Jan 9, 1984 - Gornall, A. G., C. S. Bardawill, and M. M. David. ... Henderson, G. B., E. M. Zevely, and F. M. Huennekens. ... Shane, B., and E. L. R. Stokstad.
JOURNAL OF BACTERIOLOGY, Apr. 1984, p. 202-207
Vol. 158, No. 1
0021-9193/84/040202-06$02.00/0 Copyright C 1984, American Society for Microbiology
Structural and Functional Properties of the Folate Transport Protein from a Methotrexate-Resistant Subline of Lactobacillus caseit M. ANANTHANARAYANAN, JUDY M. KOJIMA, AND GARY B. HENDERSON* Division of Biochemistry, Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037 Received 28 November 1983/Accepted 9 January 1984
A methotrexate-resistant subline of Lactobacillus casei has been isolated which transports folate at a reduced rate and contains a binding protein whose affinity for folate (Kd = 280 nM) is considerably lower than that of the corresponding protein of wild-type cells (Kd = 0.6 nM). After the addition of mercaptoethanol, however, this same protein exhibits a high affinity for folate (Kd = 1.2 nM) and transports the substrate at a normal rate. Subsequent removal of mercaptoethanol causes a rapid reversal of the activation process. Binding protein labeled covalently with carbodiimide-activated [3H]folate, solubilized with Triton X-100, and subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate had an apparent molecular weight which was approximately twofold higher than that of the corresponding protein of wild-type cells, but it could be reduced to the parental size (M, = 20,000) by prior treatment with mercaptoethanol. Purified binding protein also exhibited a similarly elevated molecular weight, and its amino acid composition was indistinguishable from that of the wild-type counterpart, except for the presence of a single cysteine residue. These findings indicate that the mutant binding protein exists in a low-affinity form due to disulfide bridge formation between two homologous protein subunits and that cleavage of this bond by mercaptoethanol generates the high-affinity state. The rapid and specific interconversion of these binding forms suggests further that the high-affinity form of the binding protein also resides in the membrane as a dimer, held together by noncovalent interactions.
The transport of folate compounds in Lactobacillus casei proceeds via a single high-affinity transport system (4, 14, 19). The uptake process is active and dependent on an energy source, and folate can be accumulated to intracellular concentrations that are several-thousand-fold higher than external levels (11). The membrane-associated binding protein which mediates folate transport is present in large amounts per cell, exhibits a very high affinity for folate, and has been purified to homogeneity (8, 9, 13). Cations are required to achieve the high-affinity state of the binding protein, suggesting that transport may proceed via a folatecation symport mechanism (5), and an affinity labeling agent has been developed which specifically reacts with the folatebinding site (6). In addition to the binding protein, a second cellular component is required for the transport of folate, as well as other vitamins (10). Various transport mutants have been isolated and used in comparative studies to show that the membrane-associated folate-binding protein of these cells is a component of the folate transport system (13). One of the later cell lines is resistant to methotrexate and has a binding protein and transport system whose activities are enhanced in parallel by mercaptoethanol. In the present study, additional information on the defect in the mutant binding protein is reported. The results indicate that the low binding affinity of this protein for folate is due to dimer formation between two identical protein subunits, attached via a disulfide bridge. Cleavage of this disulfide bridge by mercaptoethanol converts the binding protein to a high-affinity form.
MATERIALS AND METHODS Radiolabeled vitamins. [3',5',7,9-3H]folate (500 mCi/ mmol), [3',5',7-3H]methotrexate (250 mCi/mmol), and [35S]thiamine hydrochloride (189 mCi/mmol) were obtained from Amersham Corp. [3H]folate and [3H]methotrexate were both purified by thin-layer chromatography (5) and diluted to a specific activity of 250,000 dpm/nmol before use. Radioisotope solutions contained 10% ethanol (to impair radiochemical breakdown of the substrates) and were stored at -20°C. Growth of cells. L. casei rhamnosis (ATCC 7469) and the methotrexate-resistant subline were grown for 16 h at 30°C in the medium described by Flynn et al. (1) containing 20 g of vitamin-free casein hydrolysate (ICN Pharmaceuticals Inc.) per liter and 5 nM folate. For thiamine binding and transport determinations, cells were grown with high levels of folate (5 ,uM) but without added thiamine. Preparation of EDC-activated folate compounds. EDC [1ethyl-3(3-dimethylaminopropyl)-carbodiimide]-activated folate and methotrexate were prepared as described previously (6, 12) by combining 4 ,umol of either folic acid or methotrexate (free acid) and 40 ,umol of EDC in 2.0 ml of anhydrous dimethyl sulfoxide and incubating the mixtures for 1 h at 23°C. Dilutions of the reagents were performed in dimethyl sulfoxide containing 2 mM EDC. EDC-activated [3HJfolate was prepared in a similar manner, except that the commercially supplied potassium salt of [3H]folate was first converted to the acid form by dissolving it in 0.1 N HCl and evaporating the solution to dryness. Pretreatment of cells. Freshly grown cells were washed with 100 volumes of 50 mM potassium HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-5 mM MgCl2 (pH 7.5) (HEPES-MgC12 buffer), resuspended to a density of 8 x
* Corresponding author. t Manuscript no. 3243-BCR from the Research Institute of Scripps Clinic.
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VOL. 158, 1984
108/ml, and then either used directly (for transport measurements) or depleted of energy reserves (for binding determinations) by incubation for 60 min at 23°C (5, 11). Treatment with reducing agents was performed by adding the desired reductant and incubating the samples for 5 min at 30°C. Folate binding or transport measurements were then performed directly. Treatment with EDC-activated folate compounds was performed after incubation with reagent for 60 min at 4°C. Excess reagent was then removed by collecting the cells by centrifugation at 24,000 x g for 5 min at 4°C and washing with 4 ml of buffer. Binding and transport determinations. Folate- and methotrexate-binding activity was determined as described previously (5), except that measurements were performed in HEPES-MgCl2 buffer (pH 7.5). Centrifugation at 24,000 x g for 5 min at 4°C was used to collect the cells before analysis for associated radioactivity. Thiamine binding was performed by the same procedure, except that the cells were recovered by filtration on 0.3-,um nitrocellulose filters. Transport of folate (11) and thiamine (7) was measured, as described previously, in cells that had been suspended in HEPES-MgCl2 buffer (final volume, 2.0 ml) and preincubated for 2 min at 30°C with 5 mM glucose before the addition of labeled substrate (2.0 ,uM). Radioactivity was measured in Cytoscint (WestChem Products). Binding and transport results are expressed in nanomoles bound per 1010 cells and nanomoles transported per min per 1010 cells, respectively. Analysis of binding protein in cells labeled with EDCactivated [3H]folate. Wild-type and methotrexate-resistant cells (in 50 ml of 5 mM potassium HEPES-150 mM KCl [pH 7.8]) were treated for one h at 4°C with sufficient activated [3H]folate (1.2 x 106 dpm/nmol) to quantitatively label the binding protein and then lysed by probe sonication for 7 min at 4°C. After centrifugation at 32,000 x g for 5 min at 4°C, the membrane fraction was retained, and the labeled binding protein was extracted by suspending the pellet in 2 ml of 50 mM HEPES (pH 7.5) containing 2% Triton, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride and then incubating the mixture for 30 min at 23°C. After centrifugation, the supernatant fraction was combined with 2 ml of acetone and allowed to stand overnight at -20°C. The resulting precipitate was then recovered by centrifugation, dissolved in sample buffer (62.5 mM Tris-hydrochloride [pH 6.8] containing 2% sodium dodecyl sulfate [SDS] and 10% glycerol), heated at 80°C for 3 min, and subjected to SDS-polyacrylamide gel electrophoresis by the procedure of Laemmli (16). Gels were stained with 0.1% Coomassie blue for 16 h at 23°C and destained with 7% acetic acid. For autoradiography, destained gels were placed in water to remove the acetic acid, treated for 1 h at 23°C with a tritium-enhancing agent (Autofluor; National Diagnostics), dried, and exposed to Xray film (BB-1; Eastman Kodak Co.) for 8 days at -70°C. Isolation of 3H-labeled folate-binding protein. (i) Step 1: labeling with activated [3lHlfolate. Cells (100 g) that had been washed with 50 mM potassium phosphate (pH 7.8) were resuspended in 500 ml of 5 mM potassium-HEPES (pH 7.8) containing 0.15 M KCl and then mixed with 0.04 p.mol of EDC-activated [3H]folate (500 mCi/mmol). After 30 min at 4°C, unlabeled reagent (20 ,umol) was added, and the incubation was continued for an additional 30 min. To determine covalently bound [3H]folate; a sample (0.5 ml) of treated cells was incubated with 200 ,uM unlabeled folate for 20 min at 37°C, washed twice with S ml of 0.9%o NaCl, and then analyzed for associated radioactivity. (ii) Step 2: preparation of cell extracts. The cell suspension obtained from step 1 was brought to 25 mM in potassium
MECHANISM OF FOLATE TRANSPORT IN L. CASEI
203
phosphate (pH 7.5), and the binding protein was extracted by the addition of Triton X-100 (to 5%) and repeated passage of the cell mixture through a Manton-Gaulon homogenizer (9, 13). A crude extract containing the labeled binding protein was recovered by centrifugation at 24,000 x g for 30 min at 4°C and then dialyzed for 16 h at 4°C against 7 volumes of 25 mM potassium phosphate (pH 7.5). Covalently bound [3H]folate was determined in this step by adding 2 volumes of acetone to a 1-ml sample of extract, allowing the protein to precipitate for 30 min at 4°C, and analyzing the particulate fraction for radioactivity. (iii) Step 3: fractionation with DEAE-cellulose. The dialyzed extract was passed through a column (7 by 14 cm) of DEAE-cellulose that had been equilibrated at 4°C with dialysis buffer (see above). The column was then washed with 600 ml of 25 mM potassium phosphate (pH 7.5), and the eluant containing the binding protein was pooled and analyzed directly for bound [3H]folate. (iv) Step 4: silica fractionation. The DEAE-cellulose pass through obtained in the previous step was combined with 3 g of silica (QuSO G-32; Philadelphia Quartz Co.), and the mixture was stirred for 16 h at 4°C. After centrifugation at 16,000 x g for 5 min at 4°C, the adsorbent was washed three times with 300 ml of 100 mM potassium phosphate (pH 7.5) containing 0.05% Triton. The binding protein was then eluted by stirring the silica for 3 min at 4°C with 30 ml of 0.1 M Na2CO3 containing 0.05% Triton. After centrifugation at 24,000 x g for 2 min at 4°C, the eluate was recovered and immediately adjusted to a neutral pH by the dropwise addition of a saturated solution of KH2PO4. The elution procedure was then repeated two additional times, and the eluates were combined. A second portion of silica (0.3 g) was then added, and the mixture was stirred for 4 h at 4°C. The absorbent was then washed, and the binding protein was eluted as described above with three 10ml-portions of 0.1 M Na2CO3 containing 0.05% Triton. The pooled fractions were concentrated by vacuum dialysis (to ca. 3 ml) and dialyzed against 100 volumes of column buffer (200 mM KCl, 50 mM potassium phosphate, 0.05% Triton [pH 7.5]). (v) Step 5: Sephacryl S-300 chromatography. The concen-trated binding protein was applied to a column (100 by 2.5 cm) of Sephacryl S-300 and then eluted at 4°C with column buffer (see above). Fractions containing the major portion of the bound [3H]folate were pooled and concentrated (by vacuum dialysis) to ca. 2 ml. (vi) Step 6: preparative gel electrophoresis. Protein recovered from the Sephacryl column was precipitated by the addition of acetone (final concentration, 50%), recovered by centrifugation, dissolved in 62.5 mM Tris-hydrochloride (pH 6.8) containing 2% SDS and 10% glycerol, and heated at 80°C for 3 min. The sample was then applied to a preparative SDS slab gel (16), in which the well-forming comb for the stacking gel had been omitted, and then subjected to electrophoresis at 140 V until the tracking dye had reached the bottom of the gel. The binding protein was located by staining a vertical strip of gel (0.5 cm) with Coomassie blue for 5 min. The horizontal strip of the remaining gel that contained the binding protein was then excised, and the protein was eluted by overnight incubation in 4 ml of 10 mM sodium phosphate (pH 7.0) containing 0.1% SDS. Analytical gel electrophoresis. Analysis of protein samples for purity and determinations of molecular weight were performed in 11% polyacrylamide slab gels containing SDS and prepared either by the procedure of Laemmli (16) or Weber and Osborne (20). Amino acid analysis. The amino acid composition of the
204
ANANTHANARAYANAN, KOJIMA, AND HENDERSON
binding protein was determined as described previously (9, 13) with homogeneous binding protein (ca. 200 ,ug) that had been either hydrolyzed directly in 6 HCl or treated before hydrolysis with performic acid to convert cysteine to cysteic acid (18). Protein measurements. The protein content of samples containing Triton X-100 was determined by the biuret reaction (3), whereas samples in SDS were analyzed by the method of Lowry et al. (17). In both procedures, bovine serum albumin served as the standard. RESULTS Effect of mercaptoethanol on the binding of folate and methotrexate to the mutant cells. The concentration dependence for folate binding to the methotrexate-resistant subline of L. casei is shown in Fig. 1. The amount of binding protein present in these cells was in the normal range (0.32 to 0.40 nmol per 1010 cells), but its affinity for folate (Kd= 280 nM) was much lower than that of the wild-type counterpart (Kd = 0.6 nM) (5). Binding to the mutant protein was not improved by the addition of a high level (50 mM) of MgCl2 (5), but it could be increased a substantial degree by the addition of mercaptoethanol (Fig. 1). Mercaptoethanol, which had no effect on binding in the parental'cell line, decreased the Kd for folate in the mutant protein to a value (1.2 nM) which was nearly the same as that of the wild-type protein. The mutant binding protein had an even lower affinity for methotrexate (Kd - 1,000 nM) than did the wild-type counterpart (Kd = 0.8 nM), but, in this case, mercaptoethanol failed to restore binding (Kd = 65 nM) to the normal range. Concentration dependence for the increase in folate-binding affinity by mercaptoethanol. The activation process was analyzed further by performing binding measurements in cells that had been exposed to various levels of mercaptoethanol (Fig. 2). For these determinations, [3H]folate was used at a concentration of 50 nM, since these conditions produced a maximum (fourfold) difference in binding activity between the high- and low-affi'nity forms of the binding protein (Fig. 1). Conversion to the high-affinity state was half maximal at
Folate, nM FIG. 1. Concentration dependence for the binding at 4°C of folate to the mutant cells in the absence and presence of mercaptoethanol. Pretreatment with mercaptoethanol, 5 min at 30°C; mercaptoethanol concentration, 20 mM.
J. BACTERIOL.
(D3 0 3 MC 03
Dithionite
0~ 0
°0.2 Sulf ite C 0
10
20 30 40 Reductant, mM
50
FIG. 2. Effect of reductant concentration on the conversion of the mutant binding protein to the high-affinity state. Preincubation with reductant, 5 min at 30°C; [3H]folate concentration, 50 nM; MCE, mercaptoethanol.
a mercaptoethanol concentration of 0.3 mM, whereas maximum binding was observed at mercaptoethanol concentrations above 5 mM. When other reducing agents were evaluated in a similar fashion (Fig. 2), dithiothreitol was found to be equivalent to mercaptoethanol (half-maximal effect at 0.3 mM) in generating the high-affinity state. Dithionite and sulfite produced only a partial enhancement in affinity, whereas ascorbate had no effect even at concentrations as high as 50 mM. When the time dependence of the'activation process was determined, conversion of the binding protein to the high-affinity form was found to occur immediately (11/2 < 1 mmn) after exposure of the cells to 10 mM mercaptoethanol. Reversal was also rapid, since cells treated with 10 mM mercaptoethanol for 5 mm at 23°C and then washed at 4°C to remove reagent contained binding protein that had reverted (by 90%) to the low-affinity form. This reversal, however, could be blocked by exposure of the mercaptoethanoltreated cells to excess iodoacetamide (25 mM) before the wash step. Folate transport in the mutant cells. The resistant cells also exhibited a reduced capacity for folate transport (Fig. '3). When uptake was measured over a 60-mmn intervaI at 30yC andwat a saturating concentration (2.0 ,uM) of substrate, the accumulation of folate proceendd at only 65% the'rate observed in wild-type cells. However, the addition of mercaptoethanol restored folate transport to a normal level (Fig. 3). Binding and transport of thiamine. Since the L. casei transport systems for folate and thiamine have different receptor proteins bttt share a second common component (7, 10), the possibility that this shared component contains the site which is sensitive to reducing agents was assessed.'It was observed thet mercaptoethanol had no effect on either the rate or extent of thiamine transport or the amount of thiamine binding protein. Moreover, the Kd for thiamine (0.03 nM) in mstant cells was essentially the same as the
MECHANISM OF FOLATE TRANSPORT IN L. CASEI
VOL. 158, 1984
Coo
~~C/
4o 3k
E4
C ~~
~
~
~~~L/
CR(ME
0.
-5
10
20 Time, min
30
40
FIG. 3. Folate transport in the methotrexate-sensitive (LC/S) and methotrexate-resistant (LC/R) cells in the absence and presence of mercaptoethanol (MCE). [3H]folate concentration, 2 ,uM; mercaptoethanol concentration, 20 mM; incubation temperature, 30°C.
corresponding value (0.04 nM) wild-type cells (data not shown). Irreversible inhibition of the mutant binding protein with EDC-activated folate. The folate-binding protein of wild-type cells can be irreversibly inactivated by EDC-activated folate (6). This agent reacts specifically with a lysyl residue at the active site of the transport protein and leads to the covalent incorporation of an equimolar amount of folate. When EDCactivated folate was tested (in the absence of mercaptoethanol) as an irreversible inhibitor of folate binding to the mutant protein, inactivation was found to occur (Fig. 4), and surprisingly, the amount of reagent required for inactivation by 50% (15 nM) was the same as that for the wild-type parental cells, despite a 500-fold difference in folate-binding affinity between these cell lines. The reagent concentration required for half inactivation (15 nM) was also in the same range as the level of added binding protein (17 nM), indicating that inactivation in both cases was nearly stoichiometric. In contrast, EDC-activated methotrexate (12) was much less reactive towards the folate binding site. Half-maximal inactivation by this reagent occurred at a concentration of 380 nM, whereas an even higher amount (700 nM) was required to produce a comparable inactivation in the parental cells. Labeling of the binding protein with EDC-activated [3H]folate. Mutant cells exposed to excess (200 nM) EDC-activated [3H]folate contained covalently incorporated [3H]folate in an amount equivalent to the level of binding protein. Moreover, when the labeled cells were extracted with Triton X-100 and the solubilized proteins were subjected to SDS-polyacrylamide gel electrophoresis, a single radioactive band was observed after autoradiography (Fig. 5). In the absence of mercaptoethanol (lane 1), the labeled protein migrated with an apparent molecular weight of 28,000, whereas a band with a lower molecular weight (18,000) was observed with the added reductant (lane 2) (Fig. 5). In wild-type cells that had been treated similarly, a single labeled protein with a molecular weight of 18,000 was observed in both the absence (lane 3) and presence (lane 4) of mercaptoethanol (Fig. 5). Purification of the binding protein. Binding protein from
205
the mutant cells could be isolated in a homogeneous form (Table 1) by a procedure similar to that used to isolate the wild-type protein (9, 13). It was necessary, however, to label the binding protein with EDC-activated [3H]folate before extraction from the membrane and to utilize two additional purification steps (chromatography on DEAE-cellulose and preparative gel electrophoresis) that had not been used previously. The final purification was generally in the range of 200-fold, and a yield of 15% was typically achieved. A protein sample obtained after Sephacryl S-300 chromatography and then analyzed by SDS-gel electrophoresis (Laemmli procedure) is shown in Fig. 6, lane 2. This mixture contained a prominent band with an apparent molecular weight of 28,000, although other proteins of various sizes were also present. After treatment with mercaptoethanol, this major band shifted to an apparent molecular weight of 18,000 (Fig. 6, lane 3). Contaminating proteins present in this sample were not affected by mercaptoethanol, and no new proteins with a molecular weight lower than 18,000 were generated by reductant treatment. Homogeneous binding protein that had been exposed to mercaptoethanol and then isolated by preparative gel electrophoresis is shown in lane 4 (Fig. 6). The mutant binding protein exhibited a different molecular weight when analyzed by the SDS-polyacrylamide gel system of Weber and Osborne (20), even though the same protein standards were used in both cases. The binding protein migrated under these conditions with an apparent molecular weight of 38,000 in the absence of mercaptoethanol and of 20,000 with added reductant. In previous studies in which disc gel electrophoresis in this same buffer system was used, the wild-type protein was found to have a molecular weight of 25,000 (9, 13). Amino acid analysis of the binding protein. Mutant binding protein that had been isolated by preparative gel electropho-
ii 0 I-
40 c
0
m0 I-' 0
I (L.
200 300 400 500 EDC-Activated Substrate, nM
100
FIG. 4. Concentration dependence for the irreversible inhibition of folate-binding activity in wild-type and mutant cells by EDCactivated folate and EDC-activated methotrexate. Wild-type (filled symbols) or mutant (open symbols) cells were treated for 1 h at 40 with the indicated concentrations of EDC-activated folate (triangles) or EDC-activated methotrexate (circles), washed to remove excess reagent, and then assayed for folate-binding activity.
206
J. BACTERIOL.
ANANTHANARAYANAN, KOJIMA, AND HENDERSON
resis (Fig. 6, lane 4) was found to have an amino acid composition which was indistinguishable from that of the wild-type protein, except for the presence of 1.1 to 1.3 mol of cysteine per 20,000 g of protein. Moreover, the same amino acid composition was obtained when either the higher (28,000)- or lower (18,000)-molecular weight species was excised from gels and analyzed. DISCUSSION The folate-binding and folate transport properties of a methotrexate-resistant subline of L. casei have been evaluated. The results show that the folate-binding protein of these cells is present in normal amounts but has an unusually low affinity for folate (Kd = 280 nM). The distinguishing feature of this binding protein is that it can also assume a highaffinity state whose Kd value for folate (1.2 nM) is essentially the same as that of wild-type cells (Kd= 0.6 nM). Conversion to this high-affinity form is mediated by strong reducing agents, such as mercaptoethanol and dithiothreitol (Fig. 2). Growth of the mutant cell line can occur optimally at the same low level of folate (5 nM) required by wild-type cells, indicating that a portion of the mutant binding protein is probably in a high-affinity state at some stage of the growth phase. A source of reducing equivalents required for this conversion could be cysteine, which is added to the growth medium in relatively high amounts (0.25 g/liter). Full-grown cells, however, contain binding protein that is almost entirely in the low-affinity form. The mutant binding protein also exhibits a low affinity for methotrexate (Kd = 1,000 nM), and mercaptoethanol improves binding, but the dissociation constant for methotrexate after mercaptoethanol treatment (65 nM) does not approach the value observed in wild-type cells (Kd = 0.8 nM). This preferential reduction in binding of methotrexate, relative to that offolate, would thus appear to account for at least a portion of the 50-fold resistance to methotrexate that is exhibited by the mutant cells. The mutant binding protein can be covalently labeled with EDC-activated folate (Fig. 4) in the same fashion as its wild-
TABLE 1. Purification of the folate-binding protein from mutant cells
Step
Protein
2,000
5,800
1.3
100
2,370
1,774
3.6
85
3
(l (ml)(
Crude extract (from 102 g of cells) DEAE-cellulose pass through Silica G-32 fractionation Sephacryl S-300 chromatography
PurificaBound tion [3H]folate Yield M (-fold)
Volume
(m) (g)
(cpm/;Lg)
4.0
12.8
144
24
110
1.3
4.2
266
15
204
type counterpart cells (6). An interesting observation regarding this inactivation is that the loss in binding activity in treated samples is half maximal in both cell lines at the same low level of reagent (15 nM), even though the affinity of the mutant binding protein for folate is nearly 500-fold lower than that of the wild-type protein. This result suggests that either the mutation has not affected binding of reagent to the same. extent as folate or that the rate of covalent modification by reagent is much faster than its rate of decomposition, thus allowing inactivation to eventually reach the same extent with both proteins. Inactivation is also observed with EDC-activated methotrexate but only at relatively high levels (Fig. 4). Surprisingly, the latter agent is slightly more effective in the mutant cells, despite their 1,000-fold lower affinity for methotrexate. It thus appears that the receptor sites on the wild-type and mutant binding proteins differ substantially in affinity for folate and methotrexate, but they react in much the same fashion with the corresponding EDCactivated compounds. Covalent modification with EDC-activated [3H]folate provides a means for tagging the mutant binding protein so that
TOPW68Kv43 K'-
on
_
26K"22 K"-
m
__ W
_ m -0_
14K"-
1 1
2 3
4
FIG. 5. Autoradiography of membrane extracts from the mutant (lanes 1 and 2) and wild-type (lanes 3 and 4) cells labeled with EDCactivated [3H]folate and subjected to SDS-gel electrophoresis. Samples in lanes 2 and 4 were treated with mercaptoethanol (20 mM) for 1 min at 80°C before electrophoresis. Each lane contained protein with ca. 12,000 dpm of bound radioactivity. Gels were prepared by the procedure of Laemmli (16).
2
3
4
FIG. 6. Electrophoretic analysis of folate-binding protein purified from the mutant cells. Lane 1, protein standards were bovine serum albumin, ovalbumin, a-chymotrypsinogen, dihydrofolate reductase (L1210 cells), lysozyme; lane 2, binding protein purified through the Sephacryl S-300 chromatography step; lane 3, same as that described for lane 2 but chromatography was after treatment with 20 mM mercaptoethanol for 1 min at 80°C; lane 4, binding protein purified further by preparative gel electrophoresis and treated with 20 mM mercaptoethanol. Gels were prepared by the procedure of Laemmli (16).
VOL. 158, 1984
it can be detected in detergent extracts of cells (Fig. 5) and during subsequent purification steps (Table 1). The label is particularly useful in visualizing the protein after SDSpolyacrylamide gel electrophoresis, and it led to the discovery (Fig. 5) that the mutant protein has a higher molecular weight (28,000 to 38,000, depending on the buffer system used) than its wild-type counterpart (18,000 to 20,000). The lactose transport protein of Escherichia coli also migrates anomalously during SDS-polyacrylamide gel electrophoresis relative to its true molecular weight (2). Both proteins, however, had the same molecular weight (18,000 to 20,000) after exposure to mercaptoethanol. Moreover, since treatment with mercaptoethanol produces only a single protein band, the higher-molecular-weight form of the mutant binding protein appears both to consist of two subunits of identical size. These observations, along with the finding that both protein forms have comparable amino acid compositions, indicate that the mutant protein is a homologous dimer formed between two identical protein subunits and that the link between these subunits is a disulfide bridge. The single sulfhydryl residue which is present in the mutant binding protein appears to provide the structural basis for this disulfide linkage. Intramolecular disulfide bonds or oligomers of the binding protein higher than a dimer were not observed, as predicted by the fact that each protein subunit contains only a single cysteine residue. The presence and absence of a covalent bond between individual subunits of the binding protein thus correlate directly with the low- and high-affinity forms of this protein, respectively. Heterologous dimer formation between the labeled binding protein and other sulfhydryl-containing membrane components was not observed, indicating that disulfide bond formation proceeds with a high degree of specificity. A possible explanation for this finding is that the binding protein normally resides in the membrane as a noncovalent dimer and that dimerization is promoted by the close proximity of the single sulfhydryl group on each of the individual protein subunits. The retention of a noncovalent dimer structure upon exposure of the binding protein to mercaptoethanol could also explain the rapid reappearance of the low-affinity (dimeric) form of the binding protein upon removal of the reducing agent. It could also be argued that since the dimeric form of the mutant binding protein can facilitate folate transport at a relatively high rate (65% of wild-type levels), then its structure must closely resemble the active form of the wild-type binder. Dimeric structures are relatively common among other membrane proteins and have been reported for several binding proteins which participate in metabolite transport (15). The ability of the mutant binding protein to assume a lowaffinity state, yet retain transport capabilities, has possible implications for the transport mechanism. A basic question regarding this process has been how a binding protein with a very high affinity for folate (Kd = 0.6 nM at pH 7.5) could release bound substrate into the cell and subsequently facilitate the concentrative uptake of folate. One plausible explanation has been that an energy source is used at the inner membrane surface to generate a low-affinity form of the binding protein. Consistent with this hypothesis, the present results show that the transport protein of a mutant subline of L. casei is susceptible to a large change in substrate-binding affinity after the chemical modification of a single sulfhydryl residue. It is thus conceivable that the binding protein of wild-type cells is susceptible to a compa-
MECHANISM OF FOLATE TRANSPORT IN L. CASEI
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