Wortmannin Retards the Movement of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor and Its Ligand out of Endosomes*

Robin Kundra and Stuart KornfeldDagger

From the Division of Hematology, Washington University School of Medicine, St. Louis, Missouri 63110

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The effect of wortmannin on the trafficking of the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF-II receptor) and its ligand beta -glucuronidase has been determined in murine L cells and normal rat kidney cells. The drug induced a 90% decrease in the steady-state level of the Man-6-P/IGF-II receptor at the plasma membrane without affecting the rate of internalization, indicating that the return of receptor from endosomes to the plasma membrane is retarded. Wortmannin also slowed the movement of receptor from endosomes to the trans-Golgi network by about 60%. Such a kinetic block would dramatically reduce the number of Man-6-P/IGF-II receptors in the trans-Golgi network, which could account for the previously described hypersecretion of procathepsin D induced by wortmannin. In addition, the drug slowed delivery of endocytosed beta -glucuronidase from endosomes to dense lysosomes. These data, taken together with the published reports of others, indicate that wortmannin inhibits membrane trafficking out of multiple compartments of the endosomal system and suggest a role for phosphatidylinositol 3-kinase in regulating these processes.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF-II receptor)1 follows a complex itinerary as it carries out its functions of transporting newly synthesized acid hydrolases to lysosomes and of internalizing ligands at the plasma membrane (1-4). In the trans-Golgi network (TGN), the receptor binds acid hydrolases and the complex is packaged into clathrin-coated vesicles. After budding from the TGN, the vesicles deliver their contents to early endosomes, and perhaps later endosomal compartments as well (5, 6). When the receptor with its bound ligand arrives in the late endosome, the acidic pH in the lumen of this compartment promotes ligand release. The free receptor recycles to the TGN to mediate another round of sorting, and the ligand travels to dense lysosomes (7-10). Cell surface Man-6-P/IGF-II receptors bind IGF-II in addition to extracellular acid hydrolases and are internalized via clathrin-coated vesicles. The endocytic and biosynthetic routes converge at the early endosome, and both pools of receptors mix freely (11-13). At steady state, the majority of the Man-6-P/IGF-II receptor is localized to endosomes with small amounts in the TGN and at the cell surface (8, 10, 14). The source of the cell surface receptor is not fully understood, but the majority may be transported from late endosomes (15).

While the role of clathrin-coated vesicles in the transport of the receptor from the TGN and the plasma membrane to endosomal compartments is well established, the nature of the vesicular carriers that mediate subsequent steps of receptor trafficking is unknown. Recently, several reports have documented that wortmannin, a fungal metabolite known to inhibit phosphatidylinositol 3-kinases (PI 3-kinases), induces the hypersecretion of the acid hydrolase procathepsin D (16, 17). Two hypotheses have been suggested to explain this defect in lysosomal enzyme sorting. One proposes that a wortmannin-sensitive enzyme is required at the TGN either for the incorporation of Man-6-P/IGF-II receptors into clathrin-coated vesicles or the formation of these vesicles (16, 17). The second hypothesis suggests that such an enzyme is required for recycling of the Man-6-P/IGF-II receptor from late endosomes to the TGN and depletion of receptors from the TGN results in procathepsin D secretion (18). It is clear from the initial studies that wortmannin produced an altered cellular morphology, namely the appearance of swollen late endosomes containing Man-6-P/IGF-II receptors. This was associated with a loss of receptors from the TGN and the cell surface (16, 18). These findings indicate that wortmannin alters the trafficking of the Man-6-P/IGF-II receptor and suggests a role for PI 3-kinases in this process. However, since these studies primarily utilized morphologic techniques to evaluate receptor distribution, it was not possible to assess the effect of wortmannin on the actual kinetics of receptor transport between compartments. Furthermore, recent work has reported that this altered endosomal morphology does not reflect a block in receptor transport to the TGN (19). To address this issue directly, we have analyzed the effect of wortmannin on the trafficking of the Man-6-P/IGF-II receptor using a variety of kinetic assays in living cells. In addition, we have analyzed the effect of this fungal metabolite on the transport of beta -glucuronidase from the cell surface to dense lysosomes. The results of these experiments indicate that wortmannin retards the movement of both the Man-6-P/IGF-II receptor and beta -glucuronidase out of late endosomes.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- All reagents were of analytical grade. Na125I was obtained from Amersham Corp.; UDP-[6-3H]galactose was from American Radiolabeled Chemicals (St. Louis, MO); lactoperoxidase was from Calbiochem (San Diego, CA); Percoll was from Pharmacia (Uppsala, Sweden); galactosyltransferase was from Fluka (Buchs, Switzerland). All other enzymes and chemicals were obtained from Sigma. Wortmannin was prepared as a 2 mM stock in Me2SO and stored at -20 °C. Aliquots were thawed only once, diluted, and used immediately.

Cells-- Mouse L cells stably expressing the bovine Man-6-P/IGF-II receptor (Cc2 cells) or Man-6-P/IGF-II receptor with a truncated cytoplasmic tail (344 cells) have been described previously (20, 21). The mutant receptor expressed by 344 cells has an Ala substitution at Tyr24, and the cytoplasmic tail is truncated to 29 amino acids. Cell lines were grown in complete medium (alpha -minimal essential medium containing 10% heat-inactivated fetal calf serum, 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate) with 350 µg/ml G418 (Life Technologies, Inc.) at 37 °C in 5% CO2. Normal rat kidney cells were obtained from ATCC and grown in complete medium.

Endocytosis of beta -Glucuronidase-- beta -Glucuronidase was purified from the secretions of 13.2.1 cells (a gift from Dr. W. Sly, St. Louis University) as described previously (20). Cc2 cells were seeded in 22-mm wells of a 12-well tissue culture plate and grown to confluence, washed with PBS, and incubated with 10 nM beta -glucuronidase in complete medium in the absence or presence of 1 µM wortmannin for appropriate times. Medium was aspirated, and cells were washed with PBS five times, washed with 15 mM phosphate-citrate buffer (pH 5.0) twice, and lysed with PBS+1% Triton X-100. An aliquot of the lysate was assayed for beta -glucuronidase activity.

Iodination of beta -Glucuronidase-- beta -Glucuronidase (30 µg) was iodinated with 1 mCi of Na125I using soluble lactoperoxidase as described (22) and gel-filtered (20). The specific radioactivity of the pooled beta -glucuronidase was 8-16 µCi/µg, assuming complete recovery of the beta -glucuronidase.

Assay for Rapid Receptor Internalization-- The short internalization assay was performed as described previously (20) with minor modifications as described below. Briefly, 344 or Cc2 cells were seeded into 22-mm wells of a 12-well plate and grown to confluence. When determining the internalization rate in the presence of wortmannin, the cells were pretreated with complete medium containing 1 µM wortmannin for 30 min. The cells were rinsed with complete medium, and then 1 ml of complete medium containing 0.1 µg (approximately 1 × 106 cpm) of 125I-beta -glucuronidase in the absence or presence of 1 µM wortmannin was added. After a 30-min incubation on ice, the cells were rapidly washed five times with ice-cold PBS containing 1% bovine serum albumin and the plate was transferred to a 37 °C water bath. A 0.5-ml portion of a mixture of 0.035% trypsin, 0.013% EDTA, 10 mM Man-6-P in 15 mM phosphate-citrate saline, pH 5, was immediately added to each of two wells that were used for measurement of the total surface binding of ligand, while the other wells received an addition of 0.5 ml of 37 °C complete medium containing 1 µM wortmannin when appropriate. After the incubation period, the medium was collected and 0.5 ml of the pH 5 trypsin/Man-6-P mixture was added to each well. At the end of an additional 3-min incubation at 37 °C, 0.8 ml of complete medium was added to each well. The cells were then harvested, pelleted, and the supernatant aspirated. The radioactivity in the cell pellet was measured in a gamma -counter. The cells from the two wells used to determine the total surface binding were treated similarly except that the radioactivity of the harvesting medium was measured directly, prior to pelleting the cells.

Cell Surface Binding of 125I-beta -Glucuronidase-- Cc2 cells were seeded in a 24-well tissue culture plate and grown to confluence. Cells were washed with complete medium and then incubated with 0.5 ml of complete medium containing 1 µM wortmannin for up to 3 h. The wortmannin-containing medium was replaced hourly. Cells were then subjected to the same procedure described for determining total surface binding in the rapid internalization assay.

Percoll Gradient Fractionation-- Percoll gradient fractionation was performed as described previously (23) with minor modifications as described below. Briefly, confluent cultures of normal rat kidney cells in 100-mm Petri dishes were preincubated or not with 1 µM wortmannin and then incubated with 3 ml of complete medium containing 2 × 106 cpm of 125I-beta -glucuronidase in the absence or presence of 1 µM wortmannin at 37 °C for an additional 15 min. The cells were washed twice with PBS and incubated with 10 ml of complete medium for up to 90 min in the absence or presence of 1 µM wortmannin. The wortmannin-containing medium was replaced hourly. After two washes with PBS, cells were scraped into 2 ml of homogenization buffer (HB) (0.25 M sucrose, 1 mM EDTA, pH 7.5) and pelleted for 10 min at 140 × g at 4 °C. The cells were resuspended in 850 µl of HB, and passed 12 times through a ball bearing homogenizer (24) with a clearance of 51.2 µm. The homogenate was diluted to 1.7 ml and centrifuged, and the resulting postnuclear supernatant was layered over a discontinuous gradient consisting of a 1.2-ml cushion of 10 × HB and 8.5 ml of an 18% Percoll solution in 1 × HB. The gradient was centrifuged for 30 min at 20,000 rpm in a Ti 50 rotor (Beckman Instruments, Palo Alto, CA) at 4 °C. The gradient was collected from the bottom of the tube in 1-ml fractions. The amount of radioactivity in each fraction was measured in a gamma -counter, and an aliquot of each fraction was assayed for beta -hexosaminidase activity after the sample was adjusted to 1% Triton X-100 and incubated on ice for 1 h.

Enzyme Assays-- beta -Glucuronidase activity was determined by dilution of the samples in 100 mM 4-methlyumbelliferyl beta -D-glucuronide, 100 mM sodium acetate, pH 4.8. The samples were incubated for 15-60 min at 37 °C; the reaction was stopped with the addition of 0.25 M glycine, pH 10.5, and the fluorescence was measured.

beta -Hexosaminidase activity was determined by dilution of the sample in 5 mM p-nitrophenyl-N-acetyl-beta -D-glucosaminide, 50 mM citrate, pH 4.5. The samples were incubated for 30 min at 37 °C; the reaction was stopped with 0.2 M sodium carbonate, and the absorbance read at 400 nm.

Sialylation Assay-- The assay was performed as described previously (12) with a few modifications (25). Briefly, mouse L cells were incubated in complete medium containing 0.02 unit/ml Vibrio cholerae neuraminidase and 0.02 unit/ml Diplococcus pneumoniae beta -galactosidase for 1 h at 37 °C. The glycosidases were removed by washing cells three times with PBS, and 10 µM 2,3-dehydro-2-desoxy-N-acetylneuraminic acid was added to all subsequent incubations to abolish the activity of any neuraminidase that may have been internalized by pinocytosis. Cells were incubated for 30 min with 20 µCi/ml UDP-[6-3H]galactose and 0.2 unit/ml galactosyltransferase on ice to label surface glycoproteins, washed, and then incubated at 37 °C for 2 h in the absence or presence of 1 µM wortmannin. The wortmannin-containing medium was changed after the first hour. The cells were collected by scraping, washed with PBS, and sonicated in lysis buffer (50 mM imidazole HCl, pH 6.5, 150 mM NaCl, 5 mM EDTA, 2% Triton X-100, 0.5% deoxycholate, and 2 µg/ml each of pepstatin A, leupeptin, chymostatin, and antipain, and 10 trypsin inhibitory units/ml of aprotinin), followed by centrifugation at 50,000 × g for 30 min at 4 °C. The supernatant containing the solubilized Man-6-P/IGF-II receptor was applied to a phosphopentamannose-Sepharose affinity column. The column was washed with G-25 buffer (50 mM imidazole, pH 6.5, 150 mM NaCl, 0.25% Triton X-100, 5 mM EDTA, 0.5 mg/ml bovine serum albumin, and protease inhibitors) and the bound Man-6-P/IGF-II receptor was eluted with G-25 buffer containing 10 mM Man-6-P. The Man-6-P eluted fractions were pooled and digested for 24 h at 37 °C with 0.02 unit/ml D. pneumoniae beta -galactosidase. This material was applied to a Sephadex G-25 column, and the radioactivity in the void volume (Man-6-P/IGF-II receptor) and in the included peak (free [3H]galactose) was determined. The percent sialylation of the Man-6-P/IGF-II receptor was calculated by dividing the radioactivity recovered in the void volume by the sum of the radioactivity in the void and included fractions, and multiplying by 100.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Wortmannin Decreases the Uptake of beta -Glucuronidase by Mouse L Cells-- As the first step toward investigating the effects of wortmannin on the trafficking of the Man-6-P/IGF-II receptor, we measured the endocytosis of the acid hydrolase beta -glucuronidase by mouse L cells in the presence of this fungal metabolite. The uptake of this enzyme is known to be mediated by the Man-6-P/IGF-II receptor (26, 27). Mouse L cells stably expressing the bovine Man-6-P/IGF-II receptor (Cc2 cells) were incubated with beta -glucuronidase for 1 h in the absence or presence of increasing concentrations of wortmannin (10 nM to 10 µM). As shown in Fig. 1, inhibition of beta -glucuronidase uptake was detected at a wortmannin concentration of 10 nM and half-maximal inhibition occurred at 40 nM. The maximal inhibition was 91%.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Wortmannin inhibits Man-6-P/IGF-II receptor-mediated endocytosis in a dose-dependent manner. Mouse L cells stably expressing the bovine Man-6-P/IGF-II receptor were incubated with beta -glucuronidase in the absence or presence of the indicated concentrations of wortmannin for 1 h. Washed cells were collected, and the amount of internalized beta -glucuronidase was determined and expressed as a percentage of that internalized by control cells (26 units) in the absence of wortmannin. Data are the means of two independent experiments performed in triplicate.

The time course of the wortmannin effect is shown in Fig. 2. When beta -glucuronidase and 1 µM wortmannin were added to the medium simultaneously, the uptake of the beta -glucuronidase decreased progressively for the first hour (relative to the control cells) and then gradually increased during the next 2 h of incubation. If the cells were pretreated with wortmannin for 15 min prior to the addition of the ligand, the inhibition of beta -glucuronidase uptake was more pronounced during the first hour. Furthermore, in the latter experiment additional wortmannin was added at hourly intervals and the uptake of beta -glucuronidase remained at the minimal level during the 3-h incubation. The fact that inhibition was maintained when wortmannin was added at regular intervals suggested that the drug was being inactivated during the course of the 3-h experiment. To test this possibility directly, cells were incubated with beta -glucuronidase for 3 h and wortmannin was added one to four times during the incubation, such that the final concentration added was 1 µM in all cases. The results summarized in Fig. 3 confirm that repeated additions of wortmannin are required to maintain the maximal inhibiting effects and support the conclusion that the wortmannin is being inactivated over time. Therefore, all subsequent experiments were performed in the presence of 1 µM wortmannin and the wortmannin-containing medium was replaced hourly.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Time course of wortmannin inhibition of Man-6-P/IGF-II receptor-mediated endocytosis. Mouse L cells stably expressing the Man-6-P/IGF-II receptor were incubated with beta -glucuronidase for the indicated intervals. One set of cells (bullet ) was incubated in the continuous presence of 1 µM wortmannin added simultaneously with ligand. Another set of cells (open circle ) was pretreated with 1 µM wortmannin 15 min prior to ligand addition and then incubated in the continued presence of wortmannin, with fresh wortmannin applied hourly (1 µM final concentration). The amount of internalized beta -glucuronidase was determined and expressed as a percentage of that internalized by control cells in the absence of wortmannin during the same interval. Data points are the mean of three independent experiments performed in triplicate with error bars representing the standard deviation. The mean values for the controls were 6.2, 11.4, 28.7, 46, and 77 units at the times shown.


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 3.   Wortmannin inhibition of Man-6-P/IGF-II receptor-mediated endocytosis diminishes with time. Mouse L cells stably expressing the Man-6-P/IGF-II receptor were incubated with beta -glucuronidase for 3 h. Equal aliquots of wortmannin were added simultaneously with ligand and during equal intervals over the 3-h incubation such that the final concentration in the 1-ml well equaled 1 µM. One addition indicates that 1 nmol of wortmannin was added at time 0. Two additions indicates that 500 pmol of wortmannin was added at time 0 and 90 min. Three additions indicates that 333 pmol of wortmannin was added at time 0, 60, and 120 min. Four additions indicates that 250 pmol of wortmannin was added at time 0, 45, 90, and 135 min. The amount of internalized beta -glucuronidase was determined and expressed as a percentage of that internalized by control cells in the absence of wortmannin. The data represent the mean of two independent experiments performed in triplicate.

Wortmannin Induces a Decrease in Cell Surface Man-6-P/IGF-II Receptors-- The inhibition of beta -glucuronidase uptake in the presence of wortmannin could be due to a loss of cell surface receptors or a decrease in their rate of internalization. To determine if the number of cell surface Man-6-P/IGF-II receptors was diminished, Cc2 cells were treated with 1 µM wortmannin for up to 3 h at 37 °C and then the surface binding of 125I-beta -glucuronidase was measured on ice. As shown in Fig. 4, wortmannin caused a rapid decline in the amount of 125I-beta -glucuronidase that bound to the cell surface receptors. The maximal decrease of 90% was reached with a t1/2 of approximately 15 min.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   Reduction of cell surface Man-6-P/IGF-II receptor in response to wortmannin accounts for inhibition of receptor-mediated endocytosis. Mouse L cells stably expressing the Man-6-P/IGF-II receptor were pretreated with 1 µM wortmannin for the indicated times, and medium containing wortmannin was replaced hourly. Cells were incubated with 1 × 106 cpm 125I-beta -glucuronidase for 30 min on ice in the continued presence of wortmannin. Washed cells were collected, and cell-associated radioactivity was measured and expressed as a percentage of that bound by cells in the absence of wortmannin. The points represent the mean of 16 determinations from four independent experiments.

Similar results were obtained when the wortmannin-treated cells were rinsed with 5 mM Man-6-P prior to measuring 125I-beta -glucuronidase binding or when the concentration of beta -glucuronidase was increased 30-fold. This indicates that the decline in 125I-beta -glucuronidase binding was not due to occupancy of the receptor by endogenous ligand or to a decrease in the affinity of the receptor for its ligand, but rather to a reduction in the number of cell surface receptors.

Wortmannin Does Not Alter the Rate of Internalization of the Man-6-P/IGF-II Receptor-- The effect of wortmannin on the initial rate of receptor internalization was determined as follows; 125I-beta -glucuronidase was allowed to bind on ice to cells that had been pretreated or not for 30 min with 1 µM wortmannin. The unbound ligand was removed, and the cells were warmed to 37 °C. Receptor internalization was followed by determining the rate of uptake of beta -glucuronidase over a 2-min period in the absence or presence of wortmannin. This experiment was initially performed with L cells expressing the wild-type receptor, but the binding of 125I-beta -glucuronidase to the cells preincubated with wortmannin was reduced so much that valid measurements could not be obtained (Fig. 4). To overcome this technical problem, the 344 cells were used because they have more surface receptors than Cc2 cells. The Man-6-P/IGF-II receptor expressed in 344 cells has a truncated (29-amino acid) tail, but it is internalized at the same rate as the wild-type receptor (20). Furthermore, the truncated receptor is lost from the cell surface of wortmannin-treated 344 cells with kinetics identical to those described for the full-length receptor in the Cc2 cell line (data not shown). Fig. 5 shows that the presence of 1 µM wortmannin had no effect on the initial rate of beta -glucuronidase internalization. Since wortmannin causes the number of cell surface receptors to decrease without altering the rate of receptor internalization, the return of receptors to the cell surface must be inhibited.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5.   Wortmannin does not affect the rate of Man-6-P/IGF-II receptor endocytosis. Mouse L cells stably expressing the truncated Man-6-P/IGF-II receptor (344 cells) were incubated with 125I-beta -glucuronidase for 30 min on ice, washed to remove unbound ligand, and warmed to 37 °C. At the indicated times, cells were placed on ice and washed, surface-bound counts were eluted, and cells were collected. The radioactivity in the cell pellet was determined and expressed as a fraction of total surface-bound radioactivity. Control cells are indicated by open circle . Cells pretreated for 30 min and then incubated in the continued presence of 1 µM wortmannin during ligand binding and internalization are indicated by bullet . The points represent the mean of three independent experiments performed in duplicate.

Wortmannin Slows the Return of the Man-6-P/IGF-II Receptor to the TGN-- One explanation for the defective acid hydrolase sorting induced by wortmannin is that recycling of the Man-6-P/IGF-II receptor to the TGN is impaired. To measure this step, we took advantage of the fact that the receptor is a glycoprotein that can be acted upon by sialyltransferases localized in the TGN (28-30). Consequently, the actions of these glycosyltransferases can be used to mark the return of receptor molecules to the Golgi. However, the Asn-linked oligosaccharides on the receptor must express a terminal galactose to be substrates for the sialyltransferases. Therefore, we pretreated mouse L cells with neuraminidase and beta -galactosidase to remove the sialic acid and galactose residues of the cellular glycoproteins. The resulting terminal N-acetylglucosamine residues of the cell surface glycoproteins (including the Man-6-P/IGF-II receptor) were labeled with [3H]galactose by incubating the cells with exogenous galactosyltransferase and UDP-[6-3H]galactose on ice. The cells were then warmed to 37 °C in the absence or presence of wortmannin for 2 h to allow the surface-labeled receptor molecules to be internalized and return to the TGN, where sialic acid residues are transferred on to the [3H]galactose by sialyltransferase. To determine the rate of sialylation, the receptor was isolated on a phosphopentamannose-Sepharose affinity column and treated with beta -galactosidase, which releases terminal [3H]galactose residues, but not [3H]galactose residues that have been modified through the addition of sialic acid moieties. The released [3H]galactose was then separated from the receptor by gel filtration. The fraction of the [3H]galactose that is resistant to beta -galactosidase reflects the rate of return of receptor molecules to the Golgi and the efficiency of their sialylation.

The results of two typical experiments performed in duplicate are shown in Table I. In each experiment, a background value of [3H]galactose that was not cleaved during the beta -galactosidase treatment was determined by digesting receptor recovered from labeled cells that had not been warmed to 37 °C. After 2 h at 37 °C, receptor from the control cells was sialylated an average of 8.5% over the background compared with an average of 3.5% in the case of the receptor from wortmannin-treated cells. Overall, the sialylation of receptors isolated from wortmannin-treated cells was reduced by about 60%. This type of resialylation experiment was also performed with BW 5147 PHAR1.8 mouse lymphoma cells with similar results (data not shown). These data indicate that wortmannin slows the return of the Man-6-P/IGF-II receptor to the TGN.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Wortmannin inhibits sialylation of the Man-6-P/IGF-II receptor
Cell surface glycoproteins of mouse L cells stably expressing the Man-6-P/IGF-II receptor were labeled with UDP-[6-3H]galactose and exogenous galactosyltransferase on ice for 30 min. Cells were warmed to 37 °C and incubated for 2 h in the absence or presence of 1 µM wortmannin. The Man-6-P/IGF-II receptor was isolated by affinity chromatography, and the percentage of beta -galactosidase-resistant counts was calculated as described under "Experimental Procedures." Background indicates the percentage of beta -galactosidase-resistant counts associated with receptors from time zero control cells which were not warmed to 37 °C.

Wortmannin Slows the Arrival of Endocytosed beta -Glucuronidase in Dense Lysosomes-- After the Man-6-P/IGF-II receptor with its bound ligand is internalized at the plasma membrane, the complex is transported through early endosomes to a late endosomal compartment, where the acidic pH promotes the release of the ligand, which is subsequently delivered to the dense lysosome. The studies described in the previous section show that wortmannin does not affect the initial rate of internalization of beta -glucuronidase, which is mediated by the Man-6-P/IGF-II receptor. To determine if this fungal metabolite influences the later steps in this pathway, we followed the delivery of 125I-beta -glucuronidase to dense lysosomes in the presence and absence of this agent. Control and wortmannin pretreated normal rat kidney cells were allowed to take up 125I-beta -glucuronidase for 15 min. Following chase times of up to 90 min, the cells were homogenized and the postnuclear supernatants were fractionated on 18% Percoll gradients to separate dense lysosomes from other membranous compartments (including endosomes) that were recovered near the top of the gradient. Fractions were collected and the radioactivity in each fraction determined, as shown in Fig. 6. To identify the position of dense lysosomes in the gradient, each fraction was assayed for the lysosomal marker beta -hexosaminidase.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 6.   Wortmannin delays arrival of endocytosed beta -glucuronidase in dense lysosomes. Control (open circle ) and 1 µM wortmannin-treated (bullet ) normal rat kidney cells were incubated with 125I-beta -glucuronidase for 15 min and chased for 30 min (A and B), 60 min (C and D), and 90 min (E and F). Postnuclear supernatants were fractionated on 18% Percoll gradients as described under "Experimental Procedures." The amount of radioactivity in each fraction was expressed as a percentage of the total radioactivity recovered (A, C, and E). The distribution of beta -hexosaminidase activity, a lysosomal marker, is shown (B, D, and F). The data shown are from one experiment and are representative of three independent experiments.

In the absence of wortmannin, half of the internalized 125I-beta -glucuronidase was delivered to dense lysosomes by 30 min of chase (Fig. 6A). In contrast, the arrival of the 125I-beta -glucuronidase in the dense lysosomes of cells treated with 1 µM wortmannin was significantly slowed, with only 37% of the ligand accumulating there after 90 min of chase (Fig. 6E). Drug concentrations of 10 nm and greater slowed the rate of arrival of 125I-beta -glucuronidase to the dense lysosomes, with the maximal effect occurring at 1 µM wortmannin (data not shown).

Interestingly, the beta -hexosaminidase activity from cells incubated in the presence of wortmannin for more than 60 min is partially redistributed to less dense fractions of the gradient. Although this finding complicates the interpretation of results obtained after longer periods of chase, it is clear that, at 30 min of chase, significantly less 125I-beta -glucuronidase is recovered in the dense lysosomes of wortmannin-treated cells, 18% compared with 54% for control cells (Fig. 6A), with no apparent change in the beta -hexosaminidase profile (Fig. 6B). A similar lag in the delivery of 125I-beta -glucuronidase to dense lysosomes was observed in wortmannin-treated L cells (data not shown). However, redistribution of the lysosomal marker occurred more rapidly in this cell line.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The data presented in this paper demonstrate that wortmannin slows the movement of the Man-6-P/IGF-II receptor from endosomes to the TGN and the plasma membrane without affecting the rate of internalization at the plasma membrane. These changes in the kinetics of receptor trafficking would be expected to alter the steady-state distribution of the Man-6-P/IGF-II receptor such that the level at the plasma membrane and TGN would decrease, whereas it increases in endosomes. This, in fact, is what has been observed in morphologic studies examining the effect of wortmannin on the distribution of the Man-6-P/IGF-II receptor (16, 18). These investigators found that, within 30 min, wortmannin caused the receptor to accumulate in swollen late endosomes while being depleted from the TGN and plasma membrane. In our experiments, wortmannin treatment of murine L cells resulted in a 90% reduction in the level of Man-6-P/IGF-II receptor at the cell surface and slowed the return of receptor to the TGN by 60%.

These kinetic effects on the trafficking of the Man-6-P/IGF-II receptor help to explain the hypersecretion of procathepsin D that occurs in response to this agent (16, 17). By slowing the return of the Man-6-P/IGF-II receptor to the TGN, wortmannin treatment would reduce the number of receptor molecules in that compartment available for binding newly synthesized acid hydrolases, including procathepsin D. When the number of available receptor molecules becomes insufficient to bind the newly synthesized acid hydrolases, hypersecretion will result. Indeed, Gaffet et al. (31) have shown recently that clathrin-coated vesicles isolated from wortmannin-treated cells contain significantly less Man-6-P/IGF-II receptor. This finding is consistent with a loss of receptor from the TGN. Furthermore, this work demonstrates that wortmannin does not prevent the formation or consumption of TGN derived clathrin-coated vesicles. While this manuscript was in preparation, Nakajima et al. (19) reported that wortmannin does not effect recycling of the Man-6-P/IGF-II receptor from late endosomes to the TGN in Chinese hamster ovary and K562 cells. While the reason for this disparity is not clear, it may reflect differences in the cell types studied or differences in the nature of the assay systems used, such as the use of lectin chromatography to determine the fraction of receptors bearing sialic acid residues.

Wortmannin also caused a marked delay in the arrival of endocytosed beta -glucuronidase in dense lysosomes. Since wortmannin does not dissipate the endosomal pH gradient, this delay cannot be attributed to the absence of the pH-induced release of ligand from the Man-6-P/IGF-II receptor that occurs in the late endosome (16, 32). Rather, these findings provide further support for a general inhibitory effect of wortmannin on the movement of proteins out of endosomes, in this case resulting in slowed delivery to lysosomes. Since the initial processing of procathepsin D occurs in late endosomes while the final cleavage that yields the mature form requires delivery of the acid hydrolase to dense lysosomes, wortmannin treatment would be expected to slow the production of mature cathepsin D. Indeed, previous work that monitored the effect of this agent on the processing of retained procathepsin D indicates that the conversion of the intermediate form of cathepsin D to the mature form is impaired in wortmannin-treated cells, lending further evidence for reduced trafficking from late endosomes to lysosomes (17). Another study following the degradation of endocytosed Semliki Forest virus as an indicator of lysosomal delivery also found that wortmannin delays the breakdown of internalized virus (32). Although these studies demonstrate that movement of proteins from endosomes to lysosomes is slowed by wortmannin, we cannot determine whether the delivery is delayed because of a direct inhibition of fusion of late endosomal multivesicular bodies with lysosomes as described for the epidermal growth factor receptor (33) or if the formation of vesicular carriers is impaired.

It is of interest that wortmannin has recently been shown to cause a redistribution of lysosomal membrane proteins from dense lysosomes to swollen late endosomes distinct from those containing the Man-6-P/IGF-II receptor (18). This suggests that retrograde trafficking from dense lysosomes proceeds in the presence of this drug while movement of proteins from late endosomes to lysosomes is inhibited. A similar phenomenon may be occurring in our experiments, which would account for the shift of beta -hexosaminidase from dense lysosomes to lighter fractions after prolonged wortmannin treatment (Fig. 6). The lysosomal marker could also localize at the top of the gradient if the depletion of lysosomal membrane proteins from lysosomes made these organelles more susceptible to lysis during homogenization. However, the activity recovered was latent without detergent treatment, consistent with the beta -hexosaminidase being within vesicles (data not shown). Furthermore, it has been shown that beta -galactosidase and Lamp 1 redistribute from lysosomes to early endosomes when transport out of early endosomes is blocked in a mutant cell line (34).2 Such observations suggest that some soluble lysosomal enzymes, in addition to lysosomal membrane proteins, may continually cycle between lysosomes and endosomes.

Although the results of this study implicate the late endosome as a target of wortmannin action, this agent has been shown to act at additional sites within the endosomal system. For example, transferrin receptor recycling, which involves early endosomal compartments, is inhibited by wortmannin while endocytosis of this receptor has been reported to be either increased or decreased (32, 35-38). In in vitro assays, the drug inhibited early endosome fusion (36, 37, 39). Wortmannin has also been reported to inhibit ligand-induced down-regulation of the platelet-derived growth factor receptor (35, 40, 41) and transcytosis in polarized epithelial cells (42). It also inhibits the insulin-induced exocytosis of the GLUT4 transporter (43-45) and IGF-II-stimulated surface expression of the Man-6-P/IGF-II receptor (46). These effects are elicited by low nanomolar concentrations of the drug, and the p110 PI 3-kinase has been implicated as a mediator of these effects. On the other hand, micromolar concentrations of wortmannin were required to induce lysosomal enzyme hypersecretion in Chinese hamster ovary cells, suggesting that one or more enzymes with differential wortmannin sensitivity may be involved in producing this effect (32). Therefore, in comparing the concentration of wortmannin required to achieve an effect, one must bear in mind that the drug is metabolized and that the rate of metabolism may vary among cell types.

As pointed out by Reaves et al. (18), the common theme of these effects of wortmannin is inhibition of membrane traffic out of the endosomal system with traffic into this system from the plasma membrane, TGN, and lysosome being relatively unaffected or possibly not affected at all as a primary event. Our data on the trafficking of the Man-6-P/IGF-II receptor and endocytosed beta -glucuronidase are consistent with this notion and further demonstrate that wortmannin slows traffic out of endosomes rather than causing a complete block.

    ACKNOWLEDGEMENTS

We thank Dr. R. F. Murphy for sharing unpublished data from his laboratory with us. We are grateful to Dr. W. Sly for the generous gift of the 13.2.1 cells and to Dr. Ian Trowbridge for providing the BW 5147 PHAR1.8 cells. We thank Walter Gregory and Carolyn Noll for excellent technical assistance and members of the Kornfeld and Majerus laboratories for helpful discussions and critical reading of this manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA 08759-30 (to S. K.), NRSA Grant for Training in Molecular Hematology T32 HL07088 (to R. K.), and MSTP Training Grant T32 GM07200 (to R. K.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Div. of Hematology, Washington University School of Medicine, 660 S. Euclid Ave., Box 8125, St. Louis, MO 63110. Tel.: 314-362-8803; Fax: 314-362-8826; E-mail: skornfel{at}im.wustl.edu.

1 The abbreviations used are: Man-6-P/IGF-II receptor, mannose 6-phosphate/insulin-like growth factor II receptor; Man-6-P, mannose 6-phosphate; IGF-II, insulin-like growth factor II; HB, homogenization buffer; PI 3-kinase, phosphatidylinositol 3-kinase; TGN, trans-Golgi network; PBS, phosphate-buffered saline.

2 J. Wightman, J. A. Schwartz, and R. F. Murphy, personal communication.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Hille-Rehfeld, A. (1995) Biochim. Biophys. Acta 1241, 177-194[Medline] [Order article via Infotrieve]
  2. Kornfeld, S. (1992) Annu. Rev. Biochem. 61, 307-330[CrossRef][Medline] [Order article via Infotrieve]
  3. Kornfeld, S., and Mellman, I. (1989) Annu. Rev. Cell Biol. 5, 483-525[CrossRef]
  4. von Figura, K., and Hasilik, A. (1986) Annu. Rev. Biochem. 55, 167-193[CrossRef][Medline] [Order article via Infotrieve]
  5. Ludwig, T., Griffiths, G., and Hoflack, B. (1991) J. Cell Biol. 115, 1561-1572[Abstract]
  6. Rijnboutt, S., Stoorvogel, W., Geuze, H. J., Strous, G. J. (1992) J. Biol. Chem. 267, 15665-15672[Abstract/Free Full Text]
  7. Brown, W. J., Goodhouse, J., and Farquhar, M. G. (1986) J. Cell Biol. 103, 1235-1247[Abstract]
  8. Geuze, H. J., Stoorvogel, W., Strous, G. J., Slot, J. W., Bleekemolen, J. E., Mellman, I. (1988) J. Cell Biol. 107, 2491-2501[Abstract]
  9. Goda, Y., and Pfeffer, S. R. (1988) Cell 55, 309-320[Medline] [Order article via Infotrieve]
  10. Griffiths, G., Hoflack, B., Simons, K., Mellman, I., and Kornfeld, S. (1988) Cell 52, 329-341[Medline] [Order article via Infotrieve]
  11. Braulke, T., Gartung, C., Hasilik, A., and von Figura, K. (1987) J. Cell Biol. 104, 1735-1742[Abstract]
  12. Duncan, J. R., and Kornfeld, S. (1988) J. Cell Biol. 106, 617-628[Abstract]
  13. Jin, M., Sahagian, G. G., Jr., and Snider, M. D. (1989) J. Biol. Chem. 264, 7675-7680[Abstract/Free Full Text]
  14. Griffiths, G., Matteoni, R., Back, R., and Hoflack, B. (1990) J. Cell Sci. 95, 441-461[Abstract]
  15. Riederer, M. A., Soldati, T., Shapiro, A. D., Lin, J., Pfeffer, S. R. (1994) J. Cell Biol. 125, 573-582[Abstract]
  16. Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H., Balch, W. E. (1995) J. Cell Biol. 130, 781-796[Abstract]
  17. Davidson, H. W. (1995) J. Cell Biol. 130, 797-805[Abstract]
  18. Reaves, B. J., Bright, N. A., Mullock, B. M., Luzio, J. P. (1996) J. Cell Sci. 109, 749-762[Abstract/Free Full Text]
  19. Nakajima, Y., and Pfeffer, S. R. (1997) Mol. Biol. Cell 8, 577-582[Abstract]
  20. Jadot, M., Canfield, W. M., Gregory, W., and Kornfeld, S. (1992) J. Biol. Chem. 267, 11069-11077[Abstract/Free Full Text]
  21. Lobel, P., Fujimoto, K., Ye, R. D., Griffiths, G., Kornfeld, S. (1989) Cell 57, 787-796[Medline] [Order article via Infotrieve]
  22. Tait, J. F., Weinman, S. A., and Bradshaw, R. A. (1981) J. Biol. Chem. 256, 11086-11092[Abstract/Free Full Text]
  23. Rohrer, J., Schweizer, A., Johnson, K. F., Kornfeld, S. (1995) J. Cell Biol. 130, 1297-1306[Abstract]
  24. Balch, W. E., and Rothman, J. E. (1985) Arch. Biochem. Biophys. 240, 413-425[Medline] [Order article via Infotrieve]
  25. Johnson, K. F., and Kornfeld, S. (1992) J. Biol. Chem. 267, 17110-17115[Abstract/Free Full Text]
  26. Kyle, J. W., Nolan, C. M., Oshima, A., and Sly, W. S. (1988) J. Biol. Chem. 263, 16230-16235[Abstract/Free Full Text]
  27. Natowicz, M., Baenziger, J. U., and Sly, W. S. (1982) J. Biol. Chem. 257, 4412-4420[Abstract/Free Full Text]
  28. Bennett, G., and O'Shaughnessy, D. (1981) J. Cell Biol. 88, 1-15[Abstract]
  29. Roth, J., Taatjes, D. J., Weinstein, J., Paulson, J. C., Greenwell, P., Watkins, W. M. (1986) J. Biol. Chem. 261, 14307-14312[Abstract/Free Full Text]
  30. Roth, J., Taathes, D. J., Lucocq, J. M., Weinstein, J., Paulson, J. C. (1985) Cell 43, 287-295[Medline] [Order article via Infotrieve]
  31. Gaffet, P., Jones, A. T., and Clague, M. J. (1997) J. Biol. Chem. 272, 24170-24175[Abstract/Free Full Text]
  32. Martys, J. L., Wjasow, C., Gangi, D. M., Kielian, M. C., McGraw, T. E., Backer, J. M. (1996) J. Biol. Chem. 271, 10953-10962[Abstract/Free Full Text]
  33. Futter, C. E., Pearse, A., Hewlett, L. J., Hopkins, C. R. (1996) J. Cell Biol. 132, 1011-1023[Abstract]
  34. Wilson, R. B., Mastick, C. C., and Murphy, R. F. (1993) J. Biol. Chem. 268, 25357-25363[Abstract/Free Full Text]
  35. Shpetner, H., Joly, M., Hartley, D., and Corvera, S. (1996) J. Cell Biol. 132, 595-605[Abstract]
  36. Spiro, D. J., Boll, W., Kirchhausen, T., and Wessling-Resnick, M. (1996) Mol. Biol. Cell 7, 355-367[Abstract]
  37. Li, G., D'Souza-Schorey, C., Barbieri, M. A., Roberts, R. L., Klippel, A., Williams, L. T., Stahl, P. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10207-10211[Abstract]
  38. Shepherd, P. R., Soos, M. A., and Siddle, K. (1995) Biochem. Biophys. Res. Commun. 211, 533-539
  39. Jones, A. T., and Clague, M. J. (1995) Biochem. J. 311, 31-34[Medline] [Order article via Infotrieve]
  40. Joly, M., Kazlauskas, A., and Corvera, S. (1995) J. Biol. Chem. 270, 13225-13230[Abstract/Free Full Text]
  41. Joly, M., Kazlauskas, A., Fay, F. S., Corvera, S. (1994) Science 263, 684-687[Medline] [Order article via Infotrieve]
  42. Hansen, S. H., Olsson, A., and Casanova, J. E. (1995) J. Biol. Chem. 270, 28425-28432[Abstract/Free Full Text]
  43. Clark, J. F., Young, P. W., Yonezawa, K., Kasuga, M., and Holman, G. D. (1994) Biochem. J. 300, 631-635[Medline] [Order article via Infotrieve]
  44. Kotani, K., Carozzi, A. J., Sakaue, H., Hara, K., Robinson, L. J., Clark, S. F., Yonezawa, K., James, D. E., Kasuga, M. (1995) Biochem. Biophys. Res. Commun. 209, 343-348[CrossRef][Medline] [Order article via Infotrieve]
  45. Okada, T., Kawano, Y., Sakakibara, Y., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3568-3573[Abstract/Free Full Text]
  46. Korner, C., and Braulke, T. (1996) Mol. Cell. Endocrinol. 118, 201-205[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.