

Transfer of cholesterol from its site of synthesis to the plasma membrane

We have followed the transfer of newly synthesized cholesterol to the plasma membrane in cultured fibroblasts using cholesterol oxidase as a probe. Since the enzyme has access only to the plasma membrane in intact cells, it permits the discrimination of cell surface and endogenous cholesterol. Cholesterol synthesized from radiolabeled acetate was transferred to the plasma membrane in a strictly first order fashion with a half-time of 1-2 h at 37 degrees C. The rate of transfer was similar in rapidly growing and confluent cells and was not affected by preincubating the cells in lipoprotein-deficient serum which greatly stimulated cholesterol synthesis. We used equilibrium density gradient centrifugation of homogenates from cholesterol oxidase-treated cells to examine further the distribution of newly synthesized cholesterol between cellular pools. We identified membrane fractions enriched in newly synthesized cholesterol yet inaccessible to cholesterol oxidase. The cholesterol in these membranes eventually moved to the plasma membrane. The movement of exogenous radiocholesterol from the plasma membrane to the cell interior also was examined by this method. No detectable transfer was observed over several hours, during which time endogenous cholesterol moved to the plasma membrane. We conclude that the transfer of newly synthesized cholesterol to the plasma membrane is a vectorial process and is not mediated by a simple diffusional equilibrium.     

Photoaffinity labeling of insulin-sensitive hexose transporters in intact rat adipocytes. Direct evidence that latent transporters become exposed to the extracellular space in response to insulin

Irradiation of intact rat adipocytes with high intensity ultraviolet light in the presence of 0.5 microM [3H] cytochalasin B results in the labeling of Mr 43,000 and 46,000 proteins that reside in the plasma membrane fraction. In contrast to the Mr 46,000 protein, the Mr 43,000 component is not observed in the microsome fraction and exhibits lower affinity for [3H]cytochalasin B. Photolabeling of the Mr 43,000 protein is inhibited by cytochalasin D, indicating it is not a hexose transporter component. The Mr 46,000 protein exhibits characteristics expected for the glucose transporter such that D-glucose or 3-O-methylglucose but not cytochalasin D inhibits its photolabeling with [3H] cytochalasin B. Furthermore, insulin addition to intact cells either prior to or after photoaffinity labeling of the Mr 46,000 protein causes a redistribution of this component from the low density microsomes to the plasma membrane fraction, as expected for the hexose transporter. Photolabeling of transporters in both the low density microsome and plasma membrane fractions is inhibited when intact cells are equilibrated with 50 mM ethylidene glucose prior to irradiation with [3H]cytochalasin B. Incubation of intact cells with 50 mM ethylidene glucose for 1 min at 15 degrees C leads to an intracellular concentration of only 2 mM. Under these conditions, the photoaffinity labeling in intact cells of hexose transporters that fractionate with the low density microsomes is unaffected, indicating these transporters are not exposed to the extracellular medium. In contrast, photolabeling in intact insulin-treated cells of hexose transporters that fractionate with the plasma membrane is inhibited under these incubation conditions. The results demonstrate that insulin action results in the exposure to the extracellular medium of previously sequestered hexose transporters.     

Subcellular localization of binding sites for cytochalasin D: evidence from activation energies.

The activation energies for binding of tritiated cytochalasin D to HEp-2 cells and isolated plasma membrane were determined by Arrhenius plots. The higher value for intact cells (24 kcal/mol) compared to the plasma membrane fraction (4 kcal/mol at greater than 11.5 degrees C, 18 kcal/mol at less than 11.5 degrees C) was taken as evidence that [3H]cytochalasin D must penetrate the plasma membrane in order to reach its binding sites. The data support the conclusion that binding sites for [3H]cytochalasin D are intracellular, on the cytoplasmic face of the plasma membrane (rather than within the lipid bilayer), and on microsomes (endomembranes).     

Intracellular transport of phosphatidylcholine to the plasma membrane

We have used pulse-chase labeling of Chinese hamster ovary cells with choline followed by plasma membrane isolation on cationic beads to study the transport of phosphatidylcholine from the endoplasmic reticulum to the plasma membrane. We have found that the process is rapid (t1/2 [25 degrees C] = 2 min) and not affected by energy poisons or by cytochalasin B, colchicine, monensin, or carbonyl cyanide p-chlorophenylhydrazone. Cooling cells to 0 degree C effectively stops the transport process. The intracellular transport of phosphatidylcholine is distinct in several ways from the intracellular transport of cholesterol (Kaplan, M. R., and R. D. Simoni, 1985, J. Cell. Biol., 101:446-453).     

Defective plasma membrane assembly in yeast secretory mutants.

Yeast mutants that are conditionally blocked at distinctive steps in secretion and export of cell surface proteins have been used to monitor assembly of integral plasma membrane proteins. Mutants blocked in transport from the endoplasmic reticulum (sec18), from the Golgi body (sec7 and sec14), and in transport of secretory vesicles (sec1) show dramatically reduced assembly of galactose and arginine permease activities. Simultaneous induction of galactose permease and alpha-galactosidase (a secreted glycoprotein) in sec mutant cells at the nonpermissive temperature (37 degrees C) shows that both activities accumulate and can be exported coordinately when cells are returned to the permissive temperature (24 degrees C) in the presence or absence of cycloheximide. Plasma membrane fractions isolated from sec mutant cells radiolabeled at 37 degrees C have been analyzed by two-dimensional sodium dodecyl sulfate-gel electrophoresis. Although most of the major protein species seen in plasma membranes from wild-type cells are not efficiently localized in sec18 or sec7, several of these proteins appear in plasma membranes from sec1 cells. These results may be explained by contamination of plasma membrane fractions with precursor vesicles that accumulate in sec1 cells. Alternatively, some proteins may branch off during transport along the secretory pathway and be inserted into the plasma membrane by a different mechanism.     

Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane

Transport of newly synthesized cholesterol and vesicular stomatitis virus G protein from the endoplasmic reticulum to the plasma membrane is interrupted by incubation at 15 degrees C. Under this condition the newly synthesized molecules accumulate in both the endoplasmic reticulum (ER) and a subcellular vesicle fraction of low density called the lipid-rich vesicle fraction. The material in the lipid-rich vesicle fraction appears to be a post-ER intermediate in the transport process to the plasma membrane (PM). Although both newly synthesized cholesterol and G protein accumulate in this intermediate compartment at 15 degrees C, suggesting cotransport, treatment with Brefeldin A does not affect cholesterol transport to the PM, whereas it strongly inhibits G protein transport. We conclude that cholesterol and G protein leave the ER in separate vesicles, the cholesterol containing vesicles bypass the Golgi apparatus and proceed to the PM, whereas G protein containing vesicles follow the well documented Golgi route to the cell surface.     

Biosynthesis and intracellular transport of the mouse macrophage Fc receptor

The membrane insertion, processing, and intracellular transport of the mouse macrophage Fc receptor for IgG1/IgG2b was studied using specific mono- and polyclonal anti-receptor antibodies. By immunoprecipitation from Triton X-114 lysates of radiolabeled J774 cells, we determined that the mature 60-kDa receptor is a transmembrane glycoprotein which is synthesized in the rough endoplasmic reticulum as a 53-kDa precursor. Digestion of the precursor with endo-beta-N-acetylglucosaminidase F demonstrated that the receptor consisted of a 37-kDa polypeptide to which four asparagine-linked oligosaccharides were attached. Proteinase K treatment of isolated microsomes indicated that the receptor also has a putative 15-kDa cytoplasmic domain apparently recognized by at least one anti-Fc receptor monoclonal antibody. An additional 15-kDa domain was found to be inaccessible to proteolysis from either side of the membrane. Pulse-chase experiments using [35S]methionine-, [3H]mannose-, and [3H]galactose-labeled cells showed that processing of the receptor's N-linked oligosaccharides occurred rapidly (t1/2 = 15 min) and resulted in the conversion of at least three of the chains to complex endo-beta-N-acetylglucosaminidase H-resistant forms. O-Linked oligosaccharides were not detected. Fc receptor was detected on the plasma membrane 30 min after its synthesis. Transport of newly synthesized receptors to the plasma membrane was slowed but not blocked by incubation of J774 cells at 20 degrees C or by the carboxylic ionophore monensin, although monensin completely inhibited the galactosylation of the receptor.     

Ca2+-dependent translocation of hexose carrier in mouse fibroblast Swiss 3T3 cells

Ca2+-induced translocation of hexose carriers from microsomal membrane to plasma membrane was demonstrated in saponin-permeabilized Swiss 3T3 cells by a specific D-glucose-inhibitable cytochalasin B-binding assay. The number of hexose carriers in the plasma membrane and the hexose transport activity in intact cells were also compared. The incubation of permeabilized cells with 10 microM Ca2+ at 37 degrees C rapidly increased the number of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane from 13 to 40 pmol/mg protein and concomitantly decreased that in the microsomal membrane from 66 to 36 pmol/mg protein, each with a half-time of approx. 2 min. Furthermore, when Ca2+-stimulated cells were exposed to 50 microM EGTA, the effect of Ca2+ on the translocation of D-glucose-inhibitable cytochalasin B-binding sites was reversed with a half-time of approx. 5 min. The concentration of Ca2+ required for the half-maximal effect was approx 500 nM. The magnitude of the stimulatory effect of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane closely correlated with the magnitude of stimulatory action of Ca2+ on 3-O-methylglucose transport in the intact cells. These results suggest that Ca2+ regulates the activity of hexose transport across the plasma membrane through a rapid and reversible translocation of hexose carrier between microsomal and plasma membranes of mouse fibroblast Swiss 3T3 cells.     

Intracellular processing and transport of influenza-virus envelope proteins in Madin-Darby canine kidney cells. Effects of the carboxylic ionophores monensin and nigericin.

We have investigated the effects of the carboxylic ionophores monensin and nigericin on the intracellular processing and transport of the influenza-virus envelope proteins haemagglutinin and neuraminidase in Madin-Darby-canine-kidney-cell monolayers. In the presence of either ionophore, haemagglutinin acquires resistance to the enzyme endoglycosidase H more slowly than it does in untreated cells. In addition, the ionophores cause a block in oligosaccharide-processing events that are believed to occur normally in the trans elements of the Golgi complex. This block is not overcome even at long chase times. Finally, the ionophores cause a substantial slowing of the delivery of both haemagglutinin and neuraminidase to the plasma membrane. We conclude that the ionophores cause delays in the intracellular transport of these proteins both early and late in the pathway, that is, before and after passage through the trans-Golgi, and perturb the processing functions of this compartment. The possible significance of these observations with regard to the intracellular transport of newly synthesized plasma-membrane proteins in epithelial cells is discussed.     

Intracellular transport of a variant surface glycoprotein in Trypanosoma brucei

Trypanosome variant surface glycoproteins (VSGs) have a novel glycan-phosphatidylinositol membrane anchor, which is cleavable by a phosphatidylinositol-specific phospholipase C. A similar structure serves to anchor some membrane proteins in mammalian cells. Using kinetic and ultrastructural approaches, we have addressed the question of whether this structure directs the protein to the cell surface by a different pathway from the classical one described in other cell types for plasma membrane and secreted glycoproteins. By immunogold labeling on thin cryosections we were able to show that, intracellularly, VSG is associated with the rough endoplasmic reticulum, all Golgi cisternae, and tubulovesicular elements and flattened cisternae, which form a network in the area adjacent to the trans side of the Golgi apparatus. Our data suggest that, although the glycan-phosphatidylinositol anchor is added in the endoplasmic reticulum, VSG is nevertheless subsequently transported along the classical intracellular route for glycoproteins, and is delivered to the flagellar pocket, where it is integrated into the surface coat. Treatment of trypanosomes with 1 microM monensin had no effect on VSG transport, although dilation of the trans-Golgi stacks and lysosomes occurred immediately. Incubation of trypanosomes at 20 degrees C, a treatment that arrests intracellular transport from the trans-Golgi region to the cell surface in mammalian cells, caused the accumulation of VSG molecules in structures of the trans-Golgi network, and retarded the incorporation of newly synthesized VSG into the surface coat.