THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No. 13, Issue of May 5, pp. 7675-7680,1989 Printed in U. S. A.

Transport of Surface Mannose 6-Phosphate Receptor to the Golgi Complex in Cultured Human Cells*

(Received for publication, October 24, 1988)

Mingjie Jin$, G. Gary Sahagiang, and Martin D. Snider$ From the $Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 and the §Department of Physiology, Tufts Medical School, Boston, Massachusetts 021 1 1

The cation-independent mannose 6-phosphate recep- tor (MPR") functions in the packaging of both newly made and extracellular lysosomal enzymes into lyso- somes. The subcellular location of MPR" reflects these two functions; receptor is found in the Golgi complex, in endosomes, and on the cell surface. To learn about the intracellular pathway followed by surface receptor and to study the relationship between the receptor pools, we examined the entry of the surface MPR" into Golgi compartments that contain sialyltransferase. Sialic acid was removed from surface-labeled K562 cultured human erythroleukemia cells by neuramini- dase treatment. When the cells were returned to cul- ture at 37 "C, surface MPR" was resialylated by the cells with a half-time of 1-2 h. Resialylation was in- hibited by reduced temperature, a treatment that al- lows surface molecules to reach endosomes but blocks further transport. These results indicate that surface MPR" is transported to the sialyltransferase compart- ment in the Golgi complex. After culture at 37 "C, a small fraction (10-20%) of the resialylated receptor was found on the cell surface. Because a similar frac- tion of the total receptor pool is found on the cell surface, it is likely that cell surface MPR" mixes with the cellular pool after resialylation. These data also support the idea that extracellular and newly made lysosomal enzymes are transported to lysosomes through a common compartment.

In animal cells, the import of lysosomal enzymes into lysosomes is mediated by mannose 6-phosphate receptors (MPR).' These receptors recognize Man-6-P residues that are added to the asparagine-linked oligosaccharides of lysosomal enzymes during biosynthesis in the endoplasmic reticulum and Golgi complex. Enzymes bind to the receptors via these Man-6-P residues and are transported to lysosomes as a consequence (reviewed in Sahagian, 1987; von Figura and Hasilik, 1986; Kornfeld, 1987; Pfeffer, 1988).

Two distinct MPRs have been identified. One receptor is a glycoprotein of M , > 270,000 (Oshima et al., 1988; Lobel et

* This work was supported by National Institutes of Health Grant GM38183 and a Pew Scholarship in the Biomedical Sciences (to M. D. S.) and National Institutes of Health Grant DK36632 and the Searle Scholars Program/The Chicago Community Trust (to G. G. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: MPR, mannose 6-phosphate receptor; MPR'', cation-independent mannose 6-phosphate receptor; BSA, bovine serum albumin; CHO, Chinese hamster ovary; IEF, isoelectric focusing; IGF, insulin-like growth factor; Man-6-P, mannose 6-phos- phate; SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline.

al., 1988) that binds lysosomal enzymes in a cation-independ- ent manner ((MPR") Sahagian etal., 1981; Steiner and Rome, 1982). The other receptor is a glycoprotein of M , 46,000 that requires divalent cations for the high affinity binding of ligands (Hoflack and Kornfeld, 1985a, 1985b). Both receptors are transmembrane proteins, and their primary structures have been deduced from cDNA cloning (Dahms et al., 1987; Lobel et al., 1987, 1988; Pohlmann et al., 1987; Oshima et al., 1988). Our work has concentrated on MPR", which is the better understood of the two receptors.

Unlike many receptors whose sole function is the transport of extracellular ligands to lysosomes, the role of MPR" is more complex. Both newly made lysosomal enzymes within the cell and extracellular enzymes are transported to lyso- somes by this receptor. As might be expected from these complex functions, MPR" is found in a number of subcellular locations. Receptor is found in the Golgi complex, in endo- somes, in coated vesicles, as well as on the plasma membrane (Brown and Farquhar, 1984a, 1984b; Geuze et al., 1984a, 1984b; Griffiths et al., 1988; Willingham et al., 1983). At steady state, most of the receptor is in the intracellular pool with only -10% on the cell surface (Fischer et al., 1980; Sahagian and Neufeld, 1983; Shepherd et al., 1984). This protein has recently been found to have a second function. Surface MPR" is also the receptor for IGF-I1 (Morgan et al., 1987; Mac- Donald et al., 1988; Tong et al., 1988; Kiess et al., 1988).

In keeping with its complex functions, two routes of MPR" traffic have been proposed. Newly synthesized lysosomal en- zymes bind to receptor in the Golgi complex, and the receptor- ligand complex is then transported via coated vesicles (Brown and Farquhar, 1984a; Schulze-Lohoff et al., 1985; Lemansky et al., 1987) to a prelysosomal compartment (Griffiths et al., 1988) where low pH causes the complex to dissociate. Extra- cellular enzymes bind to receptor on the cell surface, and the complexes appear to be transported into the same prelysoso- mal compartment. This conclusion is supported by morpho- logical studies (Griffiths et al., 1988) and by the demonstration that surface and internal receptor pools exchange with each other (von Figura et al., 1984; Gartung et al., 1985; Sahagian, 1984; Braulke et al., 1987; Pfeffer, 1987). From this common compartment, receptors appear to recycle to the Golgi com- plex and the cell surface for additional rounds of transport (Brown et al., 1986).

To learn about the intracellular pathway of cell surface MPR" and to study the relationship between the two receptor pools, we examined the entry of cell surface MPR" into the Golgi complex. This study relies on enzymes of glycoprotein oligosaccharide synthesis that are found in this organelle. First, glycoproteins with immature oligosaccharides that are substrates for specific Golgi enzymes are generated on the cell surface. Transport of the substrate glycoproteins to enzyme- containing compartments can then be assessed by determin-

7675

7676 Transport of Surface Man-6-P Receptor to the Golgi Complex

ing whether the enzymes have modified the glycoproteins (Snider and Rogers, 1985, 1986).

In this paper, the movement of surface MPRC1 to the sialyltransferase-containing compartment was studied in K562 human erythroleukemia cells. Cells were treated with neuraminidase to remove sialic acid residues from surface receptors so that they are substrates of sialyltransferase, which has been localized to trans-Golgi cisternae and the trans-Golgi network in a number of cell types (Bennett and O'Shaughnessy, 1981; Roth et al., 1985, 1986). During recul- ture of neuraminidase-treated cells at 37 "C, nearly all the labeled asialo-MPR" was resialylated, indicating that these molecules had been transported to the sialyltransferase-con- taining compartment in the Golgi complex. This approach has been used previously to demonstrate the transport of MPR into sialyltransferase compartments in cultured ham- ster cells (Duncan and Kornfeld, 1988) and in cell-free prep- arations (Goda and Pfeffer, 1988). In our studies, some of the resialylated MPR" were found on the cell surface, showing that the receptor was cycling between the cell surface and the Golgi complex. Moreover, Man-6-P-containing ligands had no effect on MPR" resialylation, suggesting that receptor occupancy does not regulate this pathway of MPR" traffic. A preliminary report of this work has been presented (Jin et al., 1987).

MATERIALS AND METHODS

Cell Culture"K562 human erythroleukemia cells were grown in suspension in CY- minimal essential medium supplemented with 10% fetal calf serum. CHO cells were grown in the same medium supple- mented with 5% fetal calf serum.

Iodination of Cells-Surface iodination of cells was performed as previously described (Snider and Rogers, 1985) except that the glucose oxidase concentration was reduced to 50 milliunits/ml.

Neuraminidase Treatment and Reculture of Cells-Iodinated cells were used immediately after labeling. Cells were resuspended in Dulbecco's phosphate-buffered saline, 1 mg/ml BSA, 1 mg/ml Glc (PBS/BSA) at 5 X lo6 cells/ml and treated with 30 milliunits/ml Vibrio cholerae neuraminidase at 0 "C and then washed, recultured in growth medium at 37 "C, and lysed, all as previously described (Snider and Rogers, 1985). Where indicated, the following compounds were added to the culture medium: monensin (20 pM), added from a 20 mM stock in ethanol; chloroquine (100 p ~ ) ; cycloheximide (10 pg/ ml); tunicamycin (20 pg/ml), added from a 4 mg/ml stock in dimethyl sulfoxide; bovine testicular P-galactosidase ((32 pg/ml) Distler and Jourdian, 1978); and Man-6-P (5 mM).

Cell Lysis and Immunoprecipitation of MPRCr-Samples of 2.5 X lo6 cells were lysed as previously described (Snider and Rogers, 1985). In some experiments, cell lysates were treated with neuraminidase, also as reported previously (Snider and Rogers, 1985). Lysates were then incubated with 2.5 pl of fixed Staphylococcus aureus cells for 30 min and centrifuged at 13,000 X g. The pellet was discarded, and 1 pl of rabbit anti-bovine MPR" antiserum was added to the supernatant. The mixture was rocked at 4 "C overnight, and then 1.5 p1 of protein A-Sepharose beads were added. After rocking for 1 h, the beads were washed three times with 0.5% Nonidet P-40,0.5% sodium deoxycho- late, 0.05% SDS in 10 mM sodium phosphate, pH 7.2, 150 mM NaC1, 1 mM EDTA.

Isoelectric Focusing-Immunoprecipitates from 2.5 X lo6 cells were dissociated by incubating in 50 pl of 1% SDS, 1% 2-mercaptoethanol, 50 mM Tris-HC1, pH 6.8, at 37 "C for 1 h and centrifuged for 10 min at 13,000 X g. Aliquots of the supernatant (25 pl) were mixed with 10 pg of BSA as carrier. Proteins were then precipitated by adding an equal volume of 20% trichloroacetic acid (w/v). After centrifugation, the pellet was washed once with cold 80% acetone and dissolved in 20 p1 of complete sample buffer which was made immediately before use by adding 1.1 g of freshly recrystallized urea to 1 ml of 1% 2- mercaptoethanol, 3.7% Nonidet P-40, 3.7% Serva Isodalt 3-10 am- pholytes. Isoelectric focusing (IEF) and autoradiography were carried out as previously described (Snider and Rogers, 1985) except that gels contained Polybuffer 74 diluted 1:7.5 and Polybuffer 96 diluted 1:12.4 as ampholytes. The samples were loaded directly in wells on the gel, and focusing was performed at 400 V for 16-18 h.

Quantitation of Asialo-MPR" Resiulylution-Individual bands were located on dried gels by alignment with autoradiograms. Bands were then cut out and counted in a LKB y counter. The bands below the arrow in Fig. 1 were defined as basic MPR" species while the bands above the arrow were defined as acidic MPR" species. The percentage of total radioactivity in acidic species (A) was calculated to correct for the degradation of labeled MPR" during culture of labeled cells. The resialylation of asialo-MPR" was calculated from these values according to the equation: percent sialylation = ((Aaample - A,)/(A, - A,)) X 100, where A, and A, are the fraction of MPR" found in the acidic bands in neuraminidase-treated and control cells before recul- ture, respectively. This sialylation value is 0 and 100% in neuramin- idase-treated and control cells before reculture, respectively.

Binding of Lysosomal Enzymes to Control and Neuraminidase- treated Cells-Binding of lysosomal enzymes to cell surface MPR" was performed according to Robbins et al. (1983). 35S-Labeled secre- tions from ammonium chloride-treated CHO cells were prepared and used as ligands. CHO cells (1 X lo7 cells/150-cm2 tissue culture flask) were washed with Met-free minimal essential medium supplemented with 5% dialyzed fetal calf serum and nonessential amino acids, and then incubated in 10 ml of the same medium containing 1 mCi of [35S]Met (1100 Ci/mmol, Amersham Corp.), 1 pg/ml unlabeled Met, 10 mM NH4C1 for 14-16 h. The 35S-labeled CHO secretions were concentrated by ammonium sulfate precipitation, dissolved, and di- alyzed (Robbins and Myerowitz, 1981). One flask of cells yielded 1.2 x lo7 cpm radioactivity.

The binding experiment was performed at 4 "C. K562 cells were chilled, washed twice with cold PBS/BSA, and incubated in the same buffer containing 5 mM Man-6-P for 10 min to deplete receptors of endogenous ligand. Samples of lo6 cells were then washed three times and incubated in 240 p1 of PBS/BSA containing 2 X lo5 cpm of 35S- labeled CHO secretions for 1 h. Cells were then centrifuged for 5 min at 500 X g, resuspended in 0.5 ml of PBS/BSA, layered over 3 ml of 100 mg/ml BSA in PBS, and centrifuged for 5 min at 500 X g. The cell pellet was washed extensively with PBS/BSA and then eluted with PBS/BSA in the presence or absence of 5 mM Man-6-P. The eluted radioactivity was counted in a scintillation counter. Specific binding was calculated as the difference between the radioactivity eluted with and without Man-6-P. Typically, specific binding was 70% of the total. Specific binding to control cells was roughly 100 cpm; the value varied with the ligand preparation. As a test of the specificity of binding, control cells were incubated with 35S-labeled CHO secretions in the presence of 5 mM Man-6-P. In this case, specific binding was reduced by >go%.

RESULTS

Cell Surface Asialo-MPR" Was Resialylated-The trans- port of surface MPR" to the Golgi complex was assessed using sialyltransferase, which has been localized in trans- Golgi cisternae and the trans-Golgi network. Cells were sur- face-labeled with 1251 and then treated with neuraminidase to remove sialic acid from surface glycoproteins. After culture in growth medium at 37 "C for various times, cells were lysed. MPR" was then immunoprecipitated and analyzed by IEF. As shown in Fig. 1, sialic acid residues on surface MPR" were removed by treatment with neuraminidase, as indicated by the shift of receptor species to more basic positions. After 40 min of reculture, acidic species began to reappear, with a corresponding decrease in basic species. MPRC* became more acidic with increasing time and by 10-20 h of reculture resem- bled receptor from control cells that had not been treated with neuraminidase.

To prove that the reappearance of acidic MPR" species in recultured neuraminidase-treated cells was due to the addition of sialic acid, a second neuraminidase treatment was per- formed. Labeled control and neuraminidase-treated cells were recultured for 2 h. Then cell lysates were prepared, incubated with or without neuraminidase, and MPR" species were analyzed by IEF (Fig. 2). The acidic bands that appeared during reculture of neuraminidase-treated cells were modified by this second treatment to become basic ones. Thus, the reappearance of acidic MPR" species during reculture was

Transport of Surface Man-6-P Receptor to the Golgi Complex 7677

Control I Neuraminidase-Treated . . -~ - "".3"._"_ "".,.~~~.

Hours: 0 I 0 1 2 1 2 4 6 20 3 3

FIG. 1. Time course of asialo-MPRC' resialylation. Cells were iodinated and treated with neuraminidase at 0 "C and then recultured for the indicated times at 37 "C in growth medium. Control cells were treated identically except without the neuraminidase treatment. MPR" was isolated by immunoprecipitation and analyzed by IEF. An autoradiograph of the dried gel is shown, with the acidic end at the top and t,he basic end at the bottom. For quantitation of sialylation, species migrating above the horizontal arrow were scored as acidic, while species below the arrow were scored as basic.

Control Neuraminidase- Cells I Treated Cells

a b c d e FIG. 2. Retreatment of cell lysates with neuraminidase. Io-

dinated control (a , b ) and neuraminidase-treated (c-e) cells were recultured at 0 or 37 "C for 2 h in growth medium. Cell lysates were then prepared and incubated with or without neuraminidase a t 37 "C. a, b, control cell lysates incubated without ( a ) or with ( b ) neuramin- idase after cell reculture a t 37 "C. c, neuraminidase-treated cell lysate incubated without neuraminidase after reculture a t 0 "C. d, e, treated cell lysates incubated without ( d ) or with (e) neuraminidase after cell reculture a t 37 "C. MPR" was immunoprecipitated and then analyzed by IEF. An autoradiograph of the IEF gel is shown, with the acidic end at the top.

due to resialylation of asialo-MPR" and not to any other modifications.

To examine the time course of resialylation, MPR" species were quantitated as described under "Materials and Methods" by measuring the radioactivity in bands cut from the gel (Fig. 3). In neuraminidase-treated cells, there was a 20-min lag before resialylation commenced. This lag could represent the time required to transport MPR" molecules from the cell surface to the site of sialylation. The resialylation process had a half-time of 1-2 h, with the sialylation value approaching 100% after 4 h of reculture. A slow increase in the sialylation value of MPR" in control cells was also observed, with the sialylation value approaching 150% after 20 h of reculture. This suggests that receptors were also entering the sialyltrans- ferase compartment in cells that had not been treated with neuraminidase.

I I I I I

0 2 4 6 8 k k Time (hr)

FIG. 3. Quantitation of asialo-MPRC' resialylation. The ex- tent of resialylation was quantitated as described under "Materials and Methods." The sialylation value was calculated and normalized so that it equals 100 and 0% in control (m) and neuraminidase-treated cells (0) before reculture, respectively.

Control I NeuraminidaseTreated

Rearnure:- + + + + - + + + + I

conditions: - 18' 37" MOF Ch- - 18' 37" MOR Chlom -qne e n s i r q h

FIG. 4. Effects of reduced temperature, monensin, and weak bases on resialylation. Cells were iodinated at 0 "C. Control cells and neuraminidase-treated cells were recultured for 2 h under the indicated conditions. MPR" was isolated and analyzed by IEF. An autoradiograph of the gel is shown, with the acidic end at the top.

MPR" Resialylution Occurred within the Cell-To deter- mine where resialylation occurred, we examined the effect of reduced temperature on resialylation. Iodinated control and neuraminidase-treated cells were recultured in growth me- dium at 18 or 37 "C. While resialylation was observed at 37 "C, no resialylated MPR" was found after reculture at 18 "C (Fig. 4). It is known that internalization of plasma membrane proteins into endosomes occurs at 18 "C, but many intracellular transport processes are blocked. These include transport of endocytosed ligands from endosomes to lyso- somes (Dunn et al., 1980; Marsh et al., 1983; Sandvig and Olsnes, 1979) and transport of newly made glycoproteins from the Golgi to the cell surface (Matlin and Simons, 1983). These results suggest that membrane traffic is required for resialyl- ation and that MPR" is resialylated in an intracellular pos- tendosomal compartment. A second explanation, that reduced temperature inhibited sialyltransferases, has been ruled out, since we have previously shown that newly made glycopro- teins are sialylated when cells are cultured at 18 "C (Snider and Rogers, 1985).

Cell surface MPR" is transported into an intracellular compartment for resialylation. Does the receptor remain in the resialylation compartment or does it cycle back to the cell surface? To address this question, labeled control and neur- aminidase-treated cells were recultured at 0 or 37 "C for 2 h and then retreated with neuraminidase at 0 "C (Fig. 5). In control cells cultured at 0 "C, all the receptor remained on the

7678 Transport of Surface Man-6-P Receptor to the Golgi Complex Control Neurarninidase- Cells Treated Cells

Reculture: 0 37" 0 " 3 7 "

Retreatment: - + - + - + - + FIG. 5. Retreatment of cells with neuraminidase after re-

culture. Iodinated control and neuraminidase-treated cells were recultured at 0 or at 37 "C for 2 h and then incubated with or without neuraminidase at 0 "C. MPR" was then immunoprecipitated and analyzed by IEF. An autoradiograph of the gel is shown, with the acidic end at the top.

cell surface because it was completely desialylated by the neuraminidase digestion. In contrast, only 10-20% of the receptor was found on the surface of control cells cultured a t 37 "C. Because a similar fraction of the total receptor pool is found on the cell surface (Fischer et al., 1980; Sahagian and Neufeld, 1983; Shepherd et al., 1984), this suggests that sur- face and internal pools equilibrated within 2 h and supports the idea that receptor pools exchange rapidly. For neuramin- idase-treated cells, a second treatment of cells recultured at 0 "C had no effect, because no resialylation had occurred. On the other hand when cells were recultured at 37 "C, approxi- mately 10-20% of resialylated receptor could be digested by the second treatment. This indicates that a fraction of the resialylated receptor had returned to the cell surface. More- over, because similar fractions of resialylated and total MPR" were found on the cell surface, it is likely that resialylated receptor mixes with the total receptor pool.

Effects of Man-6-P-containing Ligands on MPR" Resialyl- ation-MPR" has endogenous ligands, namely newly made lysosomal enzymes that are synthesized constitutively by cells. To determine the effect of ligand binding on MPR" transport to the sialyltransferase compartment, resialylation of surface receptor was examined under conditions that alter MPR" occupancy by Man-6-P-containing ligands. To in- crease receptor occupancy, cells were incubated with saturat- ing levels of bovine testicular @-galactosidase, a ligand for MPR". In addition, weak bases and monensin were used to block the dissociation of endogenous ligands. None of these treatments had any significant effect on MPR" resialylation (Fig. 4, Table I).

To decrease receptor occupancy, cells were pretreated with cycloheximide and tunicamycin for 3 h to block the synthesis of lysosomal enzyme ligands. This pretreatment permits the transport of previously made molecules to lysosomes. Cells were then iodinated, treated with neuraminidase, and recul- tured in the presence of the same drugs. Cells were also incubated with Man-6-P to displace endogenous Man-6-P- containing ligands from surface receptor. Man-6-P has been shown to act as an agent that decreases receptor occupancy, since it reverses the accumulation of MPR" in endosomes that is induced by weak bases (Brown et al., 1986). None of the treatments had any significant effect on MPR" resialy- lation (Table I). These results suggest that receptor occupancy by Man-6-P-containing ligands is not important in determin- ing the rate of transport of surface MPR" to sialyltransferase- containing compartments.

Neuraminidase Treatment Does Not Affect MPRcl Proper-

TABLE I Effects of receptor occupancy on surface MPR" resialylation

Cells were iodinated, treated with neuraminidase, and recultured in growth medium with the indicated additions for 2 h at 37 "C. Cells treated with cycloheximide or tunicamycin were also preincubated with drug for 3 h at 37 "C prior to iodination and neuraminidase treatment. After cell lysis, MPR" was immunoprecipitated and ana- lyzed by IEF. The extent of resialylation was calculated as described under "Materials and Methods."

Neuraminidase-treated Control Treatment

Acidic MPR Sialylation Acidic MPR Sialylation % % % 95

Not recultured 44 0 77 100 Recultured

No addition 60 50 79 100 +Tunicamycin 65 65 85 107 +Cycloheximide 61 52 75 95 +Man-6-P 65 63 77 101 +@-Galactosidase 65 64 81 113

1 0 0

5 0

3 0

2 0

P .

0 1 0 2 0 3 0

Hours FIG. 6. Degradation of 1261-MPRC' in control (W) and neur-

aminidase-treated (0) cells. Cells were iodinated, treated with neuraminidase, and cultured at 37 "C. At the indicated times, MPR" was immunoprecipitated and analyzed by SDS gel electrophoresis (Laemmli, 1970). Radioactivity was measured by counting bands cut from the gel in a y counter. A semi-log plot of the radioactivity in MPR" is shown.

ties-In these studies, we have examined the intracellular traffic of surface asialo-MPR". The conclusions from these experiments can be applied to the movement of normal recep- tor only if the removal of sialic acid residues does not affect MPRC1 function. To assess the effects of neuraminidase treat- ment, we compared two properties of MPR" in control and neuraminidase-treated cells: ligand binding and receptor sta- bility.

Ligand binding was measured using [35S]Met-labeled secre- tions from ammonium chloride-treated CHO cells. Ammo- nium chloride raises the pH of acidic intracellular compart- ments and thus blocks ligand dissociation from MPR". As a result, receptors become saturated, and newly synthesized lysosomal enzymes are secreted into the medium (Gonzalez- Noriega et al., 1980). Control and neuraminidase-treated cells were incubated with labeled secretions a t 0 "C for 1 h, washed extensively, and specifically bound counts were then eluted with Man-6-P. Binding to neuraminidase-treated cells was 94 f 4% of that in control cells (mean f S.E., four determina- tions), indicating that removal of sialic acid residues did not alter the binding properties of MPR".

Receptor stability was measured by iodinating cells and determining the amount of labeled MPR" after reculture.

Transport of Surface Man-6-P Receptor to the Golgi Complex 7679

The turnover of '251-labeled MPRC1 was similar in control and treated cells (Fig. 6). The half-life of MPR" was 16 h in control cells and 21 h in treated cells. Finally, we have previously shown that neuraminidase treatment of K562 cells does not affect their viability or rates of growth and macro- molecular synthesis (Snider and Rogers, 1985).

DISCUSSION

In this study, the transport of surface MPR" through the Golgi complex has been studied using sialyltransferases as markers. These enzymes have been localized to trans-Golgi cisternae and the trans-Golgi network by several procedures. These include cell fractionation (Goldberg and Kornfeld, 1983), autoradiographic and cytochemical localization of the site of sialic acid incorporation (Bennett and O'Shaughnessy, 1981; Roth et al., 1984), and electron microscopic immuno- cytochemistry using anti-sialyltransferase antibody (Roth et al., 1985, 1986).

We have shown that asialo-MPR" generated on the cell surface is resialylated. This resialylation occurs when cells are cultured at 37 "C but not when the reculture is carried out at 18 "C. At the lower temperature, transport of surface ma- terial into endosomes can occur, but further transport is blocked (Dunn et al., 1980; Marsh et al., 1983; Sandvig and Olsnes, 1979; Griffiths et al., 1988). Thus, resialylation does not occur on the cell surface nor in endosomes, since asialo- MPR" is present in these compartments at 18 "C. The most likely site is the Golgi complex, where the bulk of the sialyl- transferase is located.

The half-time for transport of MPR" to the sialyltransfer- ase compartment is 1-2 h. This rate is similar to the rates at which surface and intracellular receptor pools mix (see below), suggesting that the resialylation compartment lies on the principal transport route for internalized MPR" in K562 cells. A lag of 20 min precedes the onset of resialylation. This lag, combined with the fact that more than 25% resialylation occurs in the 20 min following the end of the lag, suggests that the rate-limiting step in this process precedes receptor sialylation. A likely candidate for this rate-limiting step is the transport of asialo-MPR" from the cell surface to the sialyl- transferase compartment.

MPR" in control cells was also transported to the sialyl- transferase compartment, since the sialylation value of these receptors increased by 50% during reculture. This increase in sialic acid content can be explained in several ways. First it is possible that newly made MPR" are not fully sialylated when they are transported through the sialyltransferase com- partment. Additional rounds of transport through this com- partment might be required for complete sialylation. A second possibility is that sialic acid residues are lost from surface receptors in control cells and replaced after endocytosis. Be- cause most surface MPR" is exchanged into intracellular pools during reculture (Fig. 5), this would be accompanied by an increase in the sialic acid content of these receptor mole- cules.

Recently, Duncan and Kornfeld (1988) performed similar experiments to measure the transport of surface MPR to two regions of the Golgi complex. Entry into sialyltransferase compartments was examined using mutant cell lines that are deficient in the incorporation of Gal into N-linked oligosac- charides. Acceptor sites for sialyltransferase were created on surface glycoproteins by incubating cells with galactosyltrans- ferase and UDP-Gal. The sialylation of galactosylated MPR during culture was then assessed. Both MPR" and the cation- dependent MPR were sialylated with half-times of 3 h. These data are in good agreement with our data on MPR", except

that sialylation was more rapid in our study. The different rates could be due to differences in experimental design. However, a more likely explanation is that the behavior of MPR varies in the different cell lines used. Both the location of MPRC' within the Golgi complex (Brown and Farquhar, 1987) and the rate at which surface and internal receptors exchange (Pfeffer, 1987) have been shown to vary with cell type. Moreover, we found that resialylation of surface trans- ferrin receptor occurs more rapidly in K562 cells than in other human cell lines.2

Duncan and Kornfeld (1988) also examined transport of MPR into compartments that contain Golgi a-mannosidase I, which is found in earlier Golgi compartments. While trans- port of receptor through these regions did occur, the rate was much slower than for entry into the sialyltransferase com- partment. This finding is similar to our studies on transferrin receptor (Snider and Rogers, 1985,1986), which is transported into both of these Golgi regions but enters the sialyltransfer- ase compartment more rapidly.

Receptor occupancy by Man-6-P-containing ligands did not affect the transport of asialo-MPR" to the sialyltransferase compartment in our studies. This point is of some interest, since there are several apparently contradictory reports on the influence of receptor occupancy on MPR" traffic. In agreement with our finding, Braulke et al. (1987) showed that treatments which affect receptor occupancy do not have sig- nificant effects on the amount or internalization rate of surface receptor. On the other hand, a series of immunocyto- chemical studies by Farquhar and colleagues showed that these treatments alter MPR" distribution. Treatments that increase receptor occupancy cause accumulation in endo- somes, while treatments that decrease occupancy cause the receptor to accumulate in the Golgi complex (Brown et al., 1984, 1986). These results could reflect the differences in receptor behavior in different cell lines discussed above. Al- ternatively, if ligand binding causes small changes in the rates of individual steps in MPR" traffic, then receptor occupancy could affect the steady-state distribution of receptor without causing large changes in rates of receptor transport. MPR" has a second ligand, namely IGF-11. We have not examined the effect of this ligand on MPR" resialylation. However, Oka and Czech (1986) have recently reported that IGF-I1 does not affect the rate of MPR" internalization into rat adipo- cytes.

MPR" is the second surface receptor whose transport to sialyltransferase compartments has been demonstrated in K562 cells. Surface transferrin receptor was also shown to be resialylated in these cells (Snider and Rogers, 1985). In both cases resialylation required membrane traffic and the resialy- lated receptors mixed with the bulk receptor pool. However, the half-time for transferrin receptor sialylation was 2-3 h, significantly slower than for MPR". Transport of these two receptors into sialyltransferase compartments has also been observed by Goda and Pfeffer (1988) in permeabilized hamster fibroblasts. In these experiments, transferrin receptor trans- port was 4-5-fold slower than MPRC1 transport. Transferrin receptor resialylation is much slower than the rate of its cycling through endosomes, suggesting that only a fraction of the internalized receptor follows the Golgi route. In contrast, transport of MPR" to the sialyltransferase compartment is rapid enough to represent the principal transport route for internalized receptors. These differences might result from transport of the two receptors along different pathways or could be caused by differences in the rates of sorting into a common pathway.

M. D. Snider and 0. C. Rogers, unpublished results.

7680 Transport of Surface Man-6-P Receptor to the Golgi Complex

Current models for the intracellular traffic of MPR" during the packaging of lysosomal enzymes involve the mixing of surface and internal receptors in an intracellular compart- ment (Sahagian, 1984; von Figura and Hasilik, 1986; Korn- feld, 1987; Griffiths et al., 1988; Pfeffer, 1988). These models are supported by demonstrations that surface and internal MPR" pools mix with each other. While only a small fraction of the MPR" pool is on the surface at any time (Fischer et al., 1980; Sahagian and Neufeld, 1983; Shepherd et al., 1984), nearly all receptor molecules can be bound by extracellular antibody within 0.5-3 h, suggesting that the entire pool cycles to the cell surface during this time (Gartung et al., 1985; Sahagian, 1984; Braulke et al., 1987; Pfeffer, 1987). The most likely site of mixing is a prelysosomal compartment that has been shown to receive both newly made lysosomal enzymes and extracellular ligands (Griffiths et al., 1988). In this acidic compartment, lysosomal enzymes dissociate from receptor with subsequent transport of the enzymes into lysosomes. MPR" is thought to recycle from this compartment to the cell surface and the Golgi complex (Brown et al., 1986) with the steady-state distribution of receptor determined by the sorting of recycling MPR" into these two compartments.

Our data are consistent with these models of MPR" traffic. In neuraminidase-treated cells, 10-20% of the resialylated receptor was found on the cell surface. Because a similar fraction of the total MPR" pool was on the cell surface, this suggests that receptor did not remain at the site of resialyla- tion but mixed with the total MPR" pool. We also found similar mixing of surface and intracellular MPR" pools in cells that have not been treated with neuraminidase.

Acknowledgments-We wish to thank A. Tartakoff, C. Bos, C. Cooper, and P. Docherty for helpful discussions.

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