Cell, Vol. 33, 967-977, July 1983, Copyright Q 1983 by MIT 0092-8674/83/070967-l 1 $02.00/O

Fate of the Transferrin Receptor during Maturation of Sheep Reticulocytes In Vitro: Selective Externalization of the Receptor Bin-Tao Pan and Rose M. Johnstone Department of Biochemistry McGill University 3655 Drummond Street Montreal, Quebec, Canada H3G 1 Y6

Summary

The fate of the transferrin receptor during in vitro maturation of sheep reticulocytes has been followed using FITC- and ‘251-labeled anti-transferrin-receptor antibodies. Vesicles containing peptides that comi- grate with the transferrin receptor on polyacrylamide gels are released during incubation of sheep retic- ulocytes, tagged with anti-transferrin-receptor anti- bodies. Vesicle formation does not require the pres- ence of the anti-transferrin-receptor antibodies. Us- ing 1251-surface-labeled reticulocytes, it can be shown that the ‘251-labeled material which is re- leased is retained by an immunoaffinity column of the anti-transferrin-receptor antibody. Using reticu- locytes tagged with 1251-labeled anti-transferrin-re- ceptor antibodies to follow the formation of vesicles, it can be shown that at 0% or in phosphate-buffered saline the rate of vesicle release is less than that at 37% in culture medium. There is selective external- ization of the antibody-receptor complex since few other membrane proteins are found in the extemal- ized vesicles. The anti-transferrin-receptor antibod- ies cause redistribution of the receptor into patches that do not appear to be required for vesicle forma- tion.

Introduction

The transferrin receptor plays a central role in the transport of iron from the iron-transferrin complex into the cell (for revrew see Morgan, 1981). The receptor is ubiquitous on growing animal cells (Trowbridge and Omary, 1981; Suth- erland et al., 1981; Hamilton, Wada, and Sussman, 1979; Larrick and Cresswell, 1979; Galbraith et al., 1980) owing presumably to the iron requirement for proliferation. The transferrin receptor is abundant on reticulocytes (Jandl and Katz, 1963) the enucleated erythroid cells that retain the ability to synthesize hemoglobin and therefore require iron (Lowenstein, 1959). The concentration of transferrin recep- tor gradually decreases and eventually disappears during reticulocyte maturation (Van Bockxmeer and Morgan, 1979; Pan, Blostein, and Johnstone, 1983; Frazier et al., 1982).

Maturation of reticulocytes into mature cells can be followed in vitro by measuring the disappearance of retic- ulum and the decrease in amino acid transport (Benderoff, Johnstone, and Blostein, 1978; Benderoff, Blostein, and Johnstone, 1978); in nucleoside transport (Jarvis and Young, 1982); in glucose transport (Zeidler and Kim, 1982);

and in the ability of the cells to bind transferrin (Frazier et al., 1982). We have previously purified a class of antibodies against reticulocyte surface antigens and identified them mainly as anti-transferrin-receptor antibodies (Pan and Johnstone, 1980; Pan, Blostein, and Johnstone, 1983). The ability to follow the presence of the receptor on the cell surface with labeled antibody (lz51 or FITC), as well as the ability to obtain maturation during in vitro incubation, presented us with a novel way of following the fate of the transferrin receptor during maturation of sheep red cells. Little information is available on the fate of the receptor during maturation of the red cell. In fact, it has been speculated that during maturation the receptor may be altered to lose transferrin binding activity while the protein is retained in the membrane (Leibman and Aisen, 1977).

Other investigators have shown that the fate of a mem- brane receptor may be studied by using an antibody against the receptor as probe, as well as with the natural ligand (Beisiegel et al., 1981; Schlessinger, Van Ob- berghen, and Kahn, 1980). The stability of the receptor- antibody complex often makes it a more useful probe than the natural ligand for studying the fate of the receptor. The anti-transferrin-receptor antibody we obtained provided us with a probe with which we could follow the fate of the receptor during maturation in the presence of this antibody. Here, evidence is presented on the fate of the transferrin receptor during in vitro maturation of sheep reticulocytes. Using lz51- or FITC-antibody it can be shown that vesicles containing the antibody-receptor complex are externalized into the medium. Although the iodinated antibody presents a convenient ligand for following vesicle formation, forma- tion of vesicles does not require antibody. The data sug- gest that vesicle release may be the normal mechanism for shedding some reticulocyte-specific membrane pro- teins during maturation of the red cell.

Results

Externalization of Transferrin Receptors during Maturation In Vitro Sheep reticulocytes incubated at 37°C lose their capacity to bind an anti-transferrin-receptor antibody (Pan and John- stone, 1980; Pan, Blostein, and Johnstone, 1983). During this period the cells also lose their methylene-blue-stainable reticulum and take on the appearance of mature red cells (Benderoff, Blostein, and Johnstone, 1978). Using sheep reticulocytes, surface-labeled with ‘251, we observed that the amount of ‘251-labeled plasma membrane protein pre- cipitated with the specific anti-transferrin-receptor antibody decreases with time in culture in parallel with the loss of reticulum (Pan, Blostein, and Johnstone, 1983). These data suggested that the receptor might be removed from the plasma membrane. To test this possibility the cell surface was labeled with ‘251 and lactoperoxidase (Reichstein and Blostein, 1975) and the cells were incubated for 36 hr at 37°C. Initially, and after 36 hr, the cells were separated from the incubation medium. Plasma membranes were

,,. ,,

CeH 968

prepared (Dodge et al., 1963) and solubilized, and the solubilized membranes and cell-free postincubation me dium were passed through an anti-transferrin-receptor im- munoaffinity column as described (Pan, Blostein, and Johnstone, 1983).

The data in Table 1 show that initially all the antibody binding ‘251-labeled material was cell associated. With time in culture, most of the immunoreactive radioactivity was released to the medium. Moreover, this released immuno- reactive material could be centrifuged down completely at 100,000 x g. After centrifugation at 100,000 X g, no 1251- labeled immunoreactive material remained in the superna- tant.

Externalization of the Transferrin Receptor To determine the peptide composition of the released material, reticulocytes were incubated, and the post-incu- bation, cell-free pellets obtained at 100,000 X g were collected, washed, and subjected to SDS gel electropho- resis. In addition, the peptide composition of the pellet was examined after pretreating the reticulocytes with anti-trans- ferrin-receptor antibodies or after adding transferrin to the final incubation medium. For these studies the cells were first depleted of transferrin (Hemmaplardh and Morgan, 1974) and then preincubated with anti-transferrin-receptor antiserum or with nonimmune serum. The cells were washed free from serum and incubated with and without transferrin in serum-free medium. The data in Figure 1 show the peptide profile of the material released from the cells under these conditions. In all cases a 93 kilodalton (kd) peptide characteristic of the transferrin receptor and a peptide of 70 kd were seen. In cells preincubated with the immune serum, a 53 kd peptide characteristic of the heavy chain of immunoglobulin appeared in addition to the 93 and 70 kd peptides. If transferrin (MW 78,000) was present, a 78 kd peptide was also detected on the gels.

Table 1, lmmunoreactivity of ‘ZSI-Labeled Material Released durrng Incubation of ‘“l-Surface-Labeled Reticulocytes

‘?Label Recovered (total cpm)

0 hr 36 hr

Plasma Membrane 11.240 1520

Culture Medium 8340

Cells were labeled with ? by lactoperoxidase treatment, washed, and then incubated in serum-free medium for 36 hr at 37% in O&02. At zero time, membranes prepared from an aliquot of cells were dissolved and passed through an immunoaffinrty column (Pan, Blostein, and Johnstone, 1983). The column was washed with phosphate-buffered saline, pH 7.4. untrl no radioactivity was eluted. The washings were discarded. Then the retained radioactivity was released with acrdglycine buffer and counted. After 36 hr of incubatron, an equal number of cells was processed to determine the remaining surface-bound radioactivity retained by the immunoaffinity col- umn, The culture medium was also passed through an anti-transfemn- receptor column and processed rn an rdentrcal way. The data show that most of the cellular radioactivity retained by the immunoaffinity column was released into the medium during incubation. If a nonspecific immunoaffrnity column was used, no significant radioactivity was retained by the column afler washing wrth saline-phosphate. Transferin-depleted cells were used.

The data indicate that the 93 kd peptide is the transferrin receptor. The identity of the other major peptide (-70 kd) isolated with the vesicles is unknown. It is unlikely that the peptides found in the 100,000 X g pellet were due to contamination with medium, since the 93 kd and 70 kd peptides were found after incubation of cells in serum-free medium, and no peptides were found if fresh culture medium was processed in the same way. Electrophoresis of the released ‘251-labeled material reported in Table 1 gave the same peptide profile as shown in Figure 1 (lane 3) with most of the 125l located in the 93 kd peptide.

Release of ‘251-Labeled Protein into the Culture Medium The studies suggested that if the cells were tagged with the anti-transferrin-receptor antibody, the latter was re-

1 2 3 4 5

c R (93K)

- c TF(78K) + (70K)

Frgure 1. Release of the Transferrin Receptor in Presence and Absence of Antibody and Transfernn

Sheep reticulocytes were isolated and treated to remove bound transferrin, and a portion was Incubated with retrculocyte-specrfic rabbit antiserum at 0°C. Control cells were treated with nonimmune serum. After 90 min at 0°C the cells were washed, resuspended in fresh incubation medrum (serum free), with or wrthout 50 ag/ml transferrin, and incubated at 37°C under O&OZ After 21 hr, the cells were removed by centrifugatron and the cell-free culture medium was centrifuged at 100,COO x g for 90 min. The pellets thus obtained were washed once in phosphate-buffered saline (pH 7.4) recentrifuged, and lyophilized, and the material was subfected to SDS gel electrophoresis. (Lane 1) Transferrin receptor isolated by a trans- ferrin affinity column. 186K and 93K are the transferrin receptor. 78K is transfernn. (Lane 2) Peptides from the culture medium of cells preincubated with nonrmmune serum. The incubation medrum contained transfernn. (Lane 3) Is the same as lane 2, but without transfernn. (Lane 4) The cells were preincubated with immune serum, and the incubation medium contained transferrin. (Lane 5) Is the same as lane 4. but without transferrin. Note that incubation with transfernn leads to isolation of a peptide wrth a molecular weight characterrstic of transferrin (78.C~30). Incubation with antibody leads to the isolation of a peptide with a molecular weight characteristic of the heavy chain of rmmunoglobulrn (53,000). In all cases peptrdes of 93 and 186 kd appear R: receptor. TF: transferrin. IgG: heavy chain of IgG.

Fate of Transferrin Receptor in Reticulocyte Maturation 969

leased into the 100,000 X g pellet during incubation. We therefore used ‘“51-labeled anti-receptor antibody as a probe to examine the characteristics of the release proc- ess Reticulocytes bearing 1z51-labeled anti-transferrin-re- ceptor antibodies may be separated from free antibodies by filtration through Sepharose 6B. The cells eluted with the void volume. From a time course of “51-antibody binding to the cells at O°C, it was determined that a steady state was reached after 60 min (Figure 2). At the concen- tration of antibody used, about 35% of the total 1251- antibodies became cell associated at steady state at 0°C. To assess the extent of nonspecific antibody binding,

4.

I ERYTHROCYTE

. * I \ 3’0

MINUTES 60 9’0

Figure 2. Trme Course of ‘251-Antrbody Binding

A 2% suspension of sheep reticulocytes was incubated in phosphate- buffered saline (pH 7.4) with ‘251-labeled antibody, 0.25 pg/ml (specrfic activity 4.5 X lo5 cpm/pg). At various intervals 0.4 ml samples were removed and filtered through a Sepharose 6B column (1.5 X 7 cm) at 4’C. The cells eluted with the vord volume, completely separated from free ‘q- antibody. In the presence of ItDfold excess of nonlabeled antibody, the radioactivrty bound was reduced to 6% of the control and was not signrfi- cantly different from the binding in mature cells shown in the figure. Antrbody bindrng to retrculocytes failed to saturate even at concentrations up to 140 ag/ml. Representative data of antibody added (specific activity 4.5 X lo5 cpm/pg) versus amount bound (1% reticulocytes at 4°C for 90 min) IS Illustrated below:

Antibody Added (pg/ml) Amount Bound (cpm)

0.6 moo

4.0 2,500

22.0 6,600

55.0 12,200

140.0 22,cm

erythrocytes were substituted for reticulocytes in the assay. With erythrocytes cell-associated radioactivity was less than 5% of that with reticulocytes (Figure 2). This obser- vation is consistent with the fact that erythrocytes do not have (or have few) surface receptors for transferrin. To follow the fate of the cell-bound 1z51-antibody, reticulocytes bearing ‘251-labeled antibody were incubated in antibody- free culture medium at 37°C. The changes in radioactivity associated with the cell pellet and the supernatant during the course of incubation at 37’C are given in Figure 3. Radioactivity associated with the cells decreased, while that associated with the supernatant increased recipro- cally. All the components released were of high molecular weight, since all the released radioactivity was precipitable with trichloroacetic acid.

Assessment of the Binding Activity and Molecular Weight of the Released “%Antibody If free “51-antibody were released, it would be expected to retain at least part of its capacity to bind to fresh reticulo- cytes. The released, high molecular weight material con- taining ‘251 was incubated with fresh reticulocytes at 0°C and this binding was compared to that with fresh (never bound) ‘251-antibodies. The results in Table 2 show that

14. Total

I ? I

4 6 12 16 20 24 Hours

Frgure 3. Time Course of Release of Cell-Bound Radioactivity Into the Culture Medium

A 2% suspension of sheep reticulocytes in phosphate-buffered saline (pH 7.4) was incubated with ‘251-anti-transferrin-receptor antibody (0.4 fig/ml. specific activrty 4.5 X 105 cpm/ag) at 0°C for 90 mm. The ‘?-antibody labeled cells were collected In the void volume after filtration through Sepharose 6B. The cells were resuspended with culture medium containing 2% fetal calf serum to give a 1% suspension and incubated at 37C under 95% O&% COP. At Intervals, 0.4 ml samples were taken and centrifuged for 1 mm at 12,009 X g. and the cell-free supernatants and cell pellets were counted. The total applred radioactivity was recovered m the fractions shown.

Cell 970

vesicle or a soluble complex. To distinguish between these two possibilities, several experiments were carried out, the results of which are all consistent with the conclusion that a vesicle containing the antibody-transferrin receptor com- plex was released. First, the cell-free supernatant contain- ing the ‘251-labeled released material was excluded from a Sepharose 6B column, -80% of the radioactivity being eluted with the void volume. The rest of the radioactivity (-20%) coeluted with free IgG. Second, the bulk of the radioactivity found in the void volume after Sepharose gel filtration was pelleted at 100,000 x g (Table 3). The pellet so obtained was washed and subjected to SDS-polyacryl- amide gel electrophoresis. Peptides comigrating with the transferrin receptor (93 kd and 186 kd), transferrin (78 kd), and IgG (53 kd) were obtained (Figure 5; see also Figure 1). Third, the cell-free medium containing the released ‘251- complexes was centrifuged to equilibrium on a 20%~50% (w/w) sucrose density gradient. The radioactivity was found in a band containing 35%-40% sucrose, a density characteristic of vesicles derived from plasma membranes (Evans, 1978). A peak of radioactivity corresponding to free IgG was also obtained at a lower density (data not shown). Fourth, uniform membrane vesicles were detected in the cell-free postincubation culture medium after incu- bation of ‘251-antibody-labeled reticulocytes at 37°C (Figure 6). No such vesicles were found in the initial culture medium.

Two lines of evidence suggest that the antibody is on the surface of the vesicle. First, “51-antibody-containing vesicles in the cell-free culture medium were retained by a protein A-Sepharose column. About 75% of the applied radioactivity was retained but released with acid-glycine treatment, For example, an aliquot of the cell-free, postin- cubation medium containing 3 X lo3 cpm was applied to the column, and the column was washed with neutral phosphate buffer until no radioactivity was detected in the wash. After elution with acid-glycine buffer, 2.3 X 1 O3 cpm were recovered. Negative staining of the material eluted from the column after acid treatment showed vesicles similar to those seen in Figure 6. Second, the ‘*?antibody associated with vesicles was released by acid-glycine treatment. Vesicles containing ‘*?antibody were passed through Sepharose 6B columns with and without pretreat- ment with acid-glycine-buffered saline. Without acid treat- ment, most of the radioactivity eluted in the void volume, whereas after acid treatment, the radioactivity was eluted mainly with free antibody (Table 4). These experiments are consistent with the conclusion that vesicles bearing the transferrin receptor and surface-bound “51-antibody are externalized during incubation of ‘251-antibody-labeled sheep reticulocytes in vitro at 37°C.

Table 2. Reticulocyte Binding Capacity of ‘251-Antibody Released from Reticulocytes

Pretreatment of ?-Antibody

None

None

Prerncubation in cell-free medium

Preincubatton with sheep erythrocyfes

Preincubation with sheep reticulocytes

Rebinding to Fresh Reticulocytes

@pm)

21,000

600 (binding to erythro-

cytes)

1woo

20,ooo

600

To measure ‘?-antibody binding to cells, ‘251-labeled antibody was incu- bated for 90 min at 0°C with a 2% reticulocyte suspension. The cells were centrifuged, washed, and counted. The antibody used in these experiments was either fresh antrbody (no pretreatment) or antibody that had been preincubated. Preincubation was as follows. The ‘251-antibody was rncu- bated in cell-free culture medium for 24 hr at 37°C and then an aliquot was used for binding to reticutocytes (preincubation in cell-free medium). ‘251. antibody was incubated with mature cells for 24 hr at 37°C the cells were separated from the medium, and an aliquot of the cell-free medium was incubated with 2% reticulocytes at O’C (preincubation with sheep erythro cyles). ‘?antibody was incubated with reticulocytes for 530 min at 0°C. Then the cells were washed to remove unbound antibody and incubated at 37°C for 24 hr. The cell-free supematant was collected, and an aliquot was Incubated with fresh reticulocyfes at 0°C for 90 min (preincubation with sheep reticulocytes). In all cases the amount of radioactrvity used to assay bindrng to reticulocyfes was the same (74 X 103 cpm).

binding of the released ‘251-antibodies. The results in Table 2 show that binding of the released ‘251-material to fresh reticulocytes was of the same order as the nonspecific binding of fresh ‘251-antibody to the mature red cell. Prein- cubation of the antibody with or without mature red cells did not affect antibody binding to reticulocytes. The data suggest that the radioactive material released into the medium after being bound to reticulocytes had lost the capacity to rebind to reticulocytes. Loss of binding activity was not due to inactivation of the antibody by incubation at 37°C since free “51-antibody, incubated in the absence of cells, maintained its ability to bind to reticulocytes (Table 2). The “inactivation” of antibody after incubation with reticulocytes suggests that the released ‘251-material may have been altered by virtue of having been attached to the transferrin receptor. To compare the approximate mo- lecular size of the released radioactive material with that of the native ‘251-antibody, the released material was filtered through a Sephadex G-150 column. In contrast with free antibody (Figure 4) the majority of the released radioactiv- ity eluted in the void volume, suggesting that the bulk of the radioactivity released was in a higher molecular weight form (or forms) than that of free antibodies. There was no detectable degradation of ‘251-antibody to products of a lower molecular weight than that of the free antibody.

The Transferrin Receptor-Antibody Complex Is Released as a Vesicle If the ‘251-antibody were released in combination with the transferrin receptor, the released form could be either a

lmmunofluorescent Study of the Redistribution and Externalization of the Antibody-Receptor Complex FITC-labeled antibody was also used to follow the progress of the cell surface changes associated with the binding of the antibody and reticulocyte maturation, The procedures

Fate of Transfernn Receptor in Retrculocyte Maturation 971

5C

4c m

‘0 7 X

E 3c a 0

In cu -H 2C

10

0

: :-24 hrs

5 10 15 20 25 30

FRACTION NUMBER

Frgure 4. Sephadex G-150 Filtratron of the Released Radioactrvity

Cell-free postrncubation supernatants obtained as in Figure 3 were applied to Sephadex G-150 columns (1.5 X 60 cm) and eluted with phosphate-buffered saline, pH 7.4. The fractions (2.5 ml) were counted for ‘? The total radioactivity applied was recovered in the fractions shown. For these experiments the cells were preincubated with 3.5 pg/ml of ‘251-antibody at -4.5 x lo5 cpm/pg

Table 3. Distribution of the lz5 I-Labeled Materral Released from Cultured ‘2SI-Antibody-Labeled Reticulocytes

Fractions in Vord Sepharose 6B Filtration of Volume after

Total Released Cell-Free Supernatant Centrifugation

‘*‘I, Cell-Free Supernatant of 6 hr Culture Medium i!trne

;z;t;;d

(free IgG) Supernatant Pellet

12,390 9,860 1,460 783 8,835

Values are counts per minute. Reticulocytes (2% suspension) were rncu- bated in phosphate-buffered saline with ‘251-anti-transferrin-receptor antl- body at 0°C for 90 min, and the suspension was filtered through a Sepharose 6B column to separate bound from free antrbody. The cells were resuspended to give a 1% suspension in culture medium containing 2% fetal calf serum and incubated at 37’C. After 6 hr, the cells were removed by centrifugation at 12,CCO X g, and the supernatant was collected and counted. An aliquot of the supernatant was applied to a Sepharose 6B column (1.5 X 60 cm) and eluted with phosphate-buffered saline (pH 7.4). One mrllrliter fractions were collected. The vord volume obtarned was collected and centrifuged at ICQOOO X g for 1 hr, and the supernatant and pellet obtarned were counted.

were analogous to those with ‘251-antibody. The data in Figure 7a show that after binding at 0°C there was uniform fluorescence over the cell surface. However, the immuno- fluorescence started to redistribute after transfer to 37°C. Visible clusters were evident by 30 min, and after 3 hr or

longer, large patches or caps were visible (Figures 7b and 7~). Fluorescent particles appeared in the culture medium after 15 min, and their number gradually increased during the course of incubation at 37°C. The fluorescent particles from a cell-free supernatant were collected and examined by fluorescence microscopy and by negative staining. After negative staining these vesicles were similar to those shown in Figure 6. The fluorescence of the particles in the void volume were abolished by treatment with pH 2.3 glycine-buffered saline prior to chromatography on Se- pharose 66. This result is analogous to that with ‘251-labeled antibody (Table 4). These experiments are consistent with the conclusion that the antibodies (‘251-labeled or FITC- labeled) are on the surface of the vesicles.

The observation that the release of the fluorescent particles precedes patch formation and continues after patch formation suggests that patch formation is not re- quired for the externalization of the fluorescent particles (i.e., vesicles).

Moreover, in contrast with vesicle formation, which oc- curs in the absence of the antibody, patch or cap formation appears to be dependent on the presence of the anti- transferrin-receptor antibody. With reticulocytes incubated in the absence of the specific antibody, and fixed prior to exposure to the FITC-labeled antibody, the immunofluores-

Cell 972

1 23456 7

R (186K)

R (93K)

IgG (53K)

Frgure 5. Detection of the Transferrin Receptor in Released Vesicles

After 6 hr of incubation at 37°C with reticulocytes prelabeled with ? labeled antibody as in Figure 2, the cells were removed by centrifugation at 12,000 X g for 1 min. The cell-free medium was centrifuged at 100,ooO x g, and the pellet obtained was recentrifuged at lCO,OOO x g in 0.15 M NaCl containing 10% or 20% sucrose. The washed pellet obtained was subjected to SDS gel electrophoresis. (Lane 1) Fresh incubation medium only was processed: (lane 2) 106,000 x g pellet after recentrifugation In 0.15 M saline containing 10% (w/w) sucrose; (lane 3) same as lane 2, but with 20% sucrose; (lane 4) total reticulocyte plasma membranes; (lane 5) standard IgG; (lane 6) sheep transferrin standard, major peak = 76K; (lane 7) transfernn receptor (186K and 93K) isolated with immobilized sheep transferrin (Sepharose 4B-transferrin column). R should be noted that on washing with 20% sucrose (lane 3) there is loss of protein including a substantial loss of transferrin itself (78K peptide). Although the Intensity of the peptrde band at 166K is weak in this photograph (lanes 2 and 7) in other experiments it is much more apparent (see Figure 1).

cence remained uniformly distributed during incubation at 37°C although the intensity of the fluorescence decreased with time (not shown).

Kinetics of Vesicle Externalization To follow the rate of vesicle externalization in presence of the ‘z51-labeled anti-transferrin-receptor antibody, experi- ments similar to those reported in Figure 3 were conducted except that the cell-free supernatant was separated into vesicle and nonvesicle fractions by passage through Se- pharose 66. At 6 hr w-50%, and by 21 hr about -8O%, of the bound ‘251-antibodies were externalized, and the rate of externalization decreased with time (Figure 8). (The data are almost superimposable with those in Figure 3, where total, noncellular radioactivity was measured.) Cell integrity was maintained during the experimental period as k-rdi- cated by the levels of cellular potassium and ATP (Table 5). The metabolic requirements for externalization of the antibody-receptor complex were followed with FITC- and ‘251-labeled antibodies. Removal of serum from the medium

did not prevent the release of ‘251-labeled antibody (Figure 8). In contrast, incubation at 0°C or at 37°C in phosphate- buffered saline reduced antibody release. The data show that the process of externalization was dependent on cell activity. Figure 8 shows that in phosphate-buffered saline, the externalization was not immediately or completely re- duced, in contrast with the effect of lowering the temper- ature to 0°C.

Internalization of “%Antibody Most investigators believe that the transferrin-receptor complex is internalized for ion delivery (for review see Morgan, 1981). To assess whether any internalization of ‘251-antibody occurred during incubation at 37°C the acid- accessibility of the cell-bound ‘“?antibody was determined during long-term incubation at 37°C. Most of the I*?- labeled, cell-bound antibody was accessible to acid throughout the incubation period (Table 6). An increase of - 10% in the amount of acid-undissociable radioactivity was seen after the cells were transferred from 0°C to 37°C. Subsequently, the absolute amount of the acid-undissoci- able radioactivity decreased gradually and remained con- stant at - 15% of the total cell-associated radioactivity. The acid-undissociable level of radioactivity may reflect a small fraction of bound ‘251-antibody that was internalized. The gradual decrease of acid-inaccessible radioactivity may be due to the recycling of the internalized antibody- receptor complex.

Discussion

We have previously reported that an antibody prepared against sheep reticulocytes reacts specifically with the transferrin receptor in the plasma membrane. Using this antibody, it has been shown that the disappearance of the receptor can be followed during sheep reticulocyte matu- ration in vitro (Pan, Blostein, and Johnstone, 1983). The results presented here demonstrate that with the anti- transferrin-receptor antibody, it is possible to follow the redistribution and externalization of the sheep transferrin receptor in vesicular form during maturation in vitro. The released vesicles contain a peptide that is retained by an immunoaffinity column of the anti-transferrin-receptor anti- body and has a molecular weight characteristic of the transferrin receptor.

Under adverse metabolic conditions, red cells release vesicles from their surface (Allan et al., 1976; Lutz et al., 1977; Muller et al., 1981). It is unlikely that the vesicles obtained in the present work represent nonspecific mem- brane components released to the medium, since their protein composition is enriched in the transferrin receptor and their protein profile is not characteristic of that of reticulocyte plasma membranes (compare columns 2 and 4 in Figure 5). Moreover, vesicles released from red cells under adverse conditions contain the prominent red cell membrane proteins but little or no spectrin (Muller et al., 1981). We previously reported that it is difficult to distin- guish between the Coomassie-blue-stained protein profiles

Fate of Transferrin Receptor in Retrculocyte Maturation 973

Frgure 6. Vesicular Appearance of Released ‘251-Material from ‘?-Antibody-Labeled Reticulocytes

After centrifugation at 12,000 x g to remove the cells, the supernatant was filtered through a Sepharose 6B column. The vord volume was centrifuged at 100,ooO g for 1 hr. The pellet obtained was stained with 1% ammonium molybdate and examined under an electron microscope. Vesicles with surface knobs are seen. Magnification: 123,000~.

Table 4. Acrd Dissociation of ‘9.Labeled Antrbody on the Externalized Vesicles.

Acid Treatment Vord Volume Retarned Volume

- 2100 600

+ 700 2400

Values are counts per minute. ‘2sl-antibody-labeled retrculocytes were in- cubated at 37°C for 6 hr as in Table 2. The cells were removed by centrifugatron, and an aliquot of the suparnatant of the incubation medium was applied to a Sepharose 6B column (minus acid treatment). An identical sample of the supernatant was treated with 0.05 M glycine-buffered saline at pH 2.3 for IO min at 4°C neutralized with phosphate buffer, and then applred to the Sepharose 6B (plus acid treatment). The columns were eluted with phosphate-buffered saline (pH 7.4). and the eluates were counted. Two radioactive peaks were obtained, one rn the void volume and a retained portron which coeluted with free ‘?-antrbody. The radioactrvity associated wrth the void volume was considered “vescle bound.”

of plasma membranes from sheep erythrocytes and retic- ulocytes (Pan and Johnstone, 1980).

That the transferrin receptor is externalized in the form of a vesicle is supported by studies on the molecular size of the released material (exclusion by Sepharose 6B), appearance of the material in the electron microscope, banding in a sucrose gradient, and the ability to pellet the material after centrifugation at 100,000 x g.

Most of the studies reported here are based on the use of specific anti-transferrin-receptor antibodies. Parallel stud- ies show that neither antibody nor transferrin is required to obtain release of vesicles bearing the transferrin receptor. (In fact, studies in progress suggest that the antibody may slow the rate of release.) The data in Figure 1 show that vesicles isolated after incubation with the specific antibody contain a peptide corresponding to the heavy chain of IgG in addition to the 93 kd receptor peptide. If transferrin is added to the medium, a peptide with the molecular weight characteristic of sheep transferrin can also be recovered with the vesicles. These studies, therefore, suggest that during maturation of the sheep red cell, the transferrin receptor along with its ligands is physically lost, being released from the cell in vesicular form. This conclusion contrasts with an earlier suggestion that the receptor is “inactivated” but retained in the membrane during matura- tion (Leibman and Aisen, 1977).

There appears to be general agreement that the trans- ferrin-receptor complex undergoes internalization during iron delivery. There is also agreement that the receptor and transferrin are recycled back to the plasma membrane without degradation during many cycles of iron delivery (for review see Morgan, 1981; Karin and Mintz, 1981; Renswounde et al., 1982). The externalized vesicles de-

Cell 974

Figure 7. Redistrlbutron of Surface Fluorescence with Time of Incubation

Cells were incubated at 37’C in the standard culture medium with FITC- labeled anti-transferrin-receptor antibody: (a) Initial. (b) Incubation at 37OC, 30 min: (c) Incubation at 37°C 3 hr. Cells maintained at O°C for several hours marntained the distribution shown in (a).

scribed in this work could arise through exocytosis of internalized vesicular structures (Zweig, Tokuyasu, and Singer, 1981). Alternatively, they could arise from evagi- nations of the plasma membrane by a process independ-

ent of internalization. Although available data are not defin- itive enough to discriminate between these two possibili- ties, they support the possibility that the vesicles result from evaginations of the plasma membrane. This tentative conclusion is based primarily on electron microscopic evidence showing evagination of vesicles (unpublished observations). Also, vesicle formation appears to be inde- pendent of the presence of transferrin, which might have been expected to increase the rate of receptor recycling (Ciechanover et al., 1983). The data support the conclusion that externalization of vesicles is part of the maturation process, since all the detectable receptor is eventually removed from the cell and recovered in vesicles (Table I), in the time required for maturation (Pan, Blostein, and Johnstone, 1983). Kinetic studies using ?antibodies and similar studies using FITC-antibodies show that release of antibody-containing vesicles is a temperature-sensitive process (Figure 8). The rate of externalization decreases with time and does not appear to follow simple first order kinetics. It is not yet known what other factors affect externalization,

That the vesicles are enriched in the receptor relative to other membrane proteins suggests that there must be some kind of “segregation” of the receptor prior to its release from the cell. Such segregation is not evident with our immunofluorescent techniques, since in the absence of the anti-transferrin-receptor antibody, the receptors ap- pear to remain largely uniformly distributed on the cell surface.

In presence of antibody a different picture is obtained. At 37°C after addition of the anti-transferrin-receptor anti- body, the antibody-receptor complex starts to redistribute into visible patches or caps in a manner analogous to that reported with antibodies (or other ligands) in lymphocytes and other cells (Schreiner and Unanue, 1976; Salisbury, Condeelis, and Satir, 1980; Goldstein, Anderson, and Brown, 1979; Pastan and Willingham, 1981; Singer et al., 1978).

It has been proposed that lateral mobility of surface receptors in reticulocytes is limited to certain mobile do- mains in the membrane, while other areas of the membrane are immobile (Tokuyasu, Schekman, and Singer, 1979; Zweig and Singer, 1979). The present data suggest that there is substantial lateral mobility of the transferrin recep- tors in sheep reticulocyte plasma membranes.

The erythrocyte plasma membrane is believed to con- nect with the underlying cytoskeletal system through the integral (transmembrane) proteins (for review see Branton et al., 1981). Previous studies showed that the transferrin receptor spans the membrane (Pan, Blostein, Johnstone, 1983). Prior to externalization and membrane fusion, the integral membrane proteins, such as the transferrin recep- tor, must dissociate from the underlying network, and the released vesicles should be free from spectrin. The results obtained are consistent with this prediction, since no spec- trin is detected after gel electrophoresis of the released vesicles. Previous reports have also shown that membrane vesicles released from erythrocytes with increased intra- cellular Ca” (Allan et al., 1976) or ATP depletion (Lutz et

;$ of Transfernn Receptor in Retrculocyte Maturation

Figure 8. Kinetics and Metabolic Requirements for Vesicle Release

Conditions for culturing were described in Figure 3, except that, where indicated, phosphate-buff-

A Cont ro I ered saline (pH 7.4) or serum-free culture medium replaced the control, serum-containing culture me-

l Serum Free dium. The incubatron was at 37°C or at O°C. After Incubation. cells were removed by centrifugation at 12,000 x g, and the supernatant was passed through a Sepharose 6B column. The void volume contarning vesicles was counted. Percentage of extemaltzation was determined from the following relationshrp:

aPBS

00 oc

cpm in void volume

cpm in cell pellet + cpm in void volume x loo.

Table 5. Effects of Incubation with Antibody on Intracellular ATP and K+ Table 6. Dissociation of Cell-Bound Radroactivity by Acid-Glycine-buffered Levels Saline

ATP (mM) Cellular K+ (mM)

Time Tested (hr) +Antibody - Antibody + Antibody - Antibody

Total Cell-Bound ‘251-Antibody

0 2.0 2.0 115 121

1 1.4 1.5 121 121

3 1.3 1.3 121 127

6 1.1 1.1 115 121

21 1 .o 1.1 109 115

A 2% suspension of sheep reticulocytes in phosphate-buffered saline (pH 7.4) was incubated with punfred antibody (-1 pg/ml) at 0°C for 90 min. The free antibody was removed by filtratron as described in Experimental Procedures. The cells were resuspended to a 1% suspension in culture medrum containing 2% fetal calf serum and incubated at 37’C. At intervals, samples were taken and analyzed for ATP and K+ as described rn Experimental Procedures. The K’ and ATP concentrations are expressed per liter of cell water.

al., 1977); vesicles endocytosed in erythrocyte ghosts (Hardy and Schrier, 1978; Hardy, Bensch, and Schrier, 1979); and vesicles internalized in reticulocytes after ex- posure to concanavalin A (Tokuyasu, Schekman, and Singer, 1979) are all deficient in spectrin. All these obser- vations suggest that prior to externalization or internaliza- tion, the plasma membrane becomes detached from the cytoskeleton at the site of vesicle formation.

Before Acid After Acid Treatment Treatment Acid-inaccessible

Time of Incubation (hr) (cpm) (cm) ‘?-Antibody (%)

0 (freshly isolated) 12.500 782 6

0.5 10,030 I.500 15

1.0 9,070 1,530 17

3.0 7,030 I.150 16

6.0 5,030 900 12

14.0 3,330 520 16

21 .o 2,056 284 14

The cells, prelabeled at 0°C with “?-labeled anti-transferrin-receptor antr- body, were incubated at 37’C. Samples were taken at intervals and centrifuged at 12,000 x g for 1 min. The cell pellets obtained were resuspended in 0.4 ml phosphate-buffered saline at 0°C and an aliquot counted (cell-bound ‘“l-antibody). An additional 0.4 ml of ice-cold 0.2 M glycine-buffered saline (pH 2.3) was added. The sample was layered on 12 ml of 5% sucrose, dissolved in 0.1 M glycrne-buffered saline (pH 2.3) and centrifuged at 4°C at 5,860 x g for IO min, and the cell pellets obtarned were counted. The dissociable radioactivity (‘?free antibody) is the differ- ence between the cell-bound radioactrvity before and after centrifugation in the acid buffer. Control experiments show between 5% and 10% lysrs upon acid treatment, as measured by hemoglobin release.

In addition to externalization, evidence for internalization of the anti-transferrin receptor-antibody complex has also been presented. The acid dissociation experiments (Table 6) suggest that only a small percentage of ‘251-antibody is internalized at 37°C. However, since 5%-10% lysis of cells was obtained during the acid treatment, the extent of internalization may be underestimated. Nonetheless, the gradual decrease of the absolute amount of cell-bound, acid-undissociable radioactivity suggests that the internal- ized radioactivity eventually becomes surface bound, In

the present experiments, only 2% of the initial radioactivity remained undissociable after 21 hr of incubation at 37°C.

In conclusion, the data show that cultivation of sheep reticulocytes in vitro results in a loss of a transmembrane protein, the transferrin receptor, as a small extracellular vesicle. This loss is selective, since few other membrane proteins are detectable in the vesicle. Although the pres- ence of the anti-transferrin-receptor antibody in the incu- bation medium causes redistribution and patching of the receptor on the cell surface, these activities do not seem

Cell 976

to be associated with vesicle formation, a process that occurs in absence of the antibody. It is proposed that the release of the transferrin receptor in vesicular form repre- sents the normal process for ridding the cell of this receptor during red cell maturation.

Experimental Procedures

Isolation of Sheep Reticulccytes, Plasma Transfenin, and Transferrtn Receptor Reticulocyte production in sheep was induced by phlebotomy as described (Benderoff. Blostein, and Johnstone, 1976; Benderoff, Johnstone. and Blostein, 1976). For the isolation of reticulocytes, a modified differential centrifugation method was used (Benderoff, Blostein, and Johnstone, 1976; Pan, Blostein, and Johnstone, 1963).

Plasma transferrin was isolated by methods described by Morgan et al., (Morgan, 1964; Morgan, Huebers, and Finch, 1976) with the modification that before gel filtration the transferrin was fractionated with 50%-75% saturated ammonium sulfate.

The transferrin receptor was isolated by a transferrin affinity column using purified sheep plasma transferrin coupled to CNBr-Sepharose 48 (Axen, Porath, and Ernback, 1967; Pan, Blostein, and Johnstone, 1963) (see below).

Antibody Preparation Anti-transferrin-receptor antibody was isolated as previously described from sera of rabbits immunized against sheep reticulocytes (Pan, Blostein, and Johnstone, 1963). Sheep reticulocytes were used to absorb out reticulocyte- specific antibodies, which were further purified using protein A affinity columns (Ey, Prowse, and Jenkrn. 1976).

lodination of the Purified Antibodies lxfination of the purified antibodies was carried out with chloramine T (Hunter and Greenwood, 1962). The iodinated protein was separated from free ‘=I by filtration on Sephadex G-25.

lodinated Antibody Binding Reticulocytes (2% suspension) in phosphate-buffered saline, pH 7.4, were incubated with 0.25 to 1 .O pg/ml ‘“l-antibody (specific activity 1-4 x 105 cmp/Ag) at 0°C for 90 min. The cell-bound antibody was separated from free antibody by filtration at 4’C through a Sepharose 6B column of 1.5 x 7 cm or as Indicated in the legends to figures The cells were eluted in the void volume using phosphate-buffered saline as the elution medium.

Determination of ‘%Materials Released into the Cube Medium ‘q-antibody-labeled reticulocytes (1% cell suspension) were incubated at 37°C in Earle’s minimal essential medium with 2 mM glutamine, 200 U/ml of penicillin, and 0.02 pg/ml of streptomycin, with 2% fetal calf serum in 95% 02/5% CO*. At intervals samples were centrifuged at 12,000 x g for 1 min in an Eppendorf centrifuge to pellet the cells, and both the superna- tants and the cell pellets were counted. To identify and isolate the radio- activity released into the culture medium, cell-free supernatants (25-75 ml) from 6 hr cultures were applied to columns of protein A-Sepharose 48, and the columns were washed with phosphate-buffered saline, pH 7.4, until no further radioactivity was eluted (3-4 bed volumes). The washing medium was then changed to 0.1 M glycine in 0.15 M saline, pH 3.0 (elution buffer), and the eluate was collected, neutralized with phosphate buffer, and counted. The fraction of radioactivity retained by the protein A column which was recovered with elution buffer was 60%~80% of the radioactrvity applied. Before SDS gel electrophoresis, this eluate was dialyzed against drstilled H,O for 24 hr with two to three changes of Hz0 and lyophilized.

lmmunofluorescence Studies FITC-labeled antibody was prepared as previously described (The and Feltkamp, 1970). To label the reticulocytes, a 2% suspension of the cells in phosphate-buffered saline (pH 7.4) was Incubated with 5 ag/ml of FITC- antibody at 0°C for 90 min. centrifuged, and washed twrce at 4’C with phosphate-buffered saline (pH 7.4). The washed reticulocytes were resus-

pended to give a 1% cell suspension and incubated as described at 37°C. Control samples were taken before incubation at 37°C. In addition, a control suspension was fixed in 2% paraformaldehyde at room temperature for 3 min before treatment with FITC-antibody. The film used for photography was Kodacolor ASA 400, and the exposure time was 5 min.

Electron Microscopic Studies Negative Sfaining The cell-free, postincubation medium derived from incubation of antibody- labeled reticulocytes (‘251-antibody or FITC-antibody) was centrifuged at 1 OQOCO X g for 1 hr. The pellet obtained was stained with 1% ammonium molybdate and examined under a Philips 300 electron microscope. In experiments where the cell-free supernatant was first passed through a protein A affinity column, the acid eluate from the protein A column was Immediately neutralized and centrifuged to obtain a pellet for staining.

Dissociation of ‘“l-Radioactivity from the Cell Surface During the in vitro culture period, cells containing bound ‘?antibody were suspended with elution buffer (see above), layered on 5% sucrose in elution buffer, and centrifuged at 12,000 x g at 4°C. It was assumed that only surface-bound antibody would be drssociated by the acid-buffered medium.

Surface Labeling of Reticulccytes with 9 to Follow the Fate of the Receptor The procedure described by Reichstein and Blostein (1975) was used to surface label the cells. Transferrin-depleted cells were used (Hemmaplardh and Morgan, 1974). When ‘251-labeled cells were incubated, the postincu- bation medrum was passed through an antr-transferrin-receptor column and washed with phosphate-buffered saline, pH 7.4, untrl no radioactivity was released. Then the retained radioactivrty was eluted with 0.1 M glycine- buffered saline, pH 2.3. The acrd eluates were counted. The radioactivity assocrated with the cell membranes was processed as described (Pan, Blostein, and Johnstone, 1963).

ATP and K* Determination ATP was determined using the firefly lucrferase procedure (Stanley and Williams, 1969). Cellular K+ was analyzed by flame photometry using an internal Li+ standard.

Protein Determination The method of Lowry et al. (1951) was used

Electrophoresis The system of Laemmli (1970) was used with 6% running gel.

Materials Na ‘? was purchased from Frosst Company; fluorescern isothiocyanate (isomer 1) and firefly extracts were purchased from Srgma; CNBr-Sephar- ose 48, protein A-Sepharose 48, Sephadex G-25, G-150, and Sepharose 6B were purchased from Pharmacia. Culture medium and fetal calf serum were purchased from Grand Island Biolcgicals.

Acknowledgments

This work was supported by grants from the Medical Research Council of Canada, The Department of Educatron of the Province of Quebec, and the Banting Research Foundation of Canada. B. T. P. was the recipient of student scholarships from the Graduate and Medical Faculties of McGill University during the course of this work.

Our thanks are due to Anoush Cotchikian and Claire Turbide for technical assistance in pans of this work. We are grateful to Dr. A. Fuks. McGill Cancer Center, for advice and help in immunological procedures, and to Kathy Teng and Donald D’Shaughnessy for the electron microscopy.

The costs of publication of thus article were defrayed in pan by the payment of page charges. Thus article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate thus fact.

Recerved October 26, 1982; revised April 20, 1963

Fate of Transferrin Receptor in Reticulocyte Maturation 977

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