The Mannose 6-Phosphate Receptor of Chinese Hamster Ovary Cells

THEJOURNAL OF BIOLOGICAL CHEMISTRY Val. 256, No. 20. Issue of October 25. pp, 10618-10622. 1981 Printed in U S A.

The Mannose 6-Phosphate Receptorof Chinese Hamster Ovary Cells ISOLATION OF MUTANTS WITH ALTERED RECEPTORS* (Received for publication, February 2, 1981, and in revised form, May 8, 1981)

April R. Robbins and Rachel Myerowitzl From the Genetics and Biochemistry Branch, National Institute 20205 Institutes of Health, Bethesda, Maryland

of

Arthritis, Metabolismand Digestive Diseases, National

Richard J. Youle, G a r y J. Murray& and David M. Neville, Jr. From the Section on Biophysical Chemistry,Laboratory of Neurochemistry, National Institute Bethesda, Maryland20205

A method has been developed for the isolation of mutant Chinese hamster ovary cells with altered mannose 6-phosphate receptors. Mutants were selected for resistance to a conjugate composed of w-(6-phospho)pentamannose oligosaccharides covalently linked to ricin (Man 6P-ricin). Cells were incubated with this hybrid reagent in the presence of 0.1 M lactose, in order to block the normal binding of the ricin moiety to carbohydrates on the cell surface. Under conditions where binding of Man 6P-ricin occurred solely through the mannose 6-phosphate receptor, viability of the parent cells was reduced to 0.1%. To isolate Man 6P-ricinresistant mutants,mutagenized cells were treated with the conjugate, survivors were replicated, and the replicate colonies were screened for incorporation of [3sS] methionine into protein after a second incubation with Man 6P-ricin. Eleven of 20 mutants isolated according to this procedure were found to exhibit an increased resistance (2- to 9-fold) to Man 6P-ricin.Six of the resistant mutants showed decreased uptake of bovine testicular &galactosidase, which enters Chinese hamster ovary cells via the mannose 6-phosphate receptor. Studies of enzyme binding were conducted with two mutants that displayed 10% of the parental level of uptake: decreased binding of P-galactosidase to mutant A10-2-4 was found to reflect, at least in part, an increased rate of dissociation of enyzme from the receptor; mutant B4-2-1 exhibited reduced binding at low concentrations of P-galactosidase and greater thanparental binding at elevated levels of enzyme. Unexpectedly, all mutants resistantto Man 6P-ricin also showed increased resistance (1.3- to 4-fold) to ricin, although binding of ricin to the mutants wasequal to or greater than binding to the parent. Uptake of exogenous acid hydrolases into fibroblasts has been shown to depend upon the presence of a mannose 6phosphate recognition marker on those enzymes (1-6). Treatment with periodate ( 7 ) , phosphatase (1-3), or endoglycosidase H (5, 8 ) transformed the enzymes from a “high uptake”

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of National Research Service Award Postdoctoral Fellowship F32AM05893. 8 Present address, Clinical Investigations and Therapeutics Section, Developmental and Metabolic Neurology Branch, National Institute of Neurological and communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20205.

of

Mental Health,

to a “low uptake” form. Binding of acid hydrolases to both surface and intracellular membranes has been demonstrated (9-12) and amannose 6-phosphate receptor has recently been purified (13). People affected by I-cell disease synthesize hydrolases lacking the mannose 6-phosphate recognition marker (6, 14); the low intracellular and high extracellular levels of acid hydrolases associated with I-cell disease have been the basis for assigning a role to the recognition marker in the delivery of newly synthesized enzymes to lysosomes (15, 16). One might expect to find individuals with altered mannose 6phosphate receptors among patients with lysosomal storage disorders, but no such case has yet been reported. Synthetic conjugates of mannose 6-phosphate with a variety of proteins are recognized by the mannose 6-phosphate receptors for lysosomal enzymes. (17-19). The compounds examined include bovine serum albumin conjugated withp-isothiocyanatophenyl 6-phospho-mannoside (17, 18), as well as low density lipoprotein, ribonuclease, and ricin linked to a phosphorylated pentasaccharide of mannose (19, 20). Incubation of human fibroblasts with the conjugate of ricin and 4 6 phospho)-pentamannose resulted in inhibition of protein synthesis even in the presence of lactose, which blocks bindingof the conjugate to the cell via the ricin moiety; inclusion of mannose 6-phosphate eliminated inhibition by the conjugate (20). The potential value of this hybrid reagent for the selection of receptor mutants has been discussed (20). In this paper we present the isolation and characterization of Chinese hamster ovary cell mutants with altered mannose 6-phosphate receptors. The accompanying paper describes the consequences of this alteration on the compartmentdization of acid hydrolases in these mutants (21). EXPERIMENTAL PROCEDURES

Materials Fetal bovine serum and tissue-culture grade trypsin were purchased from GIBCO. Tissue culture media were prepared in the Media Supply Unit of the National Institutes of Health. Ethyl methanesulfonate was purchased from Eastman, 4-methylumbelliferyl P-galactopyranoside from Research Products International, bovine serum albumin (A grade) from Calbiochem, [%]methionine (1050 Ci/mmolf from Amersham, and ’“I-Bolton-Hunter reagent (2000 C i / m o l ) from New England Nuclear. Ricin waspurified from seeds of Ricinus communis, generously given to us by Deutzche Rizinus Oelfabrik Boley and Co., according to the method of Nicolson et al. (22). Man GP-ricin’ wasprepared by the published procedure (20).P-Galactosidase was partially purified from bovine testicles as described (23).

’ The abbreviations used are: Man GP-ricin, oligosaccharides of w (6-phospho)-pentamannosecovalently linked to ricin; CHOcells, Chinese hamster ovary cells.

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Mutants Altered Mannose with 6-Phosphate Receptors Cells and Cell Culture WTB (24), a subclone of a CHO cell line that is aproline auxotroph (25), was the gift of Dr. Raymond M. Baker, Massachusetts Institute of Technology. N211-1-8,a mutant with 15%of the wild type activity of lysosomal ,&galactosidase, was isolated from WTB using the published procedure (26) with the single modification that 4-methylumbelliferyl P-galactopyranoside was used as substrate in the screening. Cells were grown in Eagle's minimal essential medium supplemented with antibiotics, nonessential amino acids, and 10%fetal bovine serum (27) in 5%C o n a34 t "C; they were stored a t -80 "C in growth medium plus 10% dimethyl sulfoxide. Cells were kept in culture for only 2 months; a t this time frozen cells were used to initiate new stock cultures. In experiments employing serum-free medium, Waymouth medium MAB 87-3 (28), formulated as in the GIBCO catalogue, was used. For measurements of plating efficiency, cells were trypsinized, resuspended in complete growth medium, and counted in a hemocytometer; the cells were then serially diluted in complete growth medium and plated. Dilutions yielding between 200 and 450 colonies/ 100-mm dish (visualized after staining with methylene blue) were used in calculations of plating efficiencies. Duplicate values differed by less than 10%. Preparation of LZ5Z-labeled Ricin

10619 added 0.7 ml of 6.7 mM NaPi, pH 7.4, containing 0.15 M NaCl and 0.3% bovine serum albumin. ln5I-Ricin,2.5 X IO4 cpm, plus varying concentrations of unlabeled ricin, wasadded in a volume of 0.05 mlto each well. After incubation for 30 min at 4 "C, the cells were washed twice with the above buffer at 4 "C, then solubilized in 1M NaOH for counting. Assays were run in triplicate and protein was determined by the method of Lowry et al.(31). Unlike human diploid fibroblasts (32),each of the CHO cellstrains testedshowed positive cooperativity of ricin binding, with maximal binding at 1.1 X M ricin (8.3 X 10-l' mol of ricin/well). The molar amount of ricin bound was calculated as: fraction of radioactivity bound X 8.3 X 10"' mol/mg of cell protein. Greater than 90% of the bound 12511-ricin was displaced a t the highest concentration of ricin tested, 5.5 X M. Assay of P-Galactosidase Uptake CeUs were grown to an approximate density of 8 X IO5 (subconfluent) in 60-mm dishes. The cells were rinsed once in growth medium, then 4 ml of growth medium including serum and containing varying concentrations of P-galactosidase, was filtered onto the cells through a 0.22 p Swinnex filter (Millipore). After incubation a t 34 "C, growth medium was removed, the cells were washed in solution A (0.14 M NaC1/7 mM KC1/1 mM NaKPi, pH 7.4), and harvested by trypsinization. Growth medium containing 10%fetal calf serum was added to stop the action of trypsin; the cells were centrifuged, washed twice in solution A, resuspended in solution A and lysed by three cycles of freezing and thawing. P-Galactosidase activity was assayed as previously described (26).All assays of uptake were performed in duplicate; uptake increased linearly with time over a period of 12 h.

Labeling was performed by the procedure of Bolton and Hunter (29): 11.5 pg of purified ricin disolved in 10 pl of 65 mM NaPi, pH 7.5, was added to 0.5 mCiof dried '251-Bolton-Hunterreagent on ice. After 15 min, the reaction was stopped by addition of 0.2 ml of 0.2 M glycine in 0.1 M Na2B407. pH 8.5. The mixture was applied to a Sephadex G25 column (1.5 X 5 cm) that had been equilibrated with 50 mM NaPi, Assay of P-Galactosidase Binding pH 7.5, containing 0.25% gelatin. Excluded fractions containing lZ51 Specific binding of /3-galactosidase was assayed by measuring enwere pooled; assuming 100%recovery of ricin, the specific activity of zyme displaced from the cell surface by 2 mM mannose 6-phosphate Iz5I-ricinwas 9.5 X lo4 cpm/pmol. (9, IO). All steps of this procedure, a modification of that described for measuring the binding of low density lipoprotein by displacement Isolation of Man 6P-ricin-resistant Mutants with heparin (331, were carried out with media chilled to 4 "C. A newly recloned stock of N211-1-8 was grown in a T-75flask to an Binding-Cells, grown to confluence in 60-mm dishes, were placed approximate density of 2 X IO6 cells, then treated with the mutagen at 4 "C for 45 min. The cells were then washed once with growth ethyl methanesulfonate (200 pg/ml). After 25 h, the cells were harmedium and incubated in 1.5 ml of medium plus 2 mM mannose 6vested and distributed into two cultures "A" and "B"; these two phosphate. After 10 min, the cells were washed 4 times with 2 ml of populations were maintained independently throughout the remainder of the procedure. One weekafter mutagenesis, ten 100-mm dishes medium, then incubated for 60 min in 1.5 ml of medium containing each of "A" and "B" at 5 X I d cells/dish were treated with 4.3 X IO-* varying concentrations of bovine testicular &galactosidase. Binding of enzyme to the parental cells (N211-1-8)had reached its maximum M Man 6P-ricin in growth medium containing 0.1 M lactose. Eighteen h later, each dish was washed three times with growth medium plus atthis time. All incubations up to and including this one were performed in an atmosphere of 5% Con. 0.01 M lactose, the cells from each dish were harvested and replated Washing-All washes were performed with serum-free medium on 100-mm dishes at 2.5 X IO5 cells/dish. Whatman no. 50 filters, sterilized as previously described (26), were added to each dish one supplemented with 15 mM Tricine, 2 mg/ml of bovine serum albumin, day laterfor replica plating (30). The growth medium was replaced a t and adjusted with NaOH to pH 7.2. Medium was removed and the cells were washed rapidly 4 times with 2 ml. On both the fifth and 4-day intervals. After 12 days, the filter paper replicas were removed from the sixth washes, the cells were incubated for 10 min in 1.5 ml of medium; master dishes; the latter were stored in growth medium a t room the seventh and eighth washes (1.5 ml) were again rapid. The ninth temperature in an atmosphere of 5% COZ. Cells on the filters were wash (1.5 m l ) entailed incubation for 10 min; on the tenth and final treated with 4.3 X IO-' M Man 6P-ricin in growth medium containing wash the cells were incubated 10 min in 1.5 mlof medium containing 0.1 M lactose; after 18 h the fiters were washed 3 times in growth 2 mM mannose 6-phosphate. The ninth and tenth washes were saved medium lacking methionine and supplemented with 0.01 M lactose for determination of P-galactosidase activity. Enzyme Assays-0.05 ml of the collected wash was added to 0.25 and 10% dialyzed fetal calf serum. The cells were then incubated at 34 "C in 4 ml of medium, containing 50 pCi of [35S]methionine(7.7 ml of 0.1 M Na acetate, pH 4.4, containing 0.33 mM I-methylumbelliCi/mmol), and supplemented with 10% dialyzed fetal bovine serum. feryl P-galactoside. Reactions were incubated for 4 to 6 h at 37 "C. Up to this point, the procedure was carried out under sterile condi- The cells were solubilized by incubation in 0.5 ml of 0.1 M NaOH for tions. After 3 h, the replicas were transferred to Petridishes contain- 15 mina t room temperature andprotein was determined. One enzyme ing 4 ml of cold 20% trichloroacetic acid and 30 pg/ml of unlabeled unit represents one nmol of substrate hydrolyzed/h at 37 "C; 1 mmol methionine. After 15 min at 4 "C, the filters were placed on a Buchner of enzyme is equivalent to 4.6 X 10'" enzyme units. The latter value funnel, colonies facing upwards, and washed 5 times with 50 ml of was derived by fvst converting the enzyme units employed here to cold 10% trichloroacetic acid (30). The filters were then dried in an those of Distler and Jourdian (23),and then using the specific activity determined by those authors for bovine testicular /3-galactosidase oven and exposed to XR-2 film for 20 h at -80 "C. Under the conditions employed, the colonies on two filter replicas purified to homogeneity (23). All binding assays were carried out in triplicate; to minimize that were not treated with Man 6P-ricin, thus serving as positive controls, produced blackening of the f i . The majority of colonies variability in the timing of each step, no more than 4 assays were on the treated filters produced only a faint gray image; colonies, the carried out simultaneously. replicas of which darkened the f i tothe Same degree as the RESULTS untreated controls, were picked from the master dishes and cloned as previously described (26). One colony was cloned from each master Fig. 1 shows the plating efficiency of N211-1-8 (a CHO cell dish. The letter "A" and "B" in the designation of each mutant m u t a n t deficient in P-galactosidase) after treatment with difindicates from which of the original cultures, separated after mutaferent concentrations of Man 6P-ricin. After incubation with genesis, the mutant was obtained. 4.3 x lo-@M conjugate in the presence of 0.1 M lactose, 0.1% Assay of Ricin Binding of the cells were able to form colonies; inclusion of 1 mM To cells, grown to confluence in 35 mm wells of Costar trays, was mannose 6-phosphate in the incubation mixture maintained

10620

Mutants with Altered Mannose 6-Phosphate Receptors TABLEI Comparison of wild type, parent, and mutant cells with respect to toxin sensitivity and /3-galactosidase uptake Strain

ManLDloa 6P-Ricin, P-gdactosidaseb Uptake of Ricin, LDlo” Ricin bound RM

96 94 54 10 22 93

WTB N211-1-8 A9-2-1 A7-2-1 A1-0-1 B7-3-1 BG-1-3 B8-1-4 B4-2-1 A6-1-5 A10-2-4 B8-2-3 A8-1-2

5.7 5.7 11 11 14 14 14 17 29 33 36 49 49

ND‘ (1W 104 99

76

9 26

nM

pmol/mg

0.7 0.7 1.0 1.2 2.3 0.9 0.9 1.6 3.0 1.3 1.4 2.6 I .8

ND 11 ND 24 ND 21 20 15 23 24 27 ND 21 10% of the

LDlo is the dose that reduces plating efficiency to untreated cells; dose-response curves were generated for each strain and the LDlo was obtained by interpolating between concentrations Man GP-Ricin (nM) of Man GP-ricinthat yielded survivorsat frequencies lessthan 45 and FIG. 1. Viability of N211-1-8 after treatment with Man 6P- greater than 2%. The concentrations of Man 6P-ricin and ricin are based on estimated molecular weights of 70,000 and 65,000, respecricin. N211-1-8 cells,grown to 1 X lo6 in100-mmdisheswere incubated for 18 h with the indicated concentrationsof Man GP-ricin tively. Cells were incubated with 5.4 X M enzymefor 8 h; uptake is ingrowthmediumcontaining 0.1 M lactose. This incubation was conducted in the presence (0)or absence (0) of 1 mM mannose 6- expressed as percentage of uptake into N211-1-8. ND, not determined. phosphate. Cells werethen washed 3 times in medium containing0.01 M lactose, harvestedby trypsinization, counted, diluted, and replated. After 10 days, colonies were stained. Efficiency of plating was calculated with respect to that of untreated cells, which was 88% in this mannose 6-phosphate receptor(4). Six of the 11Man GP-ricinresistant mutants exhibited decreased uptakeof this enzyme experiment. I). Mutantswiththe in comparison to the parent (Table lowest levels of resistance were found to have normal uptake the viability of the cells at the level of untreated controls. At of P-galactosidase; with theexception of AS-1-2, mutants with higher concentrations of Man GP-ricin, loss of viability was 3-fold or higher resistance to Man GP-ricin all showed deobserved, even in the presence of mannose 6-phosphate, sug- creased uptake. Inclusion of mannose 6-phosphate (1 mM) gesting that lactose was nolonger saturating the sites on the abolished uptake of P-galactosidase by the parent and all of ricin moiety. Lactose at concentrations greater than 0.1 M was the mutants. itself toxic. One might have expected that some of the Man GP-ricinThe high frequency of survivors, also observed with cells resistant mutants would be resistant to ricin itself, since the it mechanism of ribosomal inactivation is the same for the previously mutagenized with ethyl methanesulfonate, made impractical to isolate the desired mutants using only a single modified and unmodified toxins. A likely candidate for ricin round of selection with ManGP-ricin. Therefore, we employed resistance was mutant A8-1-2 which exhibits a relatively high a second step in which survivors of the initial selection were level of resistance to Man GP-ricin (Table I) yet normal uptake replicated and the replicate colonies were screened for their via the mannose 6-phosphate receptor (TableSurprisingly, I). ability to incorporate [35S]methionine into protein after treat- all of the mutants exhibitedincreased resistance (1.3- to 4.3ment with Man GP-ricin (see “Experimental Procedures”). fold) to ricin (Table I); a 1.3-fold increase in LDlo corresponds Those colonies among the survivors which were capable of to double the parental plating efficiency at that concentration. protein synthesisfollowing treatment with ManGP-ricin were Little or no correlation wasobserved among the mutants picked from the master dishes andcloned. between the levels of resistance to ricin and Man GP-ricin or After treatment of 5 X lo6 cells (previously mutagenized between the level of ricin resistance and theability to interwithethylmethanesulfonate)withMan GP-ricin, 5 X lo3 nalize P-galactosidase. The presence of mannose 6-phosphate formed colonies and 4 x lo3 replicated onto the filters. Of (1mM) did not affect ricin toxicity for parent or mutantcells. these, 1.2 X lo2 incorporated [35S]methionine intoprotein Measurements of binding of lZ5I-ricinindicate that the infollowing the second treatment with ManGP-ricin. Twenty of creased resistanceof the mutants to ricin does not result from the mostvigorous colonies were chosen for cloning, and 11 of decreased binding of that toxin (Table I). these exhibited increased resistanceto Man GP-ricin. The two mutants which exhibited the least uptake of PThe efficiency of plating of each mutant was determined galactosidase, A10-2-4 and B4-2-1, were chosen for further after treatmentwith varying concentrations of the conjugate. studies. The concentration of Man GP-ricin required to reduce the a As shown in Fig. 2, uptake of enzyme into the parent was plating efficiency of each strain to10%( L D d was derived by saturable process, exhibiting half-maximal velocity at 1.5 X interpolatingbetweenconcentrations yielding survivors at lo-’ M, a value similar to that measured with human fibrofrequencies from 2 to 45% each line contained 3 or 4 points blasts (4).Uptake into the mutants continued to increase, within this range. The LDlo’s of the 11 mutants were from 2 albeit nonlinearly at high enzyme concentrations, up to the to 9 times higher than the LDlo measured for the parent highest concentration of enzyme tested (2.7 X 10” M). ManN211-1-8 (Table I). On repeated determinations thedifferent nose 6-phosphate (1mM) abolished uptake into both mutant strains always exhibited the same relative sensitivities to Man and parent cells at all concentrations of P-galactosidase emGP-ricin. ployed. Uptake of bovine testicular P-galactosidase proceeds via the Since uptakeinvolves internalization of the enzyme as well

Mutants Altered Mannose with 6-Phosphate Receptors as binding, decreased uptake of enzyme might not necessarily reflect alteration of the mannose 6-phosphate receptor. In order to measure binding of P-galactosidase to cells which have significant residual activity of that enzyme, we took advantage of the slow rate of dissociation of enzyme from the mannose 6-phosphate receptor, coupled with the rapid displacement of bound enzyme by mannose 6-phosphate (9, 10). Fig. 3 shows the procedure used: the cells must be thoroughly washed to remove loosely adhering enzyme, this is accomplished in 8 washes. Inclusion of mannose 6-phosphate (2 mM) released a small but reproducible amount of P-galactosidase. Addition of mannose 1-phosphate (2 mM) did not cause release of the enzyme (Fig. 3). If after 7 washes the cells wereshifted from 4” to 37” for 20 min, noenzymewas released upon subsequent treatment with mannose 6-phosphate. As shown in Fig. 4 binding of P-galactosidase to N211-1-8 was saturable, andhalf-maximal at 1.5 X lo-’ M. The maximal amount of enzyme bound represents 4.5 X lo3molecules/cell, one third the amount measured for human diploid fibroblasts (9). Mutant A10-2-4 appeared to bind 9 X 10’ molecules/cell at saturation, with half-maximal binding at 5 X lo-* M (but

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0.01A&-L,2 i -

1- ““.L_L”

~

25050

150 8-Galactosidase (nM)

11

450

350

FIG. 4. Binding of /&galactosidase by parentandmutant cells. N211-1-8 (O),A10-2-4 (A)and B4-2-1 (0)were pretreated with mannose &phosphate, washed, and incubated with the indicated concentrations of enzyme for 60 min at 4 “C. The cells were washed and incubated with 2 mM mannose 6-phosphate as described under “Experimental Procedures.” Each value represents the mean of three dishes of cells; values were corrected for the amount of P-galactosidase present in the ninth wash, that is the last wash prior to addition of mannose 6-phosphate.

TABLE I1 Dissociation of P-galactosidase from parent and mutantcells Twelve dishes of each cell type were pretreated with mannose 6phosphate, washed, and incubated with 1.4 X 10” M enzyme at 4 “C as described under “Experimental Procedures.” The standard washing procedure wasmodified such that mannose &phosphate was added to three dishes on the fifth wash and to three others on the tenth wash. Values were corrected for the amount of enzyme released from parallel dishes that did not receive mannose h h o s ~ h a t e . P-Galactosidase released Strain

Fifth wash

Tenth wash

pmol/mg cell protein

10

30

50

70

270

N211-1-8 A10-2-4 B4-2-1

0.051 0.031 0.043

0.044 0.010 0.038

I]-Galactosidase. nM

FIG. 2. Uptake of &galactosidase by parentandmutant cells. N211-1-8 (O),A10-2-4 (A)and B4-2-1 (0) were incubated with ,&galactosidase at the indicated concentrations for 8 h. Values are corrected for the level of enzyme activity measured in cells which did not receive exogenous enzyme, 0.90 & 0.03 pmol/mg.

see below). Binding to B4-2-1did not follow Michaelis-Menten kinetics; instead a sigmoidal pattern was obtained (Fig. 4). Saturation of binding occurred at 2.7 X M P-galactosidase, at which concentration lo4 molecules of enzyme werebound/ cell. At this same concentration of ,&galactosidase, uptake into B4-2-1 proceeded at one quarter the rate measured with parental cells (Fig. 2), although twice as much enzyme was specifically bound by the mutant. The method employed here for measuring binding relies on a low rate of dissociation of enzyme from the mannose 6phosphate receptor (9). This assumption is not valid in the case of mutant A10-2-4. As shown in Table 11, 68% of the enzyme releasable by mannose 6-phosphate was lost from A10-2-4between the fifth and tenth washes; B4-2-1and N2111-8 lost only 12 and 14%,respectively. These results indicate that the number of binding sites on A10-2-4 is significantly greater than the 9OO/cell calculated from Fig. 4. DISCUSSION

Wash

FIG. 3. Release of B-galactosidasefrom N211-1-8on sequential washes. Cells were pretreated with mannose &phosphate, washed, and incubated with 1.4 X M enzyme for 60 min a t 4 “C. Washes 1to 4,7, 8, and 11 were rapid; on washes 5, 6,9, and 10, cells were incubated in the washing medium for 10 min. Wash 10 (indicated by thearrow) contained 2 mM mannose 6-phosphate (e),2 mM mannose 1-phosphate (A),or no additions (0). The average protein content/dish was 0.50 mg.

Toxins covalently linked to a ligand for the receptor of interest have been proposed as selective agents in the isolation of receptor mutants (20).Recently, ricin linked to monoclonal antibodies directed against a cell surface antigen were shown to be toxic for cells bearing that antigen (34). In this report, we have used Man 6P-ricin (20) in the isolation of CHO cell mutants with altered mannose 6-phosphate receptors. To inhibit binding of Man 6P-ricin via the ricin moiety, treatment of the cells was conducted in the presence of 0.1 M lactose. Even under these conditions, specificity of binding of Man 6P-ricin could be maintained only at low concentrations

Mutants Altered with

10622

Mannose 6-Phosphate Receptors

of the conjugate, that yielded surviving cellsat a frequency of 0.1%. To circumvent this problem of limited toxicity without sacrificing specificity, we used a combination of selection and screening procedures: the survivors of the initial selection were plated to allow colony formation and the colonies were replicated (30); the replicas were again treated with Man 6Pricin, then tested in situ for their ability to synthesize protein. While only 1.5% of the cells surviving the selection step were actually resistant to Man 6P-ricin, half of the colonies picked and cloned followingscreening were resistant mutants. The combined procedure employed here eliminates the timeconsuming testing of individual survivors, and in some cases might prove a useful alternative to repeated rounds of selection. An unexpected finding is that all of the Man 6P-ricinresistant mutants exhibited some degree of resistance to ricin itself. Little or no correlation was observed among the mutants between the levels of resistance to the modified and unmodified toxins, or between the level of ricin-resistance and the ability to internalize enzyme via the mannose 6-phosphate receptor. We have shown that under the selection conditions employed, toxicity of Man 6P-ricin for the parental cells is dependent on the mannose 6-phosphate receptor, judged by absence of toxicity in the presence of mannose 6-phosphate. In addition, we find that the Man 6P-ricin-resistant mutants bind as much ricin as the parent. Thus, the ricin-resistance of these mutants may result from alteration in a stepsubsequent to toxin binding. It has been proposed that the galactose binding site may participate in toxicity by mediating binding to some intracellular component (32). The increased resistance to ricin observed with the mutants described here may result from alterations in this presumptive intracellular binding site. Two Man 6P-ricin-resistant mutants, A10-2-4 and B4-2-1, were shownto have markedly reduced uptake of P-galactosidase. These mutants were isolated from a CHO cell mutant deficient inlysosomal /?-galactosidase, and this facilitated measurements of uptake of that enzyme. However,the residual /?-galactosidaseactivity in these cells wastoo high for the direct determination of enzyme bindingby assay of enzymatic activity. Therefore, we used an indirect assay, based on measurements of the amount of bound 8-galactosidase that can be displaced by mannose 6-phosphate (9, 10). While this method is satisfactory for simple demonstration of altered binding of enzyme to the mutant cells, it is of limited usefulness for interpreting details of this binding: for example, the accelerated rate of dissociation of &galactosidase from the receptors of A10-2-4 makes the estimate of total binding sites on that mutant erroneously low. In the accompanying paper we examine the compartmentalization of newly synthesized acid hydrolases in the two mutants with altered mannose 6-phosphate receptors.

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Acknowledgment-We thank Dr. Elizabeth F. Neufeld for helpful discussions of this work. REFERENCES 1. Kaplan, A,, Achord, D. T., and Sly, W . S. (1977) Proc.Natl.Acad.

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