(Received for publication, May 31, 1994; and in revised form, October 17, 1994)
From the
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.
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 ()and
Golgi
proteins(16, 17, 18, 19, 20, 21, 22) .
In addition, two studies have shown that
10 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.
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
-I-10, 400 µg/ml
G418. R2 cells were grown in the same medium containing 20 µM Lovastatin and 200 µM mevalonate(34) .
To isolate biotinylated
LDLR, samples of 5 10
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.
H-Labeled O-linked oligosaccharides were
released by incubating the avidin-agarose beads with 200 µl of 1 M NaBH
, 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 (B
- B
/B
- B
) where B
is the
fraction of the radioactivity in the anionic fraction. B
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.
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 (
), 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, (
)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
10
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 (
) 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.
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 nM
I-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 (
), and total
uptake (
) are shown.
Consequently, we developed a more sensitive technique
for assessing LDLR resialylation. Cells were pulse-labeled with
[H]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. [H]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
H-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;
, 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.
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.
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.
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.
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.
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.