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Volume 270, Number 2, Issue of January 13, 1995 pp. 665-671
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of Clathrin-coated Vesicles in Glycoprotein Transport from the Cell Surface to the Golgi Complex (*)

(Received for publication, May 31, 1994; and in revised form, October 17, 1994)

Cindy R. Bos,  Samuel L. Shank,  Martin D. Snider

From the Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Plasma membrane glycoproteins recycle to the Golgi complex, but the route followed by these proteins is not known. To elucidate the pathway of transport, the involvement of clathrin-coated vesicles was tested. This was accomplished by comparing the traffic of wild type low density lipoprotein receptor (LDLR) and FH 683, a mutant receptor whose endocytosis from the cell surface in coated vesicles is reduced by 90-95%. Wild type LDLR traveled from the cell surface to the sialyltransferase compartment of the Golgi with a half-time of 2.5 h in K562 human leukemia cells expressing receptor from a transfected cDNA. In contrast, FH 683 LDLR recycled to the Golgi at 33% of the wild type rate, suggesting that wild type LDLR is largely transported to the Golgi by a pathway that involves clathrin-coated vesicles. Moreover, because clathrin-coated vesicles that bud from the plasma membrane are transported to endosomes, surface-to-Golgi transport probably involves an endosomal intermediate. Finally, because there was substantial transport of mutant LDLR to the Golgi even though its endocytosis in coated vesicles was greatly reduced, there may be a second pathway of surface-to-Golgi traffic. Our results suggest that wild type LDLR may move from plasma membrane to Golgi by two routes. Two-thirds of the traffic proceeds via a coated vesicle-mediated pathway while the remainder may follow a clathrin-independent pathway.

INTRODUCTION

Recent studies have shown that membrane glycoproteins recycle from the cell surface to the Golgi complex (reviewed in (1) ). Recycling proteins include receptors that function in receptor-mediated endocytosis(2, 3, 4, 5, 6, 7, 8, 9, 10, 11) , proteins of regulated secretory vesicles(12, 13, 14, 15) , and small synaptic vesicles(11) , as well as other PM (^1)and Golgi proteins(16, 17, 18, 19, 20, 21, 22) . In addition, two studies have shown that geq10 glycoproteins cycle from the PM to the Golgi(23, 24) . These are a distinct subset because most PM glycoproteins do not undergo this recycling(9, 24, 25, 26) . Nevertheless, the flux of proteins from PM to Golgi is substantial. Approximately 25% of the flux of PM glycoproteins through the Golgi is comprised of recycling molecules in CHO-cultured fibroblasts(24) .

Although it is clear that a subset of PM glycoproteins recycle to the Golgi, the route of transport is not known. This lack of understanding is due to the fact that recycling is typically detected by methods that provide little or no information about the transport steps. The biochemical, morphological, and cell fractionation methods used to study recycling only establish the origin of the traffic (the PM) and its destination (the Golgi), but do not allow the individual transport steps to be elucidated(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) .

Clathrin-coated vesicles that bud from the PM could be involved in PM-to-Golgi transport because many recycling proteins are internalized from the PM in these structures. However, previous investigations of this problem by us and others have failed to support a role for coated vesicles(27, 28, 29) . To test directly the role of clathrin-coated vesicles in PM-to-Golgi traffic, we studied the PM-to-Golgi transport of LDLR. These experiments have compared the traffic of wild type LDLR and FH 683, an LDLR mutant that is missing all but 2 residues of the 50-residue C-terminal cytoplasmic tail(30) . Wild type LDLR is preferentially clustered in clathrin-coated pits on the PM and is rapidly internalized. In contrast, FH 683 receptor lacks the sequence in the cytoplasmic tail that causes clustering in coated pits, so it is diffusely distributed on the PM and is taken up much more slowly.

Our experiments were performed in K562 human leukemia cell lines expressing mutant and wild type LDLR from stably transfected cDNAs. K562 cells were chosen because they have been used extensively in our studies of PM-to-Golgi transport(4, 10, 28, 29, 31) . Retrograde transport was assayed by measuring traffic into the trans-Golgi compartment that contains the glycoprotein processing enzyme, sialyltransferase. This was accomplished by removing sialic acid residues from labeled LDLR on the cell surface, reculturing the cells, and analyzing LDLR to determine if sialic acid residues had been replaced during the culture period. LDLR has 1 N-linked and 6-9 O-linked glycans, all of which have sialic acid (32) . We measured PM-to-Golgi transport by measuring the repair of O-linked glycans on desialylated LDLR.

We found that mutant LDLR was transported to the Golgi complex, although at only one-third the rate of wild type receptor. However, traffic of mutant receptor to the Golgi was affected to a much smaller extent than endocytosis from the PM, which occurred at only 10% the wild type rate. These results suggest that endocytosis via clathrin-coated vesicles is required for approximately two-thirds of PM-to-Golgi traffic of wild type LDLR. The remainder of the transport may occur by a clathrin-independent pathway.

EXPERIMENTAL PROCEDURES

Cells and Cell Culture

K562 human leukemia cells were cultured as described previously(4) . Wild type and FH 683 LDLR were expressed in these cells using constructs with receptor cDNAs under the control of the human cytomegalovirus immediate early promoter (obtained from C. G. Davis, Repligen Corp). K562 cells stably expressing these cDNAs were prepared by electroporation of 10^7 cells with receptor cDNA construct and pRSV-neo. Cells were then cultured at 4000 cells/well in 96-well plates in alpha-I-10 (alpha-minimal essential medium, 5 µg/ml bovine insulin, 5 µg/ml human transferrin, 5 ng/ml sodium selenite, 10% Nu-Serum (Collaborative Research)). After 3 days, 400 µg/ml G418 was added to select transfected cells.

G418-resistant cell clones were stained for LDLR expression using the anti-receptor monoclonal antibody, C7(33) . Cells were washed with PBS, 0.5% BSA, 0.1% sodium azide at 4 °C, incubated with C7 for 30 min, washed again, and incubated with fluorescein-labeled goat anti-mouse IgG. The cells were then analyzed on an Epics V flow cytometer (Coulter). The fluorescence of several cell lines was significantly above that of untransfected K562 cells. LDLR expression in these lines was confirmed by metabolically labeling the cells with [S]Met, immunoprecipitating the receptor as described below, and analyzing on SDS gels. One cell line expressing wild type LDLR (R2) and one cell line expressing FH 683 receptor (A8) were chosen for all subsequent studies. The A8 cell line was maintained in alpha-I-10, 400 µg/ml G418. R2 cells were grown in the same medium containing 20 µM Lovastatin and 200 µM mevalonate(34) .

Internalization and Degradation of I-C7-IgG

C7 antibody was iodinated to a specific activity of 1500-1800 cpm/fmol using NaI and IODOGEN (Pierce). To measure uptake, cells were washed twice at room temperature in HEPES-buffered alpha-minimal essential medium containing 10% lipoprotein-deficient fetal bovine serum and suspended at 1 times 10^6 cells/ml in the same medium containing 30 nMI-C7-IgG (666 cpm/fmol). After incubation at 37 °C for 1-9 h, cell-associated radioactivity in samples of 1 times 10^5 cells was determined by centrifugation through dibutylphthalate/mineral oil (9:1) (35) and counting the cell pellet in a counter. In our experiments, nearly all radioactivity in this fraction was due to ligand taken up by endocytosis; ligand bound to surface LDLR, which was measured by incubating cells with I-C7-IgG at 4 °C, was barely detectable. Degradation of I-C7-IgG was determined by measuring the amount of trichloroacetic acid-soluble radioactivity in the culture medium(36) .

Metabolic Labeling with [S]Met

K562 cells were metabolically labeled with [S]Met for 1 h at 37 °C as described previously(4) . The cells were then washed once in growth medium and chased in the same medium for the indicated times.

Immunoprecipitation of LDLR

Immunoprecipitation was carried out at 4 °C using a modification of the method of Tolleshaug et al.(37) . Cells (2.5 times 10^6) were washed in 10 mM sodium HEPES, pH 7.4, 200 mM NaCl, 2 mM CaCl(2), 2.5 mM MgCl(2), 1 mM phenylmethylsulfonyl fluoride, 0.1 mM leupeptin (buffer B) and lysed in 600 µl of buffer B containing 1.5% Triton X-100 and 10 mg/ml BSA. After 20 min, samples were centrifuged at 13,000 times g for 30 min. The supernatants were decanted, C7 anti-LDLR (5 µg) was added, and the samples were rocked overnight. Affinity-purified goat anti-mouse IgG (30 µg) was added, and the samples were rocked for 20 min. Protein A-Sepharose (45 µl of a 25% suspension) was then added, and the samples were rocked for 1.5 h. The tubes were centrifuged, the supernatants were discarded, and the pellets were washed twice with 250 µl of lysis buffer and once with 50 mM Tris, pH 8, 2 mM CaCl(2) at room temperature.

Measurement of LDLR Resialylation

For labeling with [^3H]GlcN, cells were washed twice with glucose-free minimal essential medium containing nonessential amino acids, 0.1 mg/ml glucose, and 2% lipoprotein-deficient fetal bovine serum(38) , suspended in the same medium containing 400 µCi/ml [^3H]GlcN at 1.3 times 10^7 cells/ml, and incubated at 37 °C for 4 h. The cells were then washed with alpha-I-10 and incubated in this medium at 37 °C for 2 h at 5 times 10^6 cells/ml. To biotinylate surface proteins, cells were washed 3-4 times in ice-cold PBS (pH 8) and suspended at 6.6 times 10^6 cells/ml in PBS (pH 8) containing 1 mg/ml freshly dissolved sulfo-NHS-biotin (Pierce). After 30 min on ice, 1 volume of minimal essential medium was added(39) , and the cells were then washed four times with ice-cold PBS (pH 7.4). Then, surface sialic acid residues were removed by incubating with 30 milliunits/ml Vibrio cholerae neuraminidase at 0 °C for 60 min(4) . Finally, cells were cultured in growth medium at 37 °C.

To isolate biotinylated LDLR, samples of 5 times 10^6 cells were lysed. Then, LDLR was immunoprecipitated as described above and the immune complexes were dissociated by incubation in 100 µl of Buffer B, 2% SDS, 10 mg/ml BSA at 95 °C for 10 min. Samples were centrifuged and the supernatants were mixed with 65 µl of 20% Triton X-100, 1% bovine serum albumin in buffer B. Then, 6 µl of avidin-agarose beads was added and the samples were rocked overnight at 4 °C. The beads were then washed six times with 1.5 ml of buffer B, 1% BSA, 0.1% Triton X-100.

^3H-Labeled O-linked oligosaccharides were released by incubating the avidin-agarose beads with 200 µl of 1 M NaBH(4), 50 mM NaOH for 16 h at 45 °C. After adding 16.6 µl of 4 M acetic acid, oligosaccharides were separated from peptides by passing the mixture over a 0.9-ml column of Bio-Rad AG 50-X8. The samples were lyophilized and then evaporated repeatedly from methanol(40) .

Anionic and neutral oligosaccharides were separated by ion exchange chromatography(32) . Samples were dissolved in 2 mM Tris base and loaded onto 0.625-ml columns of QAE-Sephadex equilibrated in the same buffer. Neutral oligosaccharides were eluted with 8 ml, and anionic oligosaccharides were then eluted with 4.5 ml of 2 mM Tris base, 250 mM NaCl. Radioactivity in the eluates was determined by liquid scintillation counting.

Sialylation values were calculated according to the following formula: percent sialylation = 100 times (B - B(0)/B - B(0)) where B is the fraction of the radioactivity in the anionic fraction. B(0) and B are similar values from neuraminidase-treated and control samples before reculture, respectively. The sialylation value is 0% in neuraminidase-treated cells before reculture and 100% in control cells before reculture.

Measurement of Surface and Intracellular LDLR Pools

Cells were biotinylated as described above, except that the reducible reagent NHS-SS-biotin was used instead of sulfo-NHS-biotin. Biotinylated cells were cultured in growth medium at 37 °C for the indicated times. Cell surface biotinyl groups were then cleaved by incubating with 50 mM GSH at 0 °C followed by treatment with 5 mM iodoacetamide(11) . Cells were then lysed, and LDLR was immunoprecipitated. Samples were then subjected to SDS gel electrophoresis and transferred to nitrocellulose filters. Finally, the filters were probed with horseradish peroxidase-streptavidin conjugate (Amersham), which was visualized on x-ray film using a chemiluminescent peroxidase substrate (ECL, Amersham). Data were quantified by scanning densitometry. The protected fraction was calculated as the ratio of signals from samples incubated with and without GSH. The intracellular fraction was calculated by subtracting the protected fraction of cells kept at 0 °C from the protected fraction of cells incubated at 37 °C. In all cases, the protected fraction from cells kept at 0 °C was <0.4%.

RESULTS

Characterization of LDLR in Transfected K562 Cell Lines

Our studies of LDLR transport to the Golgi utilized two K562 lines that express wild type (R2) and internalization-defective receptor (A8) from transfected cDNAs. Before these lines could be used, it was necessary to show that they expressed functional LDLR from the transfected cDNAs. This was accomplished by examining the uptake and degradation of anti-LDLR monoclonal antibody C7, which is taken up and degraded with the same kinetics as low density lipoprotein(33) . Cells were incubated with I-C7 at 37 °C, and radioactivity that was cell-associated or in protein degradation products was determined (Fig. 1). Untransfected K562 cells took up and degraded small amounts of I-C7 (Fig. 1, A and B). In contrast, cells transfected with wild type LDLR cDNA took up and degraded ligand at 10 times the rate of untransfected cells. Cell-associated radioactivity in R2 cells increased over 6-7 h and then reached a plateau. These cells also degraded I-C7 after a lag of 2 h. Finally, as expected, total uptake by R2 cells, which was calculated as the sum of cell-associated and degraded radioactivity, was linear over 9 h (Fig. 1C). Cells transfected with mutant cDNA also took up and degraded I-C7 (Fig. 1, A and B). Total uptake was linear with time (Fig. 1C), but the rate was only 12 ± 1% (± S.E.) of the rate in cells transfected with wild type cDNA. This suggests that R2 and A8 cells expressed functional wild type and internalization-defective LDLR from the transfected cDNAs, respectively. Moreover, the low level of uptake in untransfected cells suggests that geq90% of the LDLR in R2 and A8 cells is expressed from the transfected cDNAs. Therefore, our studies of PM-to-Golgi traffic in these cells have examined wild type or mutant receptors expressed from the transfected cDNAs.

Figure 1: Uptake of an LDLR ligand by R2, A8, and K562 cells. Cells were incubated with I-labeled monoclonal antibody C7 (30 nM) at 37 °C. Radioactivity that was cell-associated (A) or in degradation products in the medium (B) was determined for R2 (), A8 (bullet), and K562 () cells. C, total uptake by LDLR expressed from transfected cDNAs was calculated by summing cell-associated and degraded radioactivity in R2 or A8 cells and subtracting the corresponding values from untransfected K562 cells. Representative data from one of two experiments is shown.


To assess wild type and mutant LDLR function in our transfected cells, we determined the relative internalization rates of the two receptors. This was accomplished by dividing the relative ligand uptake rates of the two cell lines by the relative number of surface receptors in those lines(30, 41) . The latter values were determined by biotinylating cell surfaces with sulfo-NHS-biotin at 0 °C, immunoprecipitating LDLR, and probing Western blots with I-streptavidin. Cells expressing mutant receptor (A8) had 1-2 times the number of surface receptors as cells expressing wild type receptor (R2) (not shown). Using these data, we calculate that the internalization rate of FH 683 receptor was only 6-12% of the rate for wild type LDLR, (^2)in good agreement with a previous study(30) .

To further characterize the expression of LDLR polypeptide in transfected cells, metabolic labeling was performed. Cells were pulse-labeled with [S]Met for 1 h and chased for 3 h. Labeled receptor was then immunoprecipitated and analyzed on SDS gels (Fig. 2). The two transfected lines expressed high levels of the receptor. In both R2 and A8 cells, species with the sizes of mature (160-kDa) and immature (120-kDa) LDLR were present(42) . The two forms of mutant LDLR migrated faster than wild type, consistent with the smaller size of the FH 683 polypeptide(30) . LDLR was barely detectable in untransfected K562 cells. Thus, nearly all the LDLR in the R2 and A8 cell lines is expressed from the transfected cDNAs, consistent with our studies of ligand uptake.

Figure 2: Expression of LDLR in transfected and control K562 cells. Transfected cells expressing wild type (R2) and mutant (A8) LDLR and untransfected K562 cells were pulse-labeled with [S]Met and chased for 3 h. LDLR was then immunoprecipitated from samples of 2.5 times 10^6 cells and analyzed on SDS gels. An autoradiograph of the dried gel is shown. The arrow indicates the position of mature LDLR.


Both R2 and A8 cells contained significant amounts of immature LDLR (Fig. 2). A8 cells contained more immature receptor, consistent with a previous study of FH 683 LDLR(30) . This immature receptor was stable, persisting after chases of up to 15 h (Fig. 3A). The presence of immature receptors in the cells does not affect the suitability of the transfected cell lines for studying LDLR traffic. Ligand uptake by mutant and wild type receptors in transfected cells is similar to previous reports (Fig. 1). In addition, only mature receptor was expressed on the cell surface. This was shown by biotinylating cells, immunoprecipitating LDLR, and then probing Western blots with I-streptavidin (Fig. 3B). Because our experiments on PM-to-Golgi traffic study these mature cell surface receptors, we have tracked mature molecules with properties similar to LDLR in other cell lines.

Figure 3: Turnover of wild type and internalization-defective receptors. A and C, metabolically labeled LDLR. Transfected cells were pulse-labeled with [S]Met at 37 °C and chased at 37 °C for the indicated times. LDLR was then immunoprecipitated and analyzed on SDS gels. A, an autoradiograph of samples of wild type (R2) and FH 683 (A8) LDLR. C, data derived by scanning densitometry for wild type () and FH 683 (box) LDLR. B and D, biotinylated wild type LDLR. R2 cells were biotinylated with sulfo-NHS-biotin and chased at 37 °C. LDLR was then immunoprecipitated, analyzed on an SDS gel, and transferred to a nitrocellulose filter. An autoradiograph of the filter probed with I-streptavidin (B), and data derived by scanning densitometry (D) are shown. The arrows in panels A and C denote the mature form of LDLR. Representative data from one of two experiments is shown.


As a final demonstration that mutant and wild type LDLR in the transfected cells had the expected properties, we determined receptor lifetimes. Cells were pulse-labeled with [S]Met and chased for the indicated times. LDLR was then immunoprecipitated and analyzed on SDS gels (Fig. 3A). Both mature and immature receptor species were seen, and these persisted throughout the chase. Quantification of the radioactivity in mature LDLR (Fig. 3C) showed that wild type and FH 683 receptor had half-lives of 5.5 ± 1.3 and 7.2 ± 0.5 h, respectively (S.E., p > 0.1). Similar half-lives (6-7 h) have been reported for LDLR expressed from a transfected cDNA in CHO cells(41) , suggesting that mutant and wild type receptors in our transfected K562 cells have the expected lifetimes.

Effect of Biotinylation on LDLR Properties

Our experiments on cell surface-to-Golgi transport have studied biotinylated LDLR molecules. The results of these experiments are valid only if modified and unmodified receptors have similar properties. We have shown that this is the case for two properties of LDLR. First, we showed that biotinylation does not affect receptor stability. Transfected cells expressing wild type receptor were biotinylated at 0 °C and cultured for various times at 37 °C. LDLR was then immunoprecipitated, and biotinylated receptor was visualized by probing Western blots with I-streptavidin (Fig. 3B). Biotinylated receptor was degraded with a t of 4.3 ± 0.6 h (Fig. 3D) which was not significantly different (p > 0.1) from the 5.5 ± 1.3 h t of S-labeled LDLR in these cells (Fig. 3C). In a second experiment, we compared the uptake of I-C7 by wild type receptor in surface-biotinylated and control R2 cells (Fig. 4). Biotinylation did not affect the uptake or degradation of ligand by LDLR. These results show that the receptor functions normally when cells are biotinylated by the method used in our experiments.

Figure 4: Internalization and degradation of I-C7 is similar in control and biotinylated cells expressing wild type LDLR. R2 cells were biotinylated and incubated with 30 nMI-C7 at 37 °C for the indicated times. Control cells were treated identically except that the biotinylation reagent was omitted. Cell-associated radioactivity (), radioactivity in degradation products (bullet), and total uptake () are shown.


Transport of Cell Surface LDLR to the Sialyltransferase Compartment

LDLR traffic from the cell surface to the Golgi was assessed by measuring entry into the trans-Golgi region that contains sialyltransferase. This was accomplished by removing sialic acid from cell surface receptors and then measuring LDLR resialylation. In previous studies, we detected the resialylation of asialo-TfR and asialo-Man-6-P/IGF-II receptor using isoelectric focusing to separate sialylated and desialylated receptors(4, 10) . However, control and asialo-LDLR could not be separated by this technique (not shown). We were also unable to demonstrate resialylation of LDLR using mobility shifts on SDS-gel electrophoresis as an assay (not shown).

Consequently, we developed a more sensitive technique for assessing LDLR resialylation. Cells were pulse-labeled with [^3H]GlcN to label LDLR glycans and chased to allow labeled receptors to reach the cell surface. Cell surface proteins were then biotinylated, and the cells were treated with neuraminidase to desialylate surface glycoproteins. The cells were then recultured to allow transport from the cell surface to the sialyltransferase compartment. Finally, after biotinylated LDLR was isolated, O-linked glycans were prepared and analyzed by anion exchange chromatography to separate neutral (unsialylated) glycans from anionic sialylated ones.

This assay was used to show that wild type asialo-LDLR is transported from the cell surface to the sialyltransferase compartment. As shown in Fig. 5, 86% of the radioactive receptor oligosaccharides from R2 cells not treated with neuraminidase were in the anionic fraction. In contrast, only 48% of the total radioactivity was in the anionic fraction of samples from neuraminidase-treated R2 cells, indicating that this treatment removed sialic acid residues from the O-linked glycans of cell surface LDLR. We showed that this reduction in anionic oligosaccharides represents the removal of nearly all the sialic acid residues from LDLR glycans. When glycans isolated from control LDLR were treated with mild acid to remove all the sialic acid residues(43) , the anionic fraction was reduced to 39%. The acid- and neuraminidase-resistant anionic glycans probably contain sulfate residues, which are found on the N-linked glycans of LDLR from human fibroblasts(32) . These results indicate that our methods allow us to isolate cell surface LDLR and that neuraminidase efficiently removed sialic acid residues from these receptors.

Figure 5: Wild type asialo-LDLR is resialylated in transfected cells. [^3H]GlcN-labeled R2 cells were surface-biotinylated and treated with neuraminidase at 0 °C. Control cells were treated identically except that they were incubated without neuraminidase. Cells were then cultured at the indicated temperature, and biotinylated LDLR was isolated. After ^3H-labeled O-linked glycans were prepared, neutral and anionic species were separated by anion exchange chromatography. The percentage of radioactivity in anionic glycans is shown. , control cells recultured at 37 °C; , neuraminidase-treated cells recultured at 37 °C; bullet, neuraminidase-treated cells recultured at 18 °C.


When neuraminidase-treated cells expressing wild type receptor were recultured at 37 °C, the percentage of anionic glycans increased (Fig. 5), suggesting that sialic acid residues were being added to asialo-LDLR. The anionic fraction reached a maximum value of 71% after 5 h of reculture and did not change during further incubation. In order to show that the increase in the anionic glycans during the reculture of neuraminidase-treated cells was due to receptor resialylation, we treated lysates from recultured cells with neuraminidase. This treatment decreased the fraction of anionic glycans from 71% to 30% (not shown). Therefore, the reappearance of anionic glycans is due to the resialylation of LDLR during the reculture period. In control cells, the anionic fraction did not change during a similar reculture period.

To show that the resialylation of asialo-LDLR occurred within the cell, we examined the effect of reculture at 18 °C on this process. Endocytosis from the PM occurs at this temperature, but transport to other cellular compartments, including the Golgi, is blocked(4, 10, 44, 45) . When neuraminidase-treated cells expressing wild-type receptor were recultured at 18 °C, there was no resialylation (Fig. 5), in agreement with our previous results for TfR and Man 6-P/IGF-II receptor (4, 10) . This result demonstrates that resialylation of surface LDLR requires membrane traffic from the PM into postendosomal compartments. Therefore, the most likely site of LDLR resialylation is the trans-Golgi region that contains sialyltransferase.

Internalization-defective Asialo-LDLR Is Transported to the Golgi at a Reduced Rate

To test the effect of the FH 683 mutation on surface-to-Golgi transport of LDLR, the time course of resialylation was compared in R2 and A8 cells. Fig. 6shows the data expressed as sialylation values, which range from 0% in neuraminidase-treated cells before reculture to 100% in control cells before reculture. The sialylation value for wild type LDLR reached a plateau of 55%, indicating that the recovery of sialic acid was not complete. Similar observations have been reported for the resialylation of LDLR and other glycoproteins(4, 9, 10, 11, 24) . In the case of TfR, this is due to the incomplete resialylation of the entire receptor pool (4) . Assuming that the 55% value represents the maximum sialylation level, wild type receptor is transported from the cell surface to the Golgi with a t of 2.5 h. Similar half-times of recycling into the sialyltransferase compartment have been reported for TfR and Man-6-P/IGF-II receptor in K562 cells (4, 10) and for LDLR in PC12 cells(11) .

Figure 6: The FH 683 mutation slows the recycling of LDLR to the Golgi. Resialylation of wild type and FH 683 LDLR in transfected cells were measured as described in Fig. 5. Data are expressed as sialylation values, calculated as described under ``Experimental Procedures.'' Values from five separate experiments are shown.


FH 683 receptor was resialylated much more slowly than wild type (Fig. 6). The t was 7.5 h for mutant LDLR, indicating a resialylation rate one-third that of wild type. However, after 10 h, the sialylation value reached a plateau that was similar to wild type. This suggests that the truncation of the cytoplasmic tail in FH 683 LDLR affects the rate of PM-to-Golgi traffic, but not the fraction of receptors that follow this pathway.

Effect of the FH 683 Mutation on Steady-state Receptor Distribution

The FH 683 mutation had a much greater effect on LDLR endocytosis than on surface-to-Golgi transport. Whereas endocytosis by mutant LDLR occurred at 10% of the wild type rate, mutant LDLR was transported to the Golgi at 33% of the wild type rate. This is surprising because endocytosis from the cell surface must precede transport to the Golgi. One possible explanation for these findings is that the FH 683 mutation slows both the rates of endocytosis and recycling. In this case, LDLR could accumulate to nearly normal levels in an endosomal compartment from which it is transported to the Golgi.

To assess this possibility, we estimated the size of the intracellular pools of wild type and mutant LDLR in R2 and A8 cells, respectively. This was accomplished using the cleavable biotinylation reagent NHS-SS-biotin. Cells were surface-biotinylated at 0 °C and then incubated at 37 °C for 2-3 h to allow labeled receptors to equilibrate between surface and intracellular pools. Cells were then incubated on ice with GSH, which cleaves biotinyl groups only from surface receptors(46) . LDLR was immunoprecipitated, and the amount of biotinylated receptor was visualized by probing Western transfers with streptavidin-horseradish peroxidase.

In cells kept at 0 °C, a large amount of biotinylated receptor was seen (Fig. 7). If these cells were treated with GSH, the signal was reduced to 0.4% of the value in untreated cells. Because GSH does not enter the cells, this result demonstrates that all the biotinylated receptor was on the PM. In both R2 and A8 cells incubated for 3 h at 37 °C before GSH treatment, pools of protected LDLR were seen, demonstrating that biotinylated receptor was taken up from the PM during incubation at 37 °C.

Figure 7: The FH 683 mutation reduces the pool of intracellular LDLR. Transfected cells were biotinylated with NHS-SS-biotin at 0 °C and incubated for 3 h at 0 or 37 °C. Cell surface biotinyl groups were then cleaved with GSH at 0 °C. Then, LDLR was immunoprecipitated, analyzed on SDS gels, and transferred to nitrocellulose filters. After incubation with streptavidin-horseradish peroxidase and ECL, biotinylated LDLR was detected by exposure to x-ray film. Exposures of 80 min (wild type) and 5 min (FH 683) are shown. Shorter exposures were used to obtain the quantitative data described in the text.


In R2 cells, 10.4 ± 1.4% (standard error, n = 4) of the wild type LDLR was intracellular at steady state. In contrast, only 1.2 ± 0.3% of the mutant receptor was intracellular in A8 cells, which is 11% of the wild type level. Similar levels of intracellular receptor were seen in R2 and A8 cells incubated at 37 °C for 1-3 h, indicating that our measurements represent the steady state distribution of biotinylated LDLR (not shown).

The steady state distribution of receptor is largely determined by the rates of endocytosis and recycling from endosomes to the PM. Because the FH 683 mutation caused similar reductions in the endocytosis rate and the intracellular level (10% of wild type), these results suggest that the cytoplasmic tail of LDLR is required for rapid endocytosis but not for recycling from endosomes to the PM, consistent with the observations of Mayor et al.(47) . Moreover, our findings show that the relatively high levels of PM-to-Golgi transport of mutant LDLR occur despite a large reduction in the amount of intracellular receptor.

DISCUSSION

We have shown that wild type LDLR is resialylated in K562 cells, demonstrating that it recycles to the Golgi complex. The half-time of PM-to-Golgi transport was 2.5 h, which is similar to the rate reported for LDLR in PC12 pheochromocytoma cells(11) . Moreover, LDLR, TfR(4) , and Man-6-P/IGF-II receptor (10) are all transported from PM to Golgi at similar rates in K562 cells. This is consistent with the idea that this traffic occurs via a single transport pathway.

Modification of proteins during recycling has been demonstrated for many N-glycosylated proteins. However, the only previous example of repair of O-glycans is a sialomucin of rat mammary adenocarcinoma cells(19) . Our finding that the O-linked glycans of asialo-LDLR can be resialylated suggests that both N- and O-linked glycans can be repaired during recycling through the Golgi complex. In K562 cells, N-glycosylated (TfR and Man-6-P/IGF-II receptor) and O-glycosylated (LDLR) proteins were resialylated at similar rates during PM-to-Golgi transport(4, 10) . Because recycling glycoproteins enter different Golgi compartments at distinct rates(4, 24, 31) , these results suggest that sialic acids residues are added to N- and O-glycans in similar Golgi regions during recycling from the PM.

What Are the Pathways of PM-to-Golgi Traffic?

Our studies of wild type and mutant LDLR provide insights into the transport pathway that leads from PM to Golgi. We found that FH 683 LDLR was transported to the Golgi with a t of 7.5 h, 33% of the wild type rate. Because the mutant receptor is missing the cytoplasmic tail, which contains the signal for internalization in coated vesicles(30) , it is taken up from the PM much more slowly than wild type receptor. Therefore, our results suggest that clathrin-coated vesicles participate in the principal transport pathway of wild type LDLR from PM to Golgi. This is the first demonstration that coated vesicles participate in this traffic.

This role for clathrin-coated vesicles is consistent with the finding that most of the proteins known to undergo PM-to-Golgi transport are rapidly internalized from the PM via this route(2, 3, 4, 5, 6, 7, 8, 9, 10, 11) . However, this conclusion appears to contradict our previous findings using treatments that inhibit internalization via clathrin-coated vesicles. Depletion of K or depolymerization of microtubules decreased TfR endocytosis by 75-80% but did not inhibit its PM-to-Golgi transport (28, 29) . This apparent discrepancy may be explained by the fact that endocytosis from the PM is much more rapid than transport to the Golgi. Endocytosis at 20-25% of the control rate in our earlier studies may be enough to permit PM-to-Golgi traffic to continue uninhibited, whereas endocytosis of FH 683 LDLR at 10% of the wild type rate slows traffic to the Golgi.

Our results also suggest that the principal pathway of PM-to-Golgi transport involves an endosomal intermediate because coated vesicles that bud from the PM are destined for endosomes. A role for endosomes is also supported by studies from Pfeffer's laboratory showing transport from this compartment to the Golgi in vitro(8, 27) . Traffic from endosomes to the Golgi does not require clathrin-coated vesicles because the in vitro transport assay was not inhibited by antibodies to clathrin heavy chain(27) . It should be noted that receptors in endosomes have at least three possible fates: recycling to the PM, transport to the Golgi, and degradation in lysosomes. The relative rates of these processes suggest that most receptors entering endosomes from the PM recycle to the cell surface, a smaller fraction (5-10%) are transported to the Golgi, and 1% are degraded(11) .

Although the pathway of endosome-to-Golgi transport is not known, traffic from late endosomes is consistent with several lines of evidence. Pfeffer's laboratory has shown that the late endosomal protein rab9 plays a role in regulating PM-to-Golgi transport(48, 49) . Second, Man-6-P/IGF-II receptor is found at high levels in late endosomes and is probably transported from the Golgi into this compartment during the delivery of newly made lysosomal enzymes into the endocytic pathway(50) . Recycling from late endosomes into the Golgi could allow these receptors to return to the Golgi to participate in additional rounds of transport. Finally, brefeldin A causes the fusion of late endosomes and the Golgi(51, 52) , suggesting that exchange of membranes between these compartments may occur under normal circumstances. This traffic from late endosomes to the Golgi has previously been proposed by Green and Kelly(11) .

Based on these considerations, it is likely that the principal pathway of PM-to-Golgi traffic begins with the transport of membrane glycoproteins from the PM into early endosomes by clathrin-coated vesicles. Most of the glycoproteins in this compartment recycle to the PM, while a small fraction are transferred into late endosomes and then to the Golgi complex. At present, it is not clear how sorting of membrane glycoproteins from early to late endosomes or from late endosomes to the Golgi is regulated.

Is There a Second Pathway of PM-to-Golgi Transport?

FH 683 receptor was transported to the Golgi at 33% of the wild-type rate even though uptake from the PM was inhibited by 90%. There are several possible pathways that could be utilized in this residual PM-to-Golgi transport of FH 683 LDLR. First, it is possible that mutant LDLR destined for the Golgi is internalized at a slow rate by clathrin-coated vesicles. Alternatively, it is possible that mutant receptors are taken up from the PM by non-clathrin-coated vesicles which are responsible for a substantial amount of endocytosis by K562 cells(28) . It is also not clear how FH 683 LDLR reaches the Golgi following endocytosis from the PM. However, transport probably occurs via endosomes because clathrin-coated and non-clathrin-containing endocytic vesicles are thought to fuse with the same early endosomes (53) .

In either case, the FH 683 mutation must have a second effect on PM-to-Golgi transport that is independent of the inhibition of LDLR endocytosis. This conclusion is based on our observation that the mutation had a smaller effect on PM-to-Golgi transport than on the endocytosis rate and the level of intracellular receptor. One possible explanation of this finding is that the mutation increases the proportion of endosomal LDLR that reaches the Golgi and decreases return to the PM. Alternatively, it is possible that the FH 683 mutation alters the pathway of PM-to-Golgi transport. Transport of FH 683 LDLR to the Golgi through different organelles than wild type could explain the more efficient sorting of the mutant receptor into the Golgi pathway.

In summary, our studies suggest that the principal pathway of PM-to-Golgi traffic involves endocytosis in clathrin-coated vesicles and transport to the Golgi via an endosomal intermediate. At least two-thirds of the wild type receptor traffic occurs via this pathway. In addition, there may be another transport pathway that carries the remaining one-third of the traffic, and this pathway may be clathrin-independent.

FOOTNOTES

*
This work was supported by a Pew Scholarship in the Biomedical Sciences (to M. D. S.) and by Grant GM38183 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: PM, plasma membrane; BSA, bovine serum albumin; FBS, fetal bovine serum; LDLR, low density lipoprotein receptor; Man-6-P/IGF-II receptor, mannose 6-phosphate/insulin-like growth factor-II receptor; NHS-SS-biotin, sulfosuccinimidyl 2-(biotinamido)ethyl-1,3`-dithiopropionate; PBS, Dulbecco's phosphate-buffered saline; sulfo-NHS-biotin, sulfo-N-hydroxysuccinimidobiotin; TfR, transferrin receptor.

(^2)
A8 cells took up ligand at 12% the rate of R2 cells. Because A8 cells have 1-2 times the number of surface receptors, the endocytosis rate of FH 683 LDLR in A8 cells is between 6% (12%/2) and 12% of R2 cells.

ACKNOWLEDGEMENTS

We thank C. G. Davis for the LDLR cDNA constructs, J. D. Stepp for assistance with the experiments, E. Sugarman and K. Carraway for helpful discussions, and C. Carlin, C. Harding, C. Hilbert, K. Huang, and S. Lemmon for critical reading of the manuscript.

REFERENCES

  1. Snider, M. D. (1991) in Intracellular Trafficking of Proteins (Steer, C. J., and Hanover, J. A., eds) pp. 361-386, Cambridge University Press, Cambridge
  2. Regoeczi, E., Chindemi, P. A., Debanne, M. T., and Charlwood, P. A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2226-2230 [Abstract/Free Full Text]
  3. Geuze, H., Slot, J., Strous, G., Luzio, J., and Schwartz, A. (1984) EMBO J. 3, 2677-2685 [Medline] [Order article via Infotrieve]
  4. Snider, M. D., and Rogers, O. C. (1985) J. Cell Biol. 100, 826-834 [Abstract/Free Full Text]
  5. Woods, J. W., Doriaux, M., and Farquhar, M. G. (1986) J. Cell Biol. 102, 277-286
  6. Cresswell, P. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8188-8192 [Abstract/Free Full Text]
  7. Fishman, J. B., and Fine, R. E. (1987) Cell 48, 157-164 [CrossRef][Medline] [Order article via Infotrieve]
  8. Goda, Y., and Pfeffer, S. R. (1988) Cell 55, 309-320 [CrossRef][Medline] [Order article via Infotrieve]
  9. Duncan, J. R., and Kornfeld, S. (1988) J. Cell Biol. 106, 617-628 [Abstract/Free Full Text]
  10. Jin, M., Sahagian, G. G., and Snider, M. D. (1989) J. Biol. Chem. 264, 7675-7680 [Abstract/Free Full Text]
  11. Green, S. A., and Kelly, R. B. (1992) J. Cell Biol. 117, 47-56 [Abstract/Free Full Text]
  12. Hunter, A., and Phillips, J. H. (1989) Exp. Cell Res. 182, 445-460 [CrossRef][Medline] [Order article via Infotrieve]
  13. Patzak, A., and Winkler, H. (1986) J. Cell Biol. 102, 510-515 [Abstract/Free Full Text]
  14. Bonifacino, J., Yuan, L., and Sandoval, I. (1989) J. Cell Sci. 92, 701-712 [Abstract/Free Full Text]
  15. Hurtley, S. M. (1993) J. Cell Sci. 106, 649-655 [Abstract]
  16. Humphrey, J., Peters, P., Yuan, L., and Bonifacino, J. (1993) J. Cell Biol. 120, 1123-1135 [Abstract/Free Full Text]
  17. Luzio, J. P., Brake, B., Banting, G., Howell, K. E., Braghetta, P., and Stanley, K. K. (1990) Biochem. J. 270, 97-102 [Medline] [Order article via Infotrieve]
  18. Reaves, B., Horn, M., and Banting, G. (1993) Mol. Biol. Cell 4, 93-105 [Abstract]
  19. Hull, S., Sugarman, E., Spielman, J., and Carraway, K. (1991) J. Biol. Chem. 266, 13580-13586 [Abstract/Free Full Text]
  20. Kreisel, W., Hildebrandt, H., Mossner, W., Tauber, R., and Reutter, W. (1993) Biol. Chem. Hoppe Seyler 374, 255-263 [Medline] [Order article via Infotrieve]
  21. Miquelis, R., Courageot, J., Jacq, A., Blanck, O., Perrin, C., and Bastiani, P. (1993) J. Cell Biol. 123, 1695-1706 [Abstract/Free Full Text]
  22. Molloy, S., Thomas, L., Slyke, J. V., Stenberg, P., and Thomas, G. (1994) EMBO J. 13, 18-33 [Medline] [Order article via Infotrieve]
  23. Green, S. A., and Kelly, R. B. (1990) J. Biol. Chem. 265, 21269-21278 [Abstract/Free Full Text]
  24. Huang, K. M., and Snider, M. D. (1993) J. Biol. Chem. 268, 9302-9310 [Abstract/Free Full Text]
  25. Reichner, J. S., Whiteheart, S. W., and Hart, G. W. (1988) J. Biol. Chem. 263, 16316-16326 [Abstract/Free Full Text]
  26. Neefjes, J. J., Verkerk, J. M. H., Broxterman, H. J. G., van der Marel, G. A., van Boom, J. H., and Ploegh, H. L. (1988) J. Cell Biol. 107, 79-87 [Abstract/Free Full Text]
  27. Draper, R., Goda, Y., Brodsky, F., and Pfeffer, S. (1990) Science 248, 1539-1541 [Abstract/Free Full Text]
  28. Robertson, B. J., Park, R. D., and Snider, M. D. (1992) Arch. Biochem. Biophys. 292, 190-198 [CrossRef][Medline] [Order article via Infotrieve]
  29. Jin, M., and Snider, M. D. (1993) J. Biol. Chem. 268, 18390-18397 [Abstract/Free Full Text]
  30. Lehrman, M. A., Goldstein, J. L., Brown, M. S., Russell, D. W., and Schneider, W. J. (1985) Cell 41, 735-743 [CrossRef][Medline] [Order article via Infotrieve]
  31. Snider, M. D., and Rogers, O. C. (1986) J. Cell Biol. 103, 265-275 [Abstract/Free Full Text]
  32. Cummings, R. D., Kornfeld, S., Schneider, W. J., Hobgood, K. K., Tolleshaug, H., Brown, M. S., and Goldstein, J. L. (1983) J. Biol. Chem. 258, 15261-15273 [Abstract/Free Full Text]
  33. Beisiegel, U., Schneider, W. J., Goldstein, J. L., Anderson, R. G. W., and Brown, M. S. (1981) J. Biol. Chem. 256, 11923-11931 [Abstract/Free Full Text]
  34. Davis, C. G., Elhammer, Å ., Russell, D. W., Schneider, W. J., Kornfeld, S., Brown, M. S., and Goldstein, J. L. (1986) J. Biol. Chem. 261, 2828-2838 [Abstract/Free Full Text]
  35. Klausner, R. D., Van Renswoude, J., Ashwell, G., Kempf, C., Schechter, A. N., Dean, A., and Bridges, K. R. (1983) J. Biol. Chem. 258, 4715-4724 [Abstract/Free Full Text]
  36. Goldstein, J. L., and Brown, M. S. (1974) J. Biol. Chem. 249, 5153-5162 [Abstract/Free Full Text]
  37. Tolleshaug, H., Goldstein, J. L., Schneider, W. J., and Brown, M. S. (1982) Cell 30, 715-724 [CrossRef][Medline] [Order article via Infotrieve]
  38. Schneider, W. J., Goldstein, J. L., and Brown, M. S. (1980) J. Biol. Chem. 255, 11442-11447 [Abstract/Free Full Text]
  39. Lemansky, P., Fatemi, S. H., Gorican, B., Meyale, S., Rossero, R., and Tartakoff, A. (1990) J. Cell Biol. 110, 1525-1531 [Abstract/Free Full Text]
  40. Carlson, D. M. (1968) J. Biol. Chem. 243, 616-626 [Abstract/Free Full Text]
  41. Davis, C., Lehrman, M., Russell, D., Anderson, R., Brown, M., and Goldstein, J. (1986) Cell 45, 15-24 [CrossRef][Medline] [Order article via Infotrieve]
  42. Goldstein, J. L., Brown, M. S., Anderson, R. G. W., Russell, D. W., and Schneider, W. J. (1985) Annu. Rev. Cell Biol. 1, 1-39 [CrossRef]
  43. Spiro, R. (1966) Methods Enzymol. 8, 3-26
  44. Dunn, W. A., Hubbard, A. L., and Aronson, N. N., Jr. (1980) J. Biol. Chem. 255, 5971-5978 [Abstract/Free Full Text]
  45. Marsh, M., Bolzau, E., and Helenius, A. (1983) Cell 32, 931-940 [CrossRef][Medline] [Order article via Infotrieve]
  46. Bretscher, M. S., and Lutter, R. (1988) EMBO J. 7, 4087-4092 [Medline] [Order article via Infotrieve]
  47. Mayor, S., Presley, J. F., and Maxfield, F. R. (1993) J. Cell Biol. 121, 1257-1269 [Abstract/Free Full Text]
  48. Lombardi, D., Soldati, T., Riederer, M. A., Goda, Y., Zerial, M., and Pfeffer, S. R. (1993) EMBO J. 12, 677-682 [Medline] [Order article via Infotrieve]
  49. Riederer, M., Soldati, T., Shapiro, A., Lin, J., and Pfeffer, S. (1994) J. Cell Biol. 125, 573-582 [Abstract/Free Full Text]
  50. Kornfeld, S., and Mellman, I. (1989) Annu. Rev. Cell Biol. 5, 483-525 [CrossRef]
  51. Wood, S. A., Park, J. E., and Brown, W. J. (1991) Cell 67, 591-600 [CrossRef][Medline] [Order article via Infotrieve]
  52. Hunziker, W., Whitney, J. A., and Mellman, I. (1991) Cell 67, 617-627 [CrossRef][Medline] [Order article via Infotrieve]
  53. Hansen, S. H., Sandvig, K., and van Deurs, B. (1993) J. Cell Biol. 123, 89-97 [Abstract/Free Full Text]
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