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Mutant Chinese hamster ovary cells pleiotropically defective in receptor-mediated endocytosis.

Populations of Chinese hamster ovary cells selected for resistance to diphtheria toxin were found to be highly enriched for mutants deficient in the uptake of lysosomal hydrolases via the mannose 6-phosphate receptor. One doubly defective mutant, DTF 1-5-1, exhibited increased resistance to Sindbis virus, although it was able to bind and internalize virus normally. Normal production of virus was obtained when, subsequent to virus binding, the mutant was exposed for 2 min to acidic pH. Similarly, a shift to acidic pH increased the sensitivity of DTF 1-5-1 to diphtheria toxin 12-fold. Decreased uptake of lysosomal hydrolases by the mutant correlated with decreased mannose 6-phosphate receptor activity at the cell surface; results of lactoperoxidase- catalyzed iodination indicated that the surface-associated receptor was present but inactive on DTF 1-5-1. Total mannose 6-phosphate receptor activity was also decreased in the mutant and this decrease was reflected by increased secretion of lysosomal hydrolases. The phenotype of DTF 1-5-1 resembles in many ways that of cells treated with ammonia. We suggest that the defect in DTF 1-5-1 stems from an inability to deliver virus, diphtheria toxin, and lysosomal hydrolases to an acidic compartment. Other ligands may be endocytosed through a different pathway since the defect of DTF 1-5-1 did not decrease the endocytosis of ricin, modeccin, or Pseudomonas toxin and had minimal effects on uptake and degradation of low density lipoprotein.


Cells and Cell Culture:
The isolation of the parental CHO cell line has been previously described (18). All media for cell culture were prepared in the Media Supply Unit of the National Institutes of Health. Growth media (19) and Labeling media (20) contained 5% fetal bovine serum or 5% dialyzed fetal bovine serum (both from Gibco Laboratories, Grand Island Biological Co., Grand Island, NY), respectively. Procedures of cell culture have been previously described (21). Unless otherwise specified, cells were kept at 34°C.

Preparation of Labeled Secretions: Medium conditioned by
CHO cells grown in the presence of ammonia is enriched in newly synthesized lysosomal enzymes that are ligands of the mannose 6-phusphate receptor. Thus for an easily prepared, albeit crude, source of radioactive llgand, WTB cells grown to about 1 x l07 cells/T-150 flasks were washed three times with medium lacking methionine, then labeled for 12-16 h with l0 ml of medium containing l mCi [~Slmethionine, 10/,g of nonradinactive methionine and NI-LC1, 10 raM. The secretions were concentrated, dialyzed, and clarified by centrifugation as previously described (20). One flask of cells yielded 1 ml of final material containing 3 × 107 cpm; 5-8% of this radioactivity could be taken up by recipient CHO cells and of that 95% appeared to be dependent on the man 6-P receptor (22). Secretions from human diploid fibroblasts were unsatisfactory as only 2-3% of the radioactive protein was internalized by CHO cells and more than half of this uptake appeared to be independent of the man 6-P receptor.
Isolation of Mutants: A stock of WTB was distributed into three T-75 flasks, "E", "F", and "G." These cultures were grown to densities of ~2 × l0 s cells/flask, then treated with the mutagen ethyl methanesulfonate (200 txg/ml). After 18 h "E", "F", and "G" were harvested and subcultured. 5 d after mutagenesis two T-150 flasks each of "E', "F," and "G" (cell density 1.0 × l0 T cells/flask) were transferred to 39°C; after 5 h diphtheria toxin was added to each flask at 100 ng/ml. After 24 h toxin was removed, ceils were harvested, pooling pairs of flasks, cells were replated on 100-ram dishes in aliquots corresponding to 0.25, 0.5, and I of the original flasks per dish and incubated at 34°C; 1 d later the medium was changed to growth medium containing 10% fetal bovine serum and 20/xg/ml insulin (23); Whatman no. 50 fibers, sterilized as previously described (21) were added for replica plating (23).
After 10 d the filter paper replicas were separated from the master dishes; the latter were stored in growth medium at room temperature in an atmosphere of 5% CO2. The replicas were transferred to 39°C; after 5 h growth medium (4 ml) containing 7 × 105 cpm/ml of [35S] secretions from WTB ceils was filtered onto each replica through a 0.22/t Millex fdter (Millipore Corp., Bedford, MA). After 12 h medium was removed and the replicas were gently washed once with growth medium, twice with Dulbecco's phosphate buffered saline, pH 7.4, without Ca ++ or Mg ÷÷ (PBS) and once with 0.9% NaCI. The replicas were stained in a mixture of 50% methanol, 10% acetic acid, 10% trichloroacetic acid containing 0.5 mg/ml Coomassie Brilliant blue for 1 h, then destained by rinsing four times with 40% methanol, 10% acetic acid. After the stained replicas had been rinsed in water for 15 rain, they were immersed in Autofluor (National Diagnostics, Inc., Advanced Applications Institute Inc., Somerville, N J) for 2 h, dried and exposed to preexposed (24) XR-2 t'tim for 20 h at -70°C. Comparison of the stained replicas with the film revealed putative mutants; these were picked from the master dishes and cloned as previously described (21). The letters "E", "F," or "G" in the designation of the mutants indicates the mutagenized culture from which a mutant was obtained.
Protein Synthesis Assay: The assay used was that previously described (25) except that cells were washed three times in methionine-free medium then labeled for 1 h in medium containing 1 ~g,/ml of methionine and 2 t~Ci/ml [aSS]methionine. Cells were then washed and solubilized in 0.1 N NaOH for 30 rain, the lysate was neutralized with 0.1 N HCI, and aliquots were spotted on paper, protein was precipitated and radioactivity in the precipitates was counted as described (25).
Virus: Sindbis virus and vesicular stomatitis virus (VSV) were provided by Dr. Sharon S. Krag (Johns Hopkins University, Baltimore, MD). Stocks of virus were prepared by infecting WTB cells with 0.1 plaque forming unit (pfu)/ cell. Viral titers were determined on confluent monoLayers of WTB (2.5 x 106 cells/60-mm dish) under solidified growth medium containing 2% fetal bovine serum and 0.75% agarose (Bethesda Research Laboratories). After 24 h (VSV) or 40 h (Sindbis) the solidified medium was removed and the monolayers were stained with 0.5% crystal violet ~n 50% ethanol.
Sindbis labeled with [S~S]methionine was prepared by infecting cells as above with 0.1 pfu/cell. After 5 h the cells were rinsed three times in methionine-free medium then incubated for 12 h in medium containing 0.4 mCi/ml [a~S]methionine plus 1.5 #g/ml nonradioactive methionine. The medium was harvested, clarified by centrifugation at 1,000 g for 10 rain, then viruses were pelleted by centrifugation at 100,000 g for 90 man. Viruses were resuspended in 10 mM Tris, 0.1 M NaCI, 1 mM EDTA, pH 7.4 and again pelleted at 100,000 g for 90 min. Radioactivity of the virus was 1.9 cpm/l,000 pfu. Labeled virions banded as a ROBBINS ET single sharp peak after centrifugation on a gradient of potassium tartrate (26); analysis of the labeled material by electrophoresis on SDS-polyacrylamide gels (reference 27, with the modification described in reference 28) showed that all of the radioactivity was in viral proteins.
Binding to the Mannose 6-Phosphate Receptor: Binding to intact cells was conducted as previously described (10) except that labeled ammonia-induced secretions were used instead of bovine testicular fl-gaLactosidase and displacement of radioactivity rather than of enzyme activity by mannose 6-phosphate was measured. Briefly, cells were chilled to 4°C, incubated with 5 mM man 6-P to remove endogenous ligand, washed, then incubated for 60 rain in 1.5 ml containing 1.3 x 10 ~ cpm of secretious. Following binding cells were washed nine times (the ninth wash was saved), before incubation with man 6-P (5 re_M) for 10 rain. Radioactivity in the ninth and tenth washes was determined and the difference was taken. Typical values obtained with WTB cells were ninth wash: 300 cpm, tenth wash: 3,300 cpm.
Binding to membranes was also measured by displacement. Membranes were prepared as previously described (29) with the single exception that membranes were collected by centrifugation at 100,000 g for 60 rain. Membranes were treated on ice for 30 rain in buffer (25 mM Tris, 0.25 M sucrose, 5 mM EDTA) pH 7.0, with 0.5% saponin and 5 mM mamaose 6-phosphate added (29), then pelleted, resuspended and washed by centrifugation at 100,000 g for 60 rain; 1 nag total cell protein yielded -200/~g washed membranes. Samples containing 30 ~tg of membrane protein were incubated with 2.0 × 105 clam of ammonia-induced secretions in the above buffer in a final volume of 80/al for 60 rain on ice; 75/d of the assay mixture was Layered onto buffer containing 100 mg/ml bovine serum albumin (BSA), then centrifuged at 100,000 g for 20 rain at 4°C in a Beckman Airfuge (Beckman Instruments, Inc., Spinco Div., Palo Alto, CA) to separate bound from free ligand (30). The pellet was resuspended and washed twice in 175 ~1 buffer plus BSA (1 mg/ml); the superuatant from the second wash was saved. The pellet was then resuspended in buffer plus BSA (I mg/ml) containing man 6-P (5 raM), incubated on ice for 15 rain and re-centrifuged. The difference in radioactivity in washes before and after incubation with man 6-P was taken.

Radiolabeling of the Mannose &Phosphate Receptor:
For metabolic labeling with [~Slmethionine cells grown in 100-ram dishes were washed three times with medium lacking methionine then incubated for 1 h in 4 ml of medium containing 0.4 mCi [~S]methionine and 4 ~g of nonradioactive methionine. Radioactivity was chased by changing the medium to normal growth medium.
For iodination of surface receptor, cells in 100-ram dishes were chilled to 4°C for 1 h, then rinsed three times with cold PBS; cold PBS (1.5 ml) was added to each dish, followed by 0.6 mCi of 12~I and 0.32 mg lactoperoxidase. The reaction was initiated by addition of 2 ~tl of H20~ (0.03%) with subsequent additions at 2, 4, 6, and 8 rain. At 10 rain 1.5 ml of cold PBS containing BSA (10 mg/ml) was added, and the cells were rinsed twice with PBS-BSA.
Sohib'tlization of the receptor and immunoprecipitation with rabbit antiserum directed against bovine man 6-P receptor were accomplished using the procedures described by SahagLan, G. G. and E. F. Neufeld (manuscript submitted for publication).
Enzyme Assay£: Acid hydrolase activity from cell extracts, secretions, and gradient fractions was assayed with 4-methyhimbelliferyl substrates by methods previously described (20,21 ). Transglutaminase activity was assayed as the Ca++-dependent incorporation of [3H]putrescine into casein as described (31). Protein was determined by the standard procedure (32).

Mutant Isolation
Three independently mutagenized populations of wild type CHO cells (WTB) were treated with diphtheria toxin (100 ng/ hal) for 24 h, survivors were replicated, and the replicate colonies were screened for the ability to take up lysosomal hydrolases via the mannose 6-phosphate receptor (see Materials and Methods). From a total of 6.3 x 107 cells treated with toxin, 2,100 colonies were obtained. Of 1,800 colonies tested for hydrolase uptake, 130 appeared to be uptake-deficient. In contrast, no uptake-deficient colonies were observed among 3,200 colonies of mutagenized cells not previously treated with toxin. Putative mutants were picked and cloned.
Two independent mutants were tested for their response to a variety of agents that enter the cell via receptor mediatedendocytosis. As expected, both mutants exhibited increased resistance to diphtheria toxin (Table I). No increase was ob-served in the resistance of the mutants to modeccin, Pseudomonas toxin, or ricin; instead, both mutants showed an increase sensitivity to ricin and mutant DTE 1-6-4 exhibited increased sensitivity to Pseudomonas toxin.
Increased resistance to diphtheria toxin and decreased endocytosis through the mannose 6-phosphate receptor would be expected in the mutants since these were the characteristics employed in their isolation. In addition, the mutants exhibited increased resistance to Sindbis virus and vesicular stomatitis virus. Resistance was manifested in two ways: first, a two-to six-fold reduction in the number of plaques formed on monolayers of mutant versus parental cells; second, a uniform decrease in the size of plaques formed on the mutants (Table III). That plaques were obtained at fairly high frequency on the mutants does not appear to reflect the presence of revertants; identical results were obtained using freshly cloned mutants; moreover, in a test of 10 4 colonies of DTF 1-5-1 no revertants were detected.

Mechanism
The sensitivity of DTF 1-5-1 to diphtheria toxin was increased if, following incubation with toxin, the mutant was shifted to medium of pH 4.5 (Fig. 1). This procedure has  previously been shown to overcome the block in toxin sensitivity observed in the presence of ammonia (25,33). The pHinduced sensitivity of DTF 1-5-l to toxin appeared dependent on binding of toxin to receptors, since the presence of ATP, an inhibitor of toxin binding (34), during incubation with toxin, abolished the subsequent effect of the shift to acidic pH (Fig.  1). These results suggest that resistance of DTF 1-5-1 to toxin reflects a block occurring subsequent to toxin binding, but before the appearance of toxin in the cytoplasm. In similar fashion, a brief shift (2 min) to pH 5.0 following incubation of DTF 1-5-1 with Sindbis virus increased subsequent production of virus sixfold (Table IV). Thus, the mutant appeared able to bind virus and to support viral replication. Resistance to virus, like resistance to diphtheria toxin, seems to result from either a failure to internalize bound virions or to move virus from within the endocytic apparatus to the cytoplasm.
To distinguish between these possibilities, DTF 1- Table V). Lack of surface binding appears to reflect mainly decreased receptor activity, rather than decreased receptor number. Lactoperoxidase-catalyzed iodination followed by immunoprecipitation revealed that the level of surface receptor in DTF 1-5-1 was much greater than that predicted from binding studies (Fig. 3). We observed the same phenomenon in WTB cells that had been preincubated with NH4C1; no binding of ligand to man 6-P receptors at the cell surface was detected (Table V), yet on iodination signif-

TIME (min)
Binding and internalization of Sindbis virus by WTB and DTF I-5-I. Cells (grown to a density of I x 100 cells/dish in 60-ram dishes) were incubated with [35S]Sindbis, 2.9 x 10'* cpm, 10 pfu/ cell, at 34°C for the times indicated. Infected cells were washed twice with cold PBS, then incubated at 4°C with I ml of PBS containing 0.5 mg/ml bovine serum albumin, or 0.5 mg/ml proteinase K, for determination of total cell-associated virions or internalized virions, respectively (35). After 45 rain I ml PBS containing 30 mg bovine serum albumin and I mM phenylmethylsulfonylfluoride was added to each sample, cells were harvested and washed as described (35)   Binding to the receptor was determined by displacement of radioactivity bound to intact cells or membranes with 5 mM man 6-P (see Materials and Methods). Values given have been corrected for radioactivity displaced from cells or membranes when binding was conducted in the presence of 5 mM man 6-P (intact cells 250, membranes 300 cpm/mg cell protein). No inhibition of binding to intact cells or membranes was observed when binding was conducted in the presence of 5 mM man I-P. * WTB cells were grown with 10 mM NH4Cl for 12 h before the experiment; NH4CI was also present during the course of the binding assay.
icant levels of receptor were found on amine-treated cells (Fig.  3). In three experiments the level of iodinated man 6-P receptor in DTF 1-5-1 and amine-treated cells, quantitated by densitometry, ranged from 30-60% of that measured in WTB cells. Measurements of binding to total cellular mannose 6-phos- FIGURE 3 Iodination of the mannose 6-phosphate receptor on WTB and DTF 1-5-1. Cells (grown to 4 X 106 cells/ dish in 100-ram dishes) were chilled to 4°C and iodinated for 10 min at 4°C as described in Materials and Methods. Receptor was solubilized (Sahag-Jan, G. G., and E. F. Neufeld, manuscript submitted for publication) from pairs of dishes; one-half of each sample was immunoprecipitated (Sahagian, G. G., and E. F. Neufeld, manuscript submitted for publication) and the other half was taken through the immunoprecipitation procedure without antireceptor antiserum as a control for nonspecific precipitation. Precipitates were electrophoresed on SDS-polyacrylamide gels (reference 27, as modified in reference 28), and the radioactivity was detected on pre-exposed film (24). The arrow indicates the position of the man 6-P receptor. The left lane of each pair is from the nonspecific precipitation, the right lane is from the immunoprecipitation. F, DTF 1-5-1; W, WTB; W', WTB cells grown for 12 h in 10 mM NH4CI, chilled and iodinated in the presence of NH4CI. The level of radioactivity in the second band (Mr ~46,000) visible in both controls and immunoprecipitates varies unpredictably among different preparations. HOURS Turnover of the mannose 6-phosphate receptor in WTB and DTF 1-5-1. Cells (grown to 3 X 106 cells/dish in 100-mm dishes) were incubated with [aSS]methionine for I h, then label was chased for the times indicated. Precipitation and electrophoresis of the precipitates was performed as described in Fig. 3. The gels were subjected to fluorography (36) radioactivity was detected on preexposed film (24) and quantitated by densitometric scanning. Values are expressed as a percentage of the radioactivity measured in the respective cell-type following the 1-h pulse; at this point the level of radioactive receptor measured in WTB cells was 90% of that measured in DTF 1-5-1. (O) WTB; (0) DTF 1-5-1.
phate receptors indicated that the mutant had 20% of the receptor activity of parental cells (Table V). Examination of biosynthesis and degradation of receptor by metabolic labeling indicated equal rates of receptor biosynthesis in DTF 1-5-1 and WTB; the half-life of the receptor was found to be 9.5 h in the mutant and 18 h in the parent (Fig. 4). Addition of NH4C1 to WTB ceils after a radioactive pulse also resulted in increased turnover of the receptor (data not shown).
Consistent with its decreased level of mannose 6-phosphate receptor activity (20), DTF 1-5-1 exhibited reduced intracellular levels of many acid hydrolases and secreted those enzymes in correspondingly elevated amounts (Table VI). Similar results were obtained by metabolically labeling the cells and immunoprecipitating a-L-iduronidase and fl-hexosaminidase from cell extracts and media (data not shown). When cell-free extracts of mutant and parent were fractionated on 27% Percoll gradients (37), two peaks of acid hydrolase activity were obtained, consistent with previous reports (21,38). Extracts of DTF 1-5-1 showed a diminution of enzyme activity only in the denser of the two peaks, i.e., in the region corresponding to lysosomes (Fig. 5). 125 Fig. 6 shows the uptake and degradation of I-low density lipoprotein by mutant and wild type cells. At 5 #g/ml of LDL the rate of uptake into DTF 1-5-1 was 75% of that measured for WTB; degradation was 50% that of WTB. This reduction in the rate of degradation of LDL may reflect the mutant's deficiency in acid hydrolases. The kinetic parameters of LDL uptake differ slightly between DTF 1-5-1 and WTB at concentrations of LDL < 50/Lg/ml; at concentrations of ligand >50 #g/ml the kinetic parameters were identical for the two cell types (Fig. 7).
The phenotype of DTF 1-5-1 is similar in many ways to that Secreted enzymes were collected for 14 h in serum-free medium from cells grown to densities of 6-8 x 10e/100-mm dish; preparation of the secretions and assays of enzymatic activity were conducted as previously described (20). Total hydrolase activities from DTf 1-5-1 in comparison to those from WTB were fl-hexosaminidase, 116%; Ot-L-fucosidase, 116%; a-mannosidase, 97%; a-L-iduronidase, 44%; fl-glucuronidase, 130%. The decreased level of a-L-iduronidase may reflect the instability of that enzyme in medium (22).
of amine-treated ceils. One of the proposed targets in inhibition of endocytosis by the amines is transglutaminase (39), but measurements of the activity of this enzyme in crude extracts of mutant and parent cells revealed no significant differences either in the Km or rate of transfer of putrescine, or in the inhibition of enzyme activity by methylamine.

D I S C U S S I O N
The initial goal of this study was the isolation of mutants pleiotropically defective in receptor-mediated endocytosis. The mutants described here, DTE 1-6-4 and DTF 1-5-I, were found to be altered in the uptake of diphtheria toxin, lysosomal enzymes, and two lytic RNA viruses, Sindbis virus and VSV.
Preliminary studies indicate that DTF 1-5-1 also is unable to accumulate iron from complexes of transferrin-iron (Klausner,
R. D., and A. R. Robbins, unpublished data). To ensure that these multiple defects result from a single pleiotropic mutation, rather than multiple mutations, it will be necessary to isolate revertants of the mutants. The high frequency at which mutants deficient in lysosomal enzyme uptake were found among the diphtheria-toxin-resistant cells, especially in the absence of selective pressure, argues against the possibility of multiple mutations. Interestingly, cross-resistance to lytic RNA viruses was found in a number of KB cell mutants that had been selected only for resistance to diphtheria toxin (40).
Although temperature-shifts were employed in both selection and screening, neither resistance to diphtheria toxin nor the deficiency in lysosomal enzyme uptake were found to be be temperature-sensitive in any of seven mutants tested to date (data not shown). Using our procedure it may not be possible to isolate mutants with temperature-sensitive defects in steps subsequent to internalization. Toxin accumulated within the cell at the nonpermissive temperature may result in cell death on return to the permissive temperature.
The defect in DTE 1-6-4 and DTF 1-5-1 impairs endocytosis of some ligands but not of others. Resistance to ricin, modeccin, and Pseudomonas toxin was not increased in the mutants, and only marginal effects were observed on uptake of low density lipoprotein in DTF 1-5-1. Thus, while morphologic studies indicate that diphtheria toxin (9), Pseudomonas toxin (41), LDL (5), and lysosomal enzymes (8) share a common uptake pathway, endocytosis of these ligands must differ at some stage subsequent to ligand binding. CHO cell mutants defective in uptake, but not in binding, of both Pseudomonas toxin and ricin have recently been reported (16). No change was found in sensitivity of these ceils to diphtheria toxin. These results, like our own, suggest different structural or functional requirements for endocytosis of different ligands.
Our results indicate that the block in Sindbis infectivity in DTF 1-5-1 occurs at penetration into the cytosol. Our findings with diphtheria toxin are consistent with this localization of the defect. Entry of Sindbis into the cytoplasm, like that of Semliki Forest virus (35,42), appears to be induced by acidic pH; this is also the case for diphtheria toxin (25,33). One explanation for the phenotype of DTF 1-5-1 is that internalized virus and toxin never encounter an acidic environment, due either to failure of the endocytic vesicle to fuse with an acidic compartment or to a defect in acidification of the endosome (43) and/ or lysosome (44). Alteration in lysosomal pH seems least likely since DTF 1-5-1 was able to degrade LDL.
Is this postulated inability to deliver ligand to an acidic compartment consistent with our observations regarding mannose 6-phosphate receptor mediated uptake in DTF 1-5-1? Dissociation of ligand from the mannose 6-phosphate receptor has been proposed to require an acidic milieu (45); thus lack of surface-binding activity in DTF 1-5-1 and ammonia-treated normal cells (reference 45 and this paper) could result from intracellular accumulation of ligand-receptor complexes. However, iodination at 4°C revealed near-normal numbers of surface-associated receptor molecules in both the mutant and ammonia-treated cells. It is unlikely that these inactive recep-