Proteolytic cleavage of 13H]nitrobenzylthioinosine-labelied nucleoside

Biochem. J. (1985) 230, 777-784

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Proteolytic cleavage of 13H]nitrobenzylthioinosine-labelied nucleoside transporter in human erythrocytes N. Sultana JANMOHAMED,* James D. YOUNGt and Simon M. JARVIS*T *Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7, and tDepartment of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong

(Received 29 April 1985; accepted 28 May 1985) The transmembrane topology of the nucleoside transporter of human erythrocytes, which had been covalently photolabelled with [3H]nitrobenzylthioinosine, was investigated by monitoring the effect of proteinases applied to intact erythrocytes and unsealed membrane preparations. Treatment of unsealed membranes with low concentrations of trypsin and chymotrypsin at 1°C cleaved the nucleoside transporter, a band 4.5 polypeptide, apparent Mr 66000-45000, to yield two radioactive fragments with apparent Mr 38 000 and 23 000. The fragment of M, 38 000, in contrast to the Mr 23 000 fragment, migrated as a broad peak (apparent Mr 45 00031 000) suggesting that carbohydrate was probably attached to this fragment. Similar treatment of intact cells under iso-osmotic saline conditions at 1°C had no effect on the apparent Mr of the [3H]nitrobenzylthioinosine-labelled band 4.5, suggesting that at least one of the trypsin cleavage sites resulting in the apparent M, fragments of 38000 and 23000 is located at the cytoplasmic surface. However, at low ionic strengths the extracellular region of the nucleoside transporter is susceptible to trypsin proteolysis, indicating that the transporter is a transmembrane protein. In contrast, the extracellular region of the [3H]cytochalasin B-labelled glucose carrier, another band 4.5 polypeptide, was resistant to trypsin digestion. Proteolysis of the glucose transporter at the cytoplasmic surface generated a radiolabelled fragment of Mr 19000 which was distinct from the M, 23000 fragment radiolabelled with [3H]nitrobenzylthioinosine. The affinity for the reversible binding of [3H]cytochalasin B and [3H]nitrobenzylthioinosine to the glucose and nucleoside transporters, respectively, was lowered 2-3-fold following trypsin treatment of unsealed membranes, but the maximum number of inhibitor binding sites was unaffected despite the cleavage of band 4.5 to lower-Mr fragments.

Entry of nucleosides into human erythrocytes is mediated largely by a broad-specificity facilitated diffusion system inhibitable by nanomolar concentrations of nitrobenzylthioinosine (NBMPR) (Plagemann & Wohlhueter, 1980; Paterson et al. 1981; Young & Jarvis, 1983). NBMPR inhibition of nucleoside transport by human erythrocytes is competitive with respect to substrate concentration (Eilam & Cabantchik, 1977; Roger-Brown & Abbreviations used: N B M PR, nitrobenzylthioinosine

(6 -[(4- nitrobenzyl)thio]9 - ,B- D- ribofuranosylpurine};

SDS, sodium dodecyl sulphate; PMSF, phenylmethanesulphonyl fluoride. I To whom requests for reprints should be addressed.

Vol. 230

Parks, 1980) and associated with high-affinity binding of inhibitor to the transporter (apparent Kd 0.1-1.0nM) (Cass et al.,, 1974; Jarvis & Young, 1980). There is a strict proportionality between occupancy of these binding sites and the degree of transport inhibition (Cass et al., 1974). Highaffinity NBMPR binding is competitively blocked by transported nucleosides such as uridine, adenosine and deoxycytidine (Cass & Paterson, 1976; Jarvis et al., 1982, 1983). These data suggest that high-affinity NBMPR binding to human erythrocyte membranes represents interaction with an integral component of the nucleoside transporter. Additional evidence comes from studies of transporter-deficient cells. Most sheep possess erythro-

778 cytes that lack a functional nucleoside carrier and do not bind NBMPR, whereas erythrocytes from some sheep transport nucleosides and bind inhibitor (Young, 1978; Jarvis & Young, 1980). Employing an assay based upon reversible NBMPR binding activity, we demonstrated that it is possible to extract with Triton X-100 and partially purify the nucleoside transport protein (Jarvis & Young, 1981). The final partially purified preparation consisted mainly (>95%) of band 4.5 and a small amount of band 7. This preparation is capable of catalysing NBMPR-sensitive uridine transport when reconstituted into phospholipid liposomes (Tse et al., 1985). These studies raised the possibility that band 4.5 polypeptides are involved in nucleoside permeation. Further evidence to implicate band 4.5 proteins as the human erythrocyte nucleoside transporter has been provided by recent photoaffinity labelling studies with [3H]NBMPR. Exposure of erythrocyte membranes or intact cells to u.v. light in the presence of [3H]NBMPR and dithiothreitol results in the selective covalent labelling of membrane polypeptides which migrate as a single broad band on SDS/polyacrylamide gels with apparent Mr of 66000-45000, which corresponds to the band 4.5 region (Wu et al. 1983a,b). In the present paper, [3H]NBMPR covalently labelled erythrocyte membranes were subject to controlled proteolysis as a means to explore the topography in situ of the nucleoside transporter. The results obtained are compared with parallel trypsin cleavage studies of the cytochalasin B-labelled glucose transporter, another band 4.5 polypeptide.

Experimental Materials [G-3H]NBMPR (specific radioactivity 16Ci/ mmol and > 98% radiochemically pure) and [4(n)-3H]cytochalasin B (specific radioactivity 10.3Ci/mmol and >98% radiochemically pure) were purchased from Moravek Biochemicals, Brea, CA, U.S.A. and Amersham, Oakville, Ontario, respectively. NBMPR and NBTGR were generous gifts from Professor A. R. P. Paterson, University of Alberta Cancer Research Group. Electrophoresis reagents were obtained from BioRad Laboratories, Mississauga, Ontario, Canada. Trypsin (type III), a-chymotrypsin (type II), soybean trypsin inhibitor (type 1-S) and all other reagents were obtained from Sigma Chemical Co, St. Louis, MO, U.S.A. Cells and membrane preparation Fresh human blood was collected into heparinized tubes and the erythrocytes were washed three times with a medium containing 140mM-NaCl,

N. S. Janmohamed, J. D. Young and S. M. Jarvis

5 mM-KCl, 20mM-Tris/HCl (pH 7.4 at 220C), 2mM-MgCl2 and 0.1 mM-EDTA (disodium salt). The buffy coat was discarded. Haemoglobin-free erythrocyte membranes ('ghosts') and membranes depleted of peripheral proteins by treatment with 0.1 mM-EDTA (pH 11.2) were prepared as previously described (Jarvis & Young, 1980, 1981). Membranes were resuspended in 5 mM-sodium phosphate (pH7.9 at 220C).

Photoaffinity labelling with [3H]NBMPR and [3H]cytochalasin B Intact cells and freshly prepared erythrocyte membranes were photolabelled with [3H]NBMPR and [3H]cytochalasin B as described by Wu et al. (1983a) and Carter-Su et al. (1982), respectively. Non-covalently bound radioactivity was removed by washing the membrane suspensions or intact cells three times.

Trypsin treatment of red cell membranes and intact cells Photolabelled membranes resuspended in 5 mMsodium phosphate (pH 7.9) at 1 packed cell equivalent were subjected to trypsin and chymotrypsin treatment (1-100Mg/ml packed cell equivalent) at 1°C for 15min, unless noted otherwise. Proteolysis was stopped by the addition of 10 vol. of buffer containing 300!uM-PMSF followed by centrifugation at 38000g for 20min. The pellets were washed once more and then dissolved in gel sample buffer. In some instances, trypsin was inhibited by the addition of soybean trypsin inhibitor at twice the concentration of trypsin. Photolabelled intact erythrocytes were washed three times with 20 vol. of either 150mMNaCl/5mM-sodium phosphate (pH7.9) or 5mMsodium phosphate (pH7.9) in 0.3M-sucrose and then incubated at 1°C at 30% haematocrit with trypsin (3-3000yg/ml) for 15min, unless noted otherwise. Following digestion, the intact cells were washed three times with the NaCl/phosphate buffer containing 300pM-PMSF to remove trypsin. Erythrocyte ghosts were prepared from the digested cells as previously described except that the lysis buffer contained 100,uM-PMSF.

Nucleoside transport Initial rates of uridine influx were determined as previously described (Jarvis et al., 1980) using NBMPR as a transport 'stopper'. NBMPR and cytochalasin B binding Reversible NBMPR binding to erythrocyte membranes and intact cells were determined at 22°C by the method of Jarvis & Young (1980). Cytochalasin B binding to protein-depleted erythrocyte membranes in 50mM-Tris/HCl (pH7.4 1985

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Proteolytic cleavage of the nucleoside transporter at 22°C) was assayed by a centrifugation method using a Beckman Airfuge. Briefly, membrane suspensions (40 jg of protein) were incubated with [3H]cytochalasin B (initial concentration 0.031.OM) in the presence of either 0.6M-D-sorbitol or -D-glucose. Incubations were terminated 30min later by centrifugation at 130000g for 10min. Portions of the supernatant (200 p1) were retained for radioactivity determinations and the remaining supernatant was removed before assaying the membrane pellet for 3H activity by liquid scintilla-

tion counting.

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Other methods Following labelling and proteolytic digestion, samples were analysed by SDS/polyacrylamide gel electrophoresis by the procedure of Laemmli (1970) using 12% acrylamide gels. The gel lanes were cut into 2mm slices and the 3H content of these slices was measured by liquid scintillation counting in Econofluor containing 3% (v/v) Protosol (New England Nuclear) as previously described (Wu et al., 1983a). Replicate sample lanes from the same slab gels were stained with Coomassie Blue and scanned at 633nm using an LKB laser densitometer. Protein was measured by the procedure of Lowry et al. (1951).

Results Digestions of unsealed [3H]NBMPR-labelled erythrocyte ghosts with low concentrations of trypsin (1.5-7 ug/ml packed cell equivalent, 15 min at 1°C) generated two band 4.5 radioactive fragments with apparent M, values of 38000 and 23000 (Fig. 1). The magnitude of the M, 23000 fragment increased as the concentration of trypsin increased and there was a corresponding decrease in radioactivity associated with the M, 38000 polypeptide (see also Table 1). These results suggest that the lower-Mr fragment may be derived from the M, 38 000 fragment. The time dependence of the appearance of the Mr 23 000 labelled band is also consistent with this conclusion. It is evident from Table 1 that the labelled band of Mr 23000 increased with time while the original labelled band 4.5 (Mr 66000-45000) decreased. The band of Mr 38000 was seen as a transient intermediate. The Mr 38000 fragment, in contrast to the Mr 23000 fragment, migrated as a broad peak (Mr 45000-31000) indicating that carbohydrate was probably attached to this fragment. Trypsin digestion of photolabelled proteindepleted membranes gave similar radioactive profiles to those obtained with unsealed ghosts (Fig. 2). Further control experiments established that the trypsin digestion profile (22°C for 1 h) was unaffected by the buffer composition [5 mMVol. 230

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Migration (cm) a ab

Trjptic digestion of the [3H]NBMPR-labelled nucleoside transporter in unsealed ghosts Ghosts were photolabelled with 25 nM[3H]NBMPR, washed three times with buffer to remove unreacted [3H]NBMPR and then treated with trypsin for 15min at 1°C as described under 'Experimental'. Initial concentrations of trypsin (jug/ml packed cell equivalent) 0, 0; 0, 1.5; A, 7. The trypsin-treated membranes (lOOpg) were electrophoresed on 12% acrylamide gels according to Laemmli (I1970). The positions of the stacking gelrunning gel interface and the tracking dye are indicated by a and b, respectively; Mr ( x 10-3) iS indicated at the top of the Figure.

Fig. 1.

sodium phosphate, pH 7.9, compared with either 50mM-sodium phosphate (pH 7.5)/100mMNaCl/l mM-EDTA or l50mM-NaCl/5 mM-sodium phosphate (pH 7.0) (results not shown)]. Fig. 2 also demonstrates that chymotrypsin pretreated with soybean trypsin inhibitor produced a similar cleavage pattern to that of trypsin. Interestingly, the maximum number of reversible high-affinity NBMPR binding sites in protein-depleted membranes was not affected by trypsin treatment at 1Ojug/ml packed cell equivalent despite the apparent cleavage of most (>90%) of the [3H]NBMPRlabelled band 4.5 to smaller membrane fragments (7090 sites/cell in the absence of trypsin and 7460 sites/cell after trypsin incubation; mean of two experiments). In contrast, the apparent Kd value for [3H]NBMPR binding increased by two-fold following trypsin treatment (0.46 and 1 .OnM before and after trypsin incubation, respectively; mean of two experiments). The possibility that the observed sensitivity of the [3H]NBMPR-labelled

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N. S. Janmohamed, J. D. Young and S. M. Jarvis

Table 1. Concentration-dependence and time course of the ejject of trypsin on the distribution of [3H]NBMPR in membrane peptides ajter typsin treatment Protein-depleted membranes were photolabelled with [3H]NBMPR (25nM) and subjected to trypsin treatment as described under 'Experimental'. Proteolysis was stopped by the addition of trypsin inhibitor at twice the concentration of trypsin. The amount of radioactivity associated with the various membrane polypeptides was determined by SDS/polyacrylamide-gel electrophoresis. 3H incorporation into membrane peptides (% of total) Test conditions

Control Trypsin, 15min, 1°C

1 pg

5pg

10 pg 25 pg Trypsin, 5 pg, I C 2min 10min 15 min

Slices 1 1-21 (Band 4.5)

Slices 22-30 (Mr 38000 fragment)

Slices 31-40 (Mr 23000 fragment)

82.7

17.0

0.3

25.9 8.3 4.4 5.0

48.4 37.7 28.0 32.1

25.7 54.0 67.6 62.9

28.6 16.9 11.3

49.0 47.7 44.9

22.4 35.2 43.8

band 4.5 to trypsin was due to the exposure of trypsin-sensitive sites on the polypeptide following photolabelling was tested by first trypsin-treating protein-depleted membranes prior to photolysis with [3H]NBMPR. The radioactive profiles of the trypsin-treated membranes were identical with those obtained in Fig. 2, indicating that the behaviour of the [3H]NBMPR covalent-labelled band 4.5 is similar to that of the unlabelled band 4.5 protein. To determine if the proteolytic digestion of [3H]NBMPR-labelled band 4.5 in unsealed erythrocyte membranes was due to cleavage of peptide bonds accessible to the extracellular surface of the membrane, trypsin digestion of intact cells photolabelled with [3H]NBMPR was performed. Trypsin concentrations which digested [3H]NBMPRlabelled band 4.5 polypeptides in unsealed membranes at 1°C had no effect on the labelled band 4.5 proteins of intact cells suspended in phosphate-buffered saline at 1°C (Fig. 3). However, a higher concentration of trypsin (lOOpg/ml of cells, 30% haematocrit, 15 min at 1°C) yielded a radioactive fragment(s) which migrated as a broad peak (average M, 22500). This radioactive profile was different from that obtained with unsealed ghosts treated with low concentrations of trypsin. The possibility that this high concentration of trypsin was affecting the structure of the red cell membrane in a non-specific manner was examined by comparing the Coomassie Blue-stained protein profile of control and trypsin-treated cells. No difference in staining profiles were observed. Further experiments demonstrated that [3H]NBMPR-labelled band 4.5 polypeptides in intact cells suspended in phosphate-buffered saline were

digested with low concentrations of trypsin when both the incubation time and temperature were increased. For example, treatment of intact cells (30% haematocrit) with IO pg of trypsin/ml at 22°C for 60min yielded a similar radioactive pattern to that obtained after 15min at 1°C with lOOpg of trypsin/ml (results not shown). Exposure of intact cells to trypsin at low ionic strength (300 mMsucrose in 5mM-sodium phosphate buffer, pH7.9) markedly increased the sensitivity of the labelled band 4.5 to digestion by the enzyme. Trypsin concentrations as low as pg/ml of cells (30% haematocrit) at 1°C completely cleaved the labelled band 4.5 component to give a radioactive product of apparent M, 23000 (results not shown). This product had the same mobility as that obtained with intact cells at high concentrations of trypsin under iso-osmotic saline conditions (see Fig. 3). The Coomassie Blue-stained protein profile of trypsin-treated cells under low ionic strength conditions indicated that band 3, the anion carrier, had been partially digested. This result was consistent with a previous report (Jenkins & Tanner, 1977) that had demonstrated that the extracellular region of band 3 is resistant to trypsin when digestion is performed under isoosmotic ionic strength conditions but becomes susceptible to trypsin proteolysis under low ionic strength iso-osmotic conditions. Fig. 4 demonstrates that treatment of intact cells suspended in phosphate-buffered saline with high concentrations of trypsin (lOOpg/ml of cells, 30%O haematocrit) had no effect on the saturable highaffinity component of reversible NBMPR binding (apparent Kd 0.29nM for both; maximum number of NBMPR binding sites 10900 and 10200

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Proteolytic cleavage of the nucleoside transporter

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