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phosphate symporter during its solubilization and reconstitution
hf. J. Biochem. Cd
Pergamon
0020-711X(94)000644
Bid. Vol. 27, No. 3, pp. 31 l-318, 1995 Copyright 0 1995 Ekvier Science Ltd Printed in Great Britain. All rights reserved 1351-2125195 $9.50 + 0.00
Factors Afpekctimgthe Stability of the Rertal ate Sympwter During its nd Reconstitution VINCENT VACHON,’ MARIE-CLAUDE DELISLE,’ SYLVIE GIROUX,’ RAYNALD LAPRADE; RICHARD Bi?LIVEAU’* ‘Laboratoire G%membranologie mol&ulaire, Dkpartement de chimie-biochimie, Universit& du Q&bee ti Mont&al, P.O. Box 8888, Station A, Montreal, Quebec, Canada H3C 3P8 and 2Groupe de recherche en transport membranaire, UniversitC de Mont&al, P.O. Box 6128, Station A, Montreal, Quebec, Canada H3C 3J7 Phosphate is reabsorbed acrossthe brush-border membrane of the proximal t&de by a specific sodinmdependent symporter. Lie the other brush-border membrane transport protehrs of the kidney, the phosphate carrier remains to be isolated in a fdonal state. To estabgsh a set of parameters that allow to preserve its biological activity, the phosphate carrier was sohthgised under systematically varied conditions and reconstituted into proteoliposomes. Succemful reconstitution was achieved only when the extraction buffer contained lipids extracted fm the renal brush-border membrane. Glycerol, an osmolyte which reducesthe water activity of the sohttioa, was also required. It could however be replaced by 15flmM sodium or potassium te. Below this concentration and in the presenceof glycerol, the ionic strength of the solution had little effect on the stability of the transporter, but sodinm phosphate could not be @aced by sodium dilori8e. Phosphate transport in reconstituted vesicleadqended om the concentration of detergent and pH of the extraction buffer. Fhmlly, transport activity was increased when sohtbiKuathm was carried out in the presence of a reducing age& di&io&reitol. These results should be helpful during the purification and further characterization of the renal phosphate symporter. Keywordsz Stability
Reconstitution Phosphate transport
Brush-border membrane Kidney
Int. J. Biochem. Cell Biol. (1995) 27, 311-318
INTRODUCTION
Inorganic phosphate is reabsorbed from the glomerular filtrate mainly at the level of the proximal tubule. The rate-limiting step of this process is mediated by a specific sodiumdependent carrier that transports phosphate across the luminal membrane against its electrochemical gradient (Bonjour and Caverzasio, 1984; Mizgala and Quamme, 1985; Gmaj and *To whom all correspondence should be addressed. Abbreuiarions: CHAPS, 3-[(3-CholamidopropyI)-dimethylammoniol-l-propane sulfonate; CHES, Z(Cyclohexylamino)ethanesulfonic acid; Hepes, 4-(2-Hydroxyethyl)I-piperazineethane-sulfonic acid; MES, 2(N-Morpho1ino)ethanesulfonic acid. (Received 11 February 1994; accepted 26 October 1994).
Murer, 1986; Hammerman, 1986). Although the kinetics and regulation of this transport activity have been studied extensively over the last decade and a half, information concerning the identity and structure of the carrier molecule remains limited (Murer et al., 1991). The size of its functional unit, as estimated with the radiation-inactivation technique, ranges from 179 to 242 kDa in different species (Beliveau et al., 1988; Tenenhouse et al., 1990; Delisle et al., 1992). Recently, an mRNA coding for the putative sodium/phosphate symporter was isolated from rabbit kidney cortex, cloned and expressed in frog oocytes (Werner et al., 1990). The molecular mass of its predicted ammo acid sequence is 52 kDa (Werner et al., 1991). Further independent evidence indicates that 311
312
Vincent
Vachon
er al
the bovine renal phosphate carrier probably MATERIALS AND METHODS consists of a 55-kDa glycoprotein (Helps and Preparation of brush-border membrane vesicles McGivan, 1991). Taken together, these data Fresh bovine kidneys were obtained from suggest that the phosphate carrier could function as a homotetramer as was established for a local abattoir and perfused extensively with ice-cold 0.85% (w/v) NaCl immediately after the intestinal brush-border membrane sodium/ evisceration. Brush-border membrane vesicles glucose cotransporter (Stevens et al., 1990). were prepared with a MgCI, precipitation Despite these important advances, conclusive identification and further characterization of the method (Booth and Kenny, 1974) as described earlier (Vachon et al., 1991b). Purified memtransporter will also require its isolation from branes were suspended in 300mM mannitol, the brush-border membrane and reconstitution 20 mM 4-(2-Hydroxyethyl)- 1-piperazineethaneinto artificial membranes. Efficient reconstitution schemes have been reported for a variety of sulfonic acid (Hepes)/Tris, pH 7.5, and the following protease inhibitors: chymostatin A renal sodium-dependent cotransporters (Ducis and Koepsell, 1983; Koepsell et al., 1983, (10 ,ug/ml), bacitracin (I 0 pgg/ml), pepstatin 1984; Lynch and McGivan, 1987; Vachon et al., (10 pg/ml), and aprotinin (1.85 pg/ml), stored in liquid N,, and used within a month. Alkaline 1991a; Debiec et al., 1992). So far, however, phosphatase was enriched 7.4 f l.l-fold in attempts to purify functional brush-border membrane preparations membrane transport proteins have met with the brush-border limited success, presumably because these trans- compared with the cortex homogenate. porters are readily inactivated once solubilized from their native membrane (Koepsell, 1986; Solubilization and reconstitution Brush-border membrane protein extracts Koepsell and Seibicke, 1990). The renal sodium/phosphate symporter was were obtained after mixing 100 ,ul of membrane suspension (5-6 mg of protein unless indicated previously reconstituted in proteoliposomes otherwise) with 900 ~1 of the appropriate after extraction of brush-border membrane solubilization buffer as described in the figure proteins with a buffer containing 3-[(3-Chollegends. After a 10 min incubation period, the amidopropyl) - dimethylammonio] - 1 - propane mixture was centrifuged 30min at lOO,OOOg, sulfonate (CHAPS), brush-border membrane and the pellet was discarded. The supernatant lipids, glycerol, sodium phosphate, and dithio(500 ~1 unless indicated otherwise) was mixed threitol (Vachon et al., 1991a). The reconstiwith 500 ~1 of brush-border membrane lipids tuted transport activity depended strongly on (containing 7.5 prnol of phospholipids unless the presence of a transmembrane sodium gradient and shared the main properties observed indicated otherwise) previously resuspended in 150mM NaH,PO,/Na,HPO,, pH 7.5, by with intact isolated brush-border membrane vesicles (Vachon et al., 1991a). Furthermore, the sonication to clarity in a Bransonic water-bath radiation-inactivation size of the phosphate car- sonicator filled with de-aerated water at 37°C. rier was unaffected by solubilization and recon- The detergent was removed by passage through stitution (Delisle et al., 1992). During its a Sephadex G-50 column (13.0 x 1.0 cm) which purification, however, the carrier protein must was equilibrated and eluted with 150 mM be exposed to a variety of physical and chemical KNOj, 5 mM Hepes/Tris, pH 7.5. The turbid fractions eluted in the void volume were conditions without becoming irreversibly inactipooled and frozen in liquid N, for at least vated. In the present study, the conditions were finally used for the extraction and reconstitution of the 10 min. The proteoliposomes phosphate carrier were varied systematically to thawed at room temperature, concentrated by identify those parameters which could affect its centrifugation at 45,000g for 20 min and resusstability. A successful reconstitution was only pended in 150 mM KNO,, 5 mM Hepes/Tris, achieved when brush-border membrane lipids pH 7.5, at a concentration of about 3 mg of were included in the extraction buffer. The protein/ml. phosphate carrier was particularly sensitive to the detergent concentration and to high concen- Transport measurements trations of salt in the extraction medium. Its Phosphate uptake into the proteoliposomes activity was retained at high ionic strength was measured with a rapid filtration method however when NaCl was replaced by sodium or (Hopfer et al., 1973). Liposomes (5 ~1) were potassium phosphate. diluted with 25 ,~l of 5 mM Hepes/Tris, pH 7.5,
Stability of the renal sodium/phosphate
200 PM [32P]KH2P0,/K2HP0, (1 PCi) and 150 mM of either NaNO, or KNO,. After incubation at 25”C, the reaction was stopped with 1 ml of an ice-cold stop solution composed of 150 mM KC1 and 5 mM Hepes/Tris, pH 7.5. The suspension was filtered immediately under vacuum through a 0.45~pm pore size Millipore filter. The filter was rinsed with 7 ml of stop solution and the radioactivity was measured by liquid scintillation counting. Statistical analyses were done with Student’s t-test. Other methods
Brush-border membrane lipids were purified as described by Kates (1972) and lipid phosphorus was measured with the ashing technique of Ames and Dubin (1960). Protein content was determined with the bicinchoninic acid assay (Smith et al., 1985) and alkaline phosphatase was assayed as described by Kelly and Hamilton (1970). RESULTS
AND
DISCUSSION
The renal sodium/phosphate cotransporter is sensitive to the conditions under which it is extracted from the brush-border membrane (Vachon et al., 1991a). Its stability under a variety of conditions was assessed by measuring its ability to transport phosphate in a sodiumdependent manner after reconstitution into proteoliposomes. The reconstituted transport activity varied somewhat depending on the brush-border membrane vesicle preparation used as a source of protein and on the time of storage of these vesicles. To minimize the influence of this source of variability and allow meaningful comparisons between different conditions, all the proteoliposomes used in a given experiment were prepared at the same time and care was taken to avoid variations in the reconstitution procedure other than those under study. Figure 1 shows the kinetics of phosphate uptake into reconstituted liposomes. During the first few minutes of incubation, the sodium-independent uptake was rapid and accounted for a substantial proportion of total phosphate uptake. Thereafter, phosphate uptake measured in the presence of a sodium gradient continued to increase rapidly, while the sodium-independent uptake began to level off. Because the sodium-dependent uptake was near its maximum after approx. 15 min, this time point was chosen for the comparison of the transport
symporter
0
313
15
45
30
60
75
Time (min) Fig. 1. Time course of phosphate uptake into proteoliposomes prepared with different amounts of solubilized protein. Brush-border membrane proteins were extracted in a buffer composed of 150mM NaH,PO,/Na,HPO,. 5 mM Hepes/Tris, pH 7.5, 1 mM dithiothreitol, 30% (w/v) glycerol, 1.5% (w/v) CHAPS and brush-border membrane lipids (10.2 pmol of phospholipids/ml). Proteoliposomes were prepared as described in “Materials and Methods” after mixing different volumes of the protein extract with resuspended brush-border membrane lipids. The volume and lipid concentration of the suspension were adjusted so that, for each proteoliposome preparation, an equal volume of the mixture (1 ml) and an equal amount of lipids (12.6 pmol of phospholipids) were applied to the gel filtration column used for detergent removal, assuming the amount of lipids lost with the unsolubilized protein was negligible. The protein concentration during reconstitution was 3.2 (0, O), 2.1 (& Cl). 1.1 (A, ~9 and 0.5 (a, 0) mg/ml. Phosphate uptake was measured in the presence of NaNO, (0, n , A, +) or KNO, (0, q i,,!L 0). Values are means + SD of three independent experiments performed in triplicate.
=
o-,-7 0
I
3
2
CHAPS
4
(%)
Fig. 2. Comparison of phosphate uptake into proteoliposomes prepared with brush-border membrane proteins extracted with different concentrations of detergent. Proteoliposomes were prepared as described in the “Materials and Methods” section, using an extraction buffer containing 150mM NaH,PO,/Na,HPO,. 5 mM Hepes/Tris, pH 7.5, 1 mM dithiothreitol, 30% (w/v) glycerol, brush-border membrane lipids (10.2 pmol/ml of phoapholipids), and different concentrations of CHAPS (w/v). Phosphate uptake was measured in the presence of NaNO, (a) or KNO, (0). Values are means f SD of three independent experiments performed at least in triplicate.
314
activity of the liposomes different conditions.
Vincent Vachon et al.
prepared
under
Temperature
In earlier experiments, solubilization and reconstitution of the sodium/phosphate symporter were carried out at 4°C to minimize possible enzymatic degradation of the transporter molecule (Vachon et al., 1991a). Similar transport activities were observed, however, when the proteoliposomes were prepared at 4°C or at room temperature (20-22°C). All further experiments were therefore performed at room temperature. Protein concentration
The influence of the protein concentration was studied during both the reconstitution and solubilization steps. Phosphate uptake into proteoliposomes prepared with different amounts of solubilized protein is shown in Fig. 1. Although the ratio of phospholipid and protein used to form the proteoliposomes varied from 3.9 to 25.2 pmol/mg, the specific rates of phosphate uptake were similar in proteoliposomes prepared at the higher protein concentrations (1 .l, 2.1 and 3.2 mg/ml) and only slightly smaller in proteoliposomes prepared with the lowest protein concentration (0.5 mg/ml) (Fig. 1). The amount of protein added to the extraction buffer also had only a slight effect on the sodium-dependent phosphate-transport activity of the resulting proteoliposomes, at least over the range of 2 to 16 mg of protein/ml. Detergent concentration
In contrast, the reconstituted transport activity was markedly influenced by the detergent concentration used to solubilize the membranes (Fig. 2). Both the sodiumdependent transport and sodium-independent uptake of phosphate were highest in proteoliposomes prepared with protein extracted with 1.5% CHAPS, a value which corresponds to 3 times its critical micellar concentration (Chattopadhyay and London, 1984). Decreasing or doubling this concentration resulted in a significant loss of transport activity (Fig. 2). Brush-border membrane lipids
Including brush-border membrane lipids in the extraction buffer also had a strong effect on the reconstituted phosphate transport activity of the proteoliposomes (Fig. 3). Without added lipids, the uptake of phosphate in the presence
of a transmembrane sodium gradient was only slightly higher than in its absence. This weak transport activity could not be attributed to a reduced intravesicular volume due to the absence of lipids since the same total amount of lipids, including those present in the extraction buffer and those subsequently added to the protein extract, was used for each of the proteoliposome preparations. The sodium-dependent transport activity was also greatly reduced in proteoliposomes prepared by adding lipids only to the extraction buffer (25.2 ,umol of phospholipids/ml), suggesting that some lipids may precipitate along with the unsolubilized material during ultracentrifugation of the protein extract or that solubilization of the carrier protein could be hindered at high lipid concentrations. In agreement with the latter explanation, the sodium-independent uptake of phosphate, which is probably due to binding of phosphate onto the surface of the proteoliposomes, because of the presence of phosphate-binding proteolipids extracted with the brush-border membrane lipids (Kessler et al., 1982; Debiec
Ob
30
Lipids (vmol phospholipid.s/ml) Fig. 3. Comparison of phosphate uptake into proteoliposomes prepared with brush-border membrane proteins extracted in the presence of different concentrations of added brush-border membrane lipids. Proteoliposomes were prepared as described in the “Materials and Methods” section, using an extraction buffer containing 150mM NaH,POJNa,HPO, 5mM Hepes/Tris, pH 7.5, 1 mM dithiothreitol, 30% (w/v) glycerol, 1.5% (w/v) CHAPS, and different lipid concentrations. The concentrations indicated are those of the phospholipids even though the lipids used were those present in an unfractionated lipid extract of purified brush-border membranes. The amount of lipid subsequently added to the protein extract before removal of the detergent by gel filtration chromatography was adjusted so that an equal amount of lipids (12.6 pmol of phospholipids) was used for the formation of each proteoliposome preparation, assuming the amount of lipids lost with the unsolubilized protein was negligible. Phosphate uptake was measured in the presence of NaNO, (0) or KNO, (0). Values are means + SD of two independent experiments performed at least in triplicate.
Stability of the renal sodium/phosphate
and Lorenc, 1988), or to diffusion of phosphate across the lipid bilayer, was not affected significantly by the lipid concentration of the extraction buffer (Fig. 3). The renal phosphate carrier thus appears to resist well to variations in the detergent-toprotein ratio in the extraction medium as long as the lipid concentration is sufficiently high; its sensitivity to a high concentration of detergent (Fig. 2) is therefore mostly attributable to an unfavorable detergent-to-lipid ratio. Together with our earlier observation showing that the reconstituted sodium-dependent transport activity is almost completely lost when the brush-border membrane lipids used to prepare the proteoliposomes are replaced with asolectin or egg-yolk phospholipids supplemented with cholesterol (Vachon et al., 1991a), these results indicate that endogenous membrane lipids play an important role in maintaining the stability of the renal sodium/phosphate symporter during its solubilization and reconstitution as was observed for the mitochondrial phosphate carrier (Kadenbach et al., 1982) and a variety of other transport proteins (Koepsell,
315
symporter
phosphate was replaced by potassium phosphate, but the sodium-dependent phosphate transport activity was completely lost when phosphate salts were replaced with sodium chloride without changing the ionic strength of the solution (Table 1). Variations in the sodium phosphate concentration of the extraction medium, at least within the range of 0 to 150 mM, had little influence on the reconstituted phosphate transport activity. Although glycerol and phosphate salts could be removed from the extraction buffer without significantly affecting the phosphate transport activity of the resulting proteoliposomes, this
6l
A
1986). Glycerol concentration
The reconstitution of a variety of membrane transport proteins has been shown to be greatly improved by including glycerol or another osmolyte in the solubilization buffer (Ambudkar and Maloney, 1986; Maloney and Ambudkar, 1989). In the case of the renal phosphate carrier, however, similar transport activities were observed when brush-border membrane proteins were extracted in the presence of high concentrations of glycerol (30 and 50%) or in its absence. Moreover, extracting the protein at intermediate glycerol concentrations ( 10 and 15 %) resulted in a decrease in the ability of the resulting proteoliposomes to transport phosphate in a sodium-dependent fashion. The reason for this decreased transport activity remains unknown although it was consistently observed. Ionic strength
At high ionic strength, the stability of the sodium/phosphate symporter depended on the salt used in the extraction buffer. A strong sodium-dependent phosphate transport activity was reconstituted when the protein was extracted with sodium phosphate (Table 1). Similar results were obtained when sodium
01’ 4
I 5
‘,I 6
I’ 7
I’ 8
I 9
‘: 10
PH
Fig. 4. Comparison of phosphate uptake into proteoliposomes prepared with brush-border membrane proteins extracted at different pH in buffers of high (A) and low (B) ionic strength. (A) Proteoliposomes were prepared as described in “Materials and Methods”, using an extraction buffer composed of brush-border membrane lipids (10.2 pmol of phospholipids/ml), 30% (w/v) glycerol, 1.5% (w/v) CHAPS, 1 mM dithiothreitol and 1SOmM NaH,PO,/Na,HPO,, pH 5, 6, 7, 8, or 9. (B) Proteoliposomes were prepared with an extraction buffer composed of brush-border membrane lipids (10.2 ymol of phospholipids/ml), 30% (w/v) glycerol, 1.5% (w/v) CHAPS, 1 mM dithiothreitol and 20 mM 2(N-Morpholino)ethanesulfonic acid (MES)/Tris, pH 5, MES/Tris, pH 6, Hepes/Tris, pH 7, HepeqTris, pH 8, or 2(Cyclohexylamino)ethanesulfonic acid (CHES)/Tris, pH 9. Phosphate uptake was measured at pH 7.5 in the presence of NaN4 (a) or KNO, (0). Values are means f SD of two (B) or three (A) independent experiments performed in triplicate.
316
Vincent Vachon et al. Table 1. Comparison of phosphate uptake into proteohposomes prepared with brush-border membrane proteins extracted at high ionic strength with different salts Phosphate uptake (nmol/mg protein/l5 min) Salt NaH,PO,/Na,HPO, KH2P0,/K,HP0, NaCl
Concentration (mW
NaNO,
KNO,
150 150 225
5.11 kO.41 4.76 f 0.23 2.25 + 0.21
1.94kO.12 2.01 kO.13 2.02 + 0.16
Brush-border membrane proteins were extracted with a buffer composed of the indicated salts plus 5mM Hepes/Tris, pH 7.5, I mM dithiothreitol, 30% (w/v) glycerol, 1.5% (w/v) CHAPS, and brush-border membrane lipids (10.2 pmol of phospholipids/ml), and reconstituted as described in “Materials and Methods”. Phosphate uptake was measured in the presence of NaNO, or KNO,. Values are means k SD of five independent experiments performed at least in triplicate.
activity was completely lost when both were omitted (Table 2). The fact that glycerol and phosphate can compensate for one another in stabilizing the phosphate carrier during its solubilization suggests that they may act similarly by restricting access of free water to the protein molecule and thus favor inter-subunit or intramolecular interactions which are important for stabilizing its conformation.
pH 6 and 7 (Fig. 4A). At low ionic strength, the carrier was irreversibly inactivated at pH 5, but retained its activity equally well when extracted at pH 6-9 (Fig. 4B). Dithiothreitol
PH
The effect of the pH of the extraction buffer on the stability of the sodium/phosphate symporter depended on the ionic strength. When the extraction was carried out at high ionic strength, in the presence of 150emM sodium phosphate, the phosphate transport activity was similar in the proteoliposomes prepared with protein extracted at pH 5, 8 and 9, and significantly increased in the proteoliposomes prepared with protein extracted at
The sodium/phosphate symporter also appears to be sensitive to oxidation since including 1 mM dithiothreitol in the extraction buffer resulted in a 25% increase in the reconstituted phosphate transport activity measured in the presence of a sodium gradient (6.37 + 0.38 vs 4.98 +_0.38 nmol/mg protein/l5 min; P < 0.01) without affecting the sodium-independent phosphate uptake. This effect was probably due to an increase in the activity of the transporter rather than to an increase in its stability during extraction since a similar stimulation of phosphate transport activity has been observed previously with renal brush-border membrane vesicles preincubated with dithiothreitol (Suzuki et al., 1990).
Table 2. Comparison of phosphate uptake into proteoliposomes prepared with brush-border membrane proteins extracted with or without glycerol and sodium phosphate Phosphate uptake (nmol/mg protein/l5 min) Extraction buffer Complete Without glycerol Without sodium phosphate Without glycerol nor sodium phosphate
NaNO,
KNO,
2.71 + 0.17 2.83 + 0.24 2.96 + 0.16 1.66&-0.13
1.31 kO.11 1.33_+0.12 1.35 kO.13 1.38 + 0.23
Brush-border membrane proteins were extracted with a buffer composed 5 mM Hepes/Tris, pH 7.5, 1 mM dithiothreitol, I .5% (w/v) CHAPS, brush-border membrane lipids (10.2 pmol of phosphohpids/ml), with or without 150 mM NaH,POJNa2HP0, and 30% (w/v) glycerol, and reconstituted as described in “Materials and Methods”. Phosphate uptake was measured in the presence of NaNO, or KNO1. Values are means f SD of three independent experiments performed in triplicate.
Stability of the renal sodium/phosphate symporter
6-
0-l 0
, IO
c
, 20
I 30
u
I 40
Cholesterol added (mol%) Fig. 5. Effect of added cholesterol on the uptake of phosphate into reconstituted membrane vesicles. Brush-border membrane proteins were extracted in a buffer composed of 150mM NaH,PO,/Na,HPO,, 5 mM Hepes/Tris, pH 7.5, I mM dithiothreitol, 30% (w/v) glycerol, 1.5% (w/v) CHAPS, and brush-border membrane lipids (10.2 pmol of phospholipids/ml) supplemented with different amounts of cholesterol. Because the lipid composition of the bovine brush-border membrane is unknown, the percentages indicated are based on a cholesterol-to-phospholipid ratio of 0.64 mol/mol, a value close to those reported for the renal brush-border membrane of rat (0.67) (Hise et al., 1984), dog (0.64) (Carmel et al., 1985) and rabbit (0.61) (Venien and Le Grimellec, 1988). Proteoliposomes were prepared as described in “Materials and Methods” except that the lipid suspension was supplemented with the same proportion of cholesterol as the corresponding extraction medium. Uptake of phosphate was measured in the presence of NaNO, (0) or KNO, (0). Values are means + SD of three independent experiments performed at least in triplicate.
Added cholesterol
In vitro enrichment of renal brush-border membranes with cholesterol was shown to result in a significant decrease in phosphate transport activity (Mohtoris et al., 1985; Yusufi et al., 1989; Levi et al., 1990). The sodium-dependent phosphate transport into proteoliposomes prepared with brush-border membrane lipids supplemented with different amounts of cholesterol was also decreased by such additions (Fig. 5), although to a slightly smaller extent than in brush-border membranes enriched with similar amounts of cholesterol (Levi et al., 1990). SUMMARY
AND
CONCLUSIONS
During its extraction from the brush-border membrane, the stability of the renal sodium/ phosphate cotransporter depends mainly on the concentration of the detergent, the presence of brush-border membrane lipids, and, at high ionic strength, on the presence of phosphate in the solubilization buffer. A knowledge of the importance of these parameters should be useful in designing a purification scheme for this
317
transporter which has proved to be quite readily inactivated upon extraction from its native membrane. Despite its sensitivity, purification remains, along with the recent cloning of its structural gene (Werner et al., 1990, 199i), an essential step for the identification and structural characterization of the carrier molecule, and for a detailed understanding of the molecular mechanism by which phosphate is reabsorbed by the kidney. Acknowledgements-This work was supported by grants from the Medical Research Council (MRC) of Canada to R. Beliveau, and from the Natural Sciences and Engineering Research Council (NSERC) of Canada to R. Laprade. M.-C. Delisle received a scholarship for doctoral studies from the Fonds de la recherche en Sante du Quebec (FRSQ).
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Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J. and Klenk D. C. (1985) Measurement of protein using bicinchoninic acid. Analyr. Biochem.
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Stevens B. R., Fernandez A., Hirayama B., Wright E. M. and Kempner E. S. (1990) Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer. Proc. Natn. Acad. Sci U.S.A. 87, 1456-1460. Suzuki M., Capparelli A. W., Jo 0. D. and Yanagawa N. (1990) Thiol redox and phosphate transport in renal brush-border membrane. Effect of nicotinamide. Biochim. biophys.
Acta
1021,
85-90.
Tenenhouse H. S., Lee J., Harvey N., Potier M., Jetti M. and Beliveau R. (1990) Normal molecular size of the Na+-phosphate cotransporter and normal Na*dependent binding of phosphonoformic acid in renal brush border membranes of X-linked Hyp mice. Biochem. biophys. Res. Commun. 170, 1288-1293. Vachon V., Delisle M.-C., Laprade R. and Beliveau R. (1991a) Reconstitution of the renal brush-border membrane sodium/phosphate co-transporter. Biochem. J. 278, 543-548.
Vachon V., Pouliot J.-F., Laprade R. and Btliveau R. (1991b) Fractionation of renal brush border membrane proteins with Triton X-l 14 phase partitioning. Biochem. Cell Biol. 69, 206-211. Venien C. and Le Grimellec C. (1988) Phospholipid asymmetry in renal brush-border membranes. Biochim. biophys. Acta 942, 159-168. Werner A., Biber J., Forgo J., Palacin M. and Murer H. (1990) Expression of renal transport systems for inorganic phosphate and sulfate in Xenopus laevis oocytes. J. biol. Chem. 265, 12331-12336. Werner A., Moore M. L., Mantei N., Biber J., Semenza G. and Murer H. (1991) Cloning and expression of cDNA for a Na/P, cotransport system of kidney cortex. Proc. Natn. Acad. Sci. U.S.A. 88, 96089612. Yusufi A. N. K., Szczepanska-Konkel M., Hoppe A. and Dousa T. P. (1989) Different mechanisms of adaptive increase in Na+-P, cotransport across renal brush-border membrane. Am. J. Physiol. 256, F852-F861.
Pergamon
0020-711X(94)000644
Bid. Vol. 27, No. 3, pp. 31 l-318, 1995 Copyright 0 1995 Ekvier Science Ltd Printed in Great Britain. All rights reserved 1351-2125195 $9.50 + 0.00
Factors Afpekctimgthe Stability of the Rertal ate Sympwter During its nd Reconstitution VINCENT VACHON,’ MARIE-CLAUDE DELISLE,’ SYLVIE GIROUX,’ RAYNALD LAPRADE; RICHARD Bi?LIVEAU’* ‘Laboratoire G%membranologie mol&ulaire, Dkpartement de chimie-biochimie, Universit& du Q&bee ti Mont&al, P.O. Box 8888, Station A, Montreal, Quebec, Canada H3C 3P8 and 2Groupe de recherche en transport membranaire, UniversitC de Mont&al, P.O. Box 6128, Station A, Montreal, Quebec, Canada H3C 3J7 Phosphate is reabsorbed acrossthe brush-border membrane of the proximal t&de by a specific sodinmdependent symporter. Lie the other brush-border membrane transport protehrs of the kidney, the phosphate carrier remains to be isolated in a fdonal state. To estabgsh a set of parameters that allow to preserve its biological activity, the phosphate carrier was sohthgised under systematically varied conditions and reconstituted into proteoliposomes. Succemful reconstitution was achieved only when the extraction buffer contained lipids extracted fm the renal brush-border membrane. Glycerol, an osmolyte which reducesthe water activity of the sohttioa, was also required. It could however be replaced by 15flmM sodium or potassium te. Below this concentration and in the presenceof glycerol, the ionic strength of the solution had little effect on the stability of the transporter, but sodinm phosphate could not be @aced by sodium dilori8e. Phosphate transport in reconstituted vesicleadqended om the concentration of detergent and pH of the extraction buffer. Fhmlly, transport activity was increased when sohtbiKuathm was carried out in the presence of a reducing age& di&io&reitol. These results should be helpful during the purification and further characterization of the renal phosphate symporter. Keywordsz Stability
Reconstitution Phosphate transport
Brush-border membrane Kidney
Int. J. Biochem. Cell Biol. (1995) 27, 311-318
INTRODUCTION
Inorganic phosphate is reabsorbed from the glomerular filtrate mainly at the level of the proximal tubule. The rate-limiting step of this process is mediated by a specific sodiumdependent carrier that transports phosphate across the luminal membrane against its electrochemical gradient (Bonjour and Caverzasio, 1984; Mizgala and Quamme, 1985; Gmaj and *To whom all correspondence should be addressed. Abbreuiarions: CHAPS, 3-[(3-CholamidopropyI)-dimethylammoniol-l-propane sulfonate; CHES, Z(Cyclohexylamino)ethanesulfonic acid; Hepes, 4-(2-Hydroxyethyl)I-piperazineethane-sulfonic acid; MES, 2(N-Morpho1ino)ethanesulfonic acid. (Received 11 February 1994; accepted 26 October 1994).
Murer, 1986; Hammerman, 1986). Although the kinetics and regulation of this transport activity have been studied extensively over the last decade and a half, information concerning the identity and structure of the carrier molecule remains limited (Murer et al., 1991). The size of its functional unit, as estimated with the radiation-inactivation technique, ranges from 179 to 242 kDa in different species (Beliveau et al., 1988; Tenenhouse et al., 1990; Delisle et al., 1992). Recently, an mRNA coding for the putative sodium/phosphate symporter was isolated from rabbit kidney cortex, cloned and expressed in frog oocytes (Werner et al., 1990). The molecular mass of its predicted ammo acid sequence is 52 kDa (Werner et al., 1991). Further independent evidence indicates that 311
312
Vincent
Vachon
er al
the bovine renal phosphate carrier probably MATERIALS AND METHODS consists of a 55-kDa glycoprotein (Helps and Preparation of brush-border membrane vesicles McGivan, 1991). Taken together, these data Fresh bovine kidneys were obtained from suggest that the phosphate carrier could function as a homotetramer as was established for a local abattoir and perfused extensively with ice-cold 0.85% (w/v) NaCl immediately after the intestinal brush-border membrane sodium/ evisceration. Brush-border membrane vesicles glucose cotransporter (Stevens et al., 1990). were prepared with a MgCI, precipitation Despite these important advances, conclusive identification and further characterization of the method (Booth and Kenny, 1974) as described earlier (Vachon et al., 1991b). Purified memtransporter will also require its isolation from branes were suspended in 300mM mannitol, the brush-border membrane and reconstitution 20 mM 4-(2-Hydroxyethyl)- 1-piperazineethaneinto artificial membranes. Efficient reconstitution schemes have been reported for a variety of sulfonic acid (Hepes)/Tris, pH 7.5, and the following protease inhibitors: chymostatin A renal sodium-dependent cotransporters (Ducis and Koepsell, 1983; Koepsell et al., 1983, (10 ,ug/ml), bacitracin (I 0 pgg/ml), pepstatin 1984; Lynch and McGivan, 1987; Vachon et al., (10 pg/ml), and aprotinin (1.85 pg/ml), stored in liquid N,, and used within a month. Alkaline 1991a; Debiec et al., 1992). So far, however, phosphatase was enriched 7.4 f l.l-fold in attempts to purify functional brush-border membrane preparations membrane transport proteins have met with the brush-border limited success, presumably because these trans- compared with the cortex homogenate. porters are readily inactivated once solubilized from their native membrane (Koepsell, 1986; Solubilization and reconstitution Brush-border membrane protein extracts Koepsell and Seibicke, 1990). The renal sodium/phosphate symporter was were obtained after mixing 100 ,ul of membrane suspension (5-6 mg of protein unless indicated previously reconstituted in proteoliposomes otherwise) with 900 ~1 of the appropriate after extraction of brush-border membrane solubilization buffer as described in the figure proteins with a buffer containing 3-[(3-Chollegends. After a 10 min incubation period, the amidopropyl) - dimethylammonio] - 1 - propane mixture was centrifuged 30min at lOO,OOOg, sulfonate (CHAPS), brush-border membrane and the pellet was discarded. The supernatant lipids, glycerol, sodium phosphate, and dithio(500 ~1 unless indicated otherwise) was mixed threitol (Vachon et al., 1991a). The reconstiwith 500 ~1 of brush-border membrane lipids tuted transport activity depended strongly on (containing 7.5 prnol of phospholipids unless the presence of a transmembrane sodium gradient and shared the main properties observed indicated otherwise) previously resuspended in 150mM NaH,PO,/Na,HPO,, pH 7.5, by with intact isolated brush-border membrane vesicles (Vachon et al., 1991a). Furthermore, the sonication to clarity in a Bransonic water-bath radiation-inactivation size of the phosphate car- sonicator filled with de-aerated water at 37°C. rier was unaffected by solubilization and recon- The detergent was removed by passage through stitution (Delisle et al., 1992). During its a Sephadex G-50 column (13.0 x 1.0 cm) which purification, however, the carrier protein must was equilibrated and eluted with 150 mM be exposed to a variety of physical and chemical KNOj, 5 mM Hepes/Tris, pH 7.5. The turbid fractions eluted in the void volume were conditions without becoming irreversibly inactipooled and frozen in liquid N, for at least vated. In the present study, the conditions were finally used for the extraction and reconstitution of the 10 min. The proteoliposomes phosphate carrier were varied systematically to thawed at room temperature, concentrated by identify those parameters which could affect its centrifugation at 45,000g for 20 min and resusstability. A successful reconstitution was only pended in 150 mM KNO,, 5 mM Hepes/Tris, achieved when brush-border membrane lipids pH 7.5, at a concentration of about 3 mg of were included in the extraction buffer. The protein/ml. phosphate carrier was particularly sensitive to the detergent concentration and to high concen- Transport measurements trations of salt in the extraction medium. Its Phosphate uptake into the proteoliposomes activity was retained at high ionic strength was measured with a rapid filtration method however when NaCl was replaced by sodium or (Hopfer et al., 1973). Liposomes (5 ~1) were potassium phosphate. diluted with 25 ,~l of 5 mM Hepes/Tris, pH 7.5,
Stability of the renal sodium/phosphate
200 PM [32P]KH2P0,/K2HP0, (1 PCi) and 150 mM of either NaNO, or KNO,. After incubation at 25”C, the reaction was stopped with 1 ml of an ice-cold stop solution composed of 150 mM KC1 and 5 mM Hepes/Tris, pH 7.5. The suspension was filtered immediately under vacuum through a 0.45~pm pore size Millipore filter. The filter was rinsed with 7 ml of stop solution and the radioactivity was measured by liquid scintillation counting. Statistical analyses were done with Student’s t-test. Other methods
Brush-border membrane lipids were purified as described by Kates (1972) and lipid phosphorus was measured with the ashing technique of Ames and Dubin (1960). Protein content was determined with the bicinchoninic acid assay (Smith et al., 1985) and alkaline phosphatase was assayed as described by Kelly and Hamilton (1970). RESULTS
AND
DISCUSSION
The renal sodium/phosphate cotransporter is sensitive to the conditions under which it is extracted from the brush-border membrane (Vachon et al., 1991a). Its stability under a variety of conditions was assessed by measuring its ability to transport phosphate in a sodiumdependent manner after reconstitution into proteoliposomes. The reconstituted transport activity varied somewhat depending on the brush-border membrane vesicle preparation used as a source of protein and on the time of storage of these vesicles. To minimize the influence of this source of variability and allow meaningful comparisons between different conditions, all the proteoliposomes used in a given experiment were prepared at the same time and care was taken to avoid variations in the reconstitution procedure other than those under study. Figure 1 shows the kinetics of phosphate uptake into reconstituted liposomes. During the first few minutes of incubation, the sodium-independent uptake was rapid and accounted for a substantial proportion of total phosphate uptake. Thereafter, phosphate uptake measured in the presence of a sodium gradient continued to increase rapidly, while the sodium-independent uptake began to level off. Because the sodium-dependent uptake was near its maximum after approx. 15 min, this time point was chosen for the comparison of the transport
symporter
0
313
15
45
30
60
75
Time (min) Fig. 1. Time course of phosphate uptake into proteoliposomes prepared with different amounts of solubilized protein. Brush-border membrane proteins were extracted in a buffer composed of 150mM NaH,PO,/Na,HPO,. 5 mM Hepes/Tris, pH 7.5, 1 mM dithiothreitol, 30% (w/v) glycerol, 1.5% (w/v) CHAPS and brush-border membrane lipids (10.2 pmol of phospholipids/ml). Proteoliposomes were prepared as described in “Materials and Methods” after mixing different volumes of the protein extract with resuspended brush-border membrane lipids. The volume and lipid concentration of the suspension were adjusted so that, for each proteoliposome preparation, an equal volume of the mixture (1 ml) and an equal amount of lipids (12.6 pmol of phospholipids) were applied to the gel filtration column used for detergent removal, assuming the amount of lipids lost with the unsolubilized protein was negligible. The protein concentration during reconstitution was 3.2 (0, O), 2.1 (& Cl). 1.1 (A, ~9 and 0.5 (a, 0) mg/ml. Phosphate uptake was measured in the presence of NaNO, (0, n , A, +) or KNO, (0, q i,,!L 0). Values are means + SD of three independent experiments performed in triplicate.
=
o-,-7 0
I
3
2
CHAPS
4
(%)
Fig. 2. Comparison of phosphate uptake into proteoliposomes prepared with brush-border membrane proteins extracted with different concentrations of detergent. Proteoliposomes were prepared as described in the “Materials and Methods” section, using an extraction buffer containing 150mM NaH,PO,/Na,HPO,. 5 mM Hepes/Tris, pH 7.5, 1 mM dithiothreitol, 30% (w/v) glycerol, brush-border membrane lipids (10.2 pmol/ml of phoapholipids), and different concentrations of CHAPS (w/v). Phosphate uptake was measured in the presence of NaNO, (a) or KNO, (0). Values are means f SD of three independent experiments performed at least in triplicate.
314
activity of the liposomes different conditions.
Vincent Vachon et al.
prepared
under
Temperature
In earlier experiments, solubilization and reconstitution of the sodium/phosphate symporter were carried out at 4°C to minimize possible enzymatic degradation of the transporter molecule (Vachon et al., 1991a). Similar transport activities were observed, however, when the proteoliposomes were prepared at 4°C or at room temperature (20-22°C). All further experiments were therefore performed at room temperature. Protein concentration
The influence of the protein concentration was studied during both the reconstitution and solubilization steps. Phosphate uptake into proteoliposomes prepared with different amounts of solubilized protein is shown in Fig. 1. Although the ratio of phospholipid and protein used to form the proteoliposomes varied from 3.9 to 25.2 pmol/mg, the specific rates of phosphate uptake were similar in proteoliposomes prepared at the higher protein concentrations (1 .l, 2.1 and 3.2 mg/ml) and only slightly smaller in proteoliposomes prepared with the lowest protein concentration (0.5 mg/ml) (Fig. 1). The amount of protein added to the extraction buffer also had only a slight effect on the sodium-dependent phosphate-transport activity of the resulting proteoliposomes, at least over the range of 2 to 16 mg of protein/ml. Detergent concentration
In contrast, the reconstituted transport activity was markedly influenced by the detergent concentration used to solubilize the membranes (Fig. 2). Both the sodiumdependent transport and sodium-independent uptake of phosphate were highest in proteoliposomes prepared with protein extracted with 1.5% CHAPS, a value which corresponds to 3 times its critical micellar concentration (Chattopadhyay and London, 1984). Decreasing or doubling this concentration resulted in a significant loss of transport activity (Fig. 2). Brush-border membrane lipids
Including brush-border membrane lipids in the extraction buffer also had a strong effect on the reconstituted phosphate transport activity of the proteoliposomes (Fig. 3). Without added lipids, the uptake of phosphate in the presence
of a transmembrane sodium gradient was only slightly higher than in its absence. This weak transport activity could not be attributed to a reduced intravesicular volume due to the absence of lipids since the same total amount of lipids, including those present in the extraction buffer and those subsequently added to the protein extract, was used for each of the proteoliposome preparations. The sodium-dependent transport activity was also greatly reduced in proteoliposomes prepared by adding lipids only to the extraction buffer (25.2 ,umol of phospholipids/ml), suggesting that some lipids may precipitate along with the unsolubilized material during ultracentrifugation of the protein extract or that solubilization of the carrier protein could be hindered at high lipid concentrations. In agreement with the latter explanation, the sodium-independent uptake of phosphate, which is probably due to binding of phosphate onto the surface of the proteoliposomes, because of the presence of phosphate-binding proteolipids extracted with the brush-border membrane lipids (Kessler et al., 1982; Debiec
Ob
30
Lipids (vmol phospholipid.s/ml) Fig. 3. Comparison of phosphate uptake into proteoliposomes prepared with brush-border membrane proteins extracted in the presence of different concentrations of added brush-border membrane lipids. Proteoliposomes were prepared as described in the “Materials and Methods” section, using an extraction buffer containing 150mM NaH,POJNa,HPO, 5mM Hepes/Tris, pH 7.5, 1 mM dithiothreitol, 30% (w/v) glycerol, 1.5% (w/v) CHAPS, and different lipid concentrations. The concentrations indicated are those of the phospholipids even though the lipids used were those present in an unfractionated lipid extract of purified brush-border membranes. The amount of lipid subsequently added to the protein extract before removal of the detergent by gel filtration chromatography was adjusted so that an equal amount of lipids (12.6 pmol of phospholipids) was used for the formation of each proteoliposome preparation, assuming the amount of lipids lost with the unsolubilized protein was negligible. Phosphate uptake was measured in the presence of NaNO, (0) or KNO, (0). Values are means + SD of two independent experiments performed at least in triplicate.
Stability of the renal sodium/phosphate
and Lorenc, 1988), or to diffusion of phosphate across the lipid bilayer, was not affected significantly by the lipid concentration of the extraction buffer (Fig. 3). The renal phosphate carrier thus appears to resist well to variations in the detergent-toprotein ratio in the extraction medium as long as the lipid concentration is sufficiently high; its sensitivity to a high concentration of detergent (Fig. 2) is therefore mostly attributable to an unfavorable detergent-to-lipid ratio. Together with our earlier observation showing that the reconstituted sodium-dependent transport activity is almost completely lost when the brush-border membrane lipids used to prepare the proteoliposomes are replaced with asolectin or egg-yolk phospholipids supplemented with cholesterol (Vachon et al., 1991a), these results indicate that endogenous membrane lipids play an important role in maintaining the stability of the renal sodium/phosphate symporter during its solubilization and reconstitution as was observed for the mitochondrial phosphate carrier (Kadenbach et al., 1982) and a variety of other transport proteins (Koepsell,
315
symporter
phosphate was replaced by potassium phosphate, but the sodium-dependent phosphate transport activity was completely lost when phosphate salts were replaced with sodium chloride without changing the ionic strength of the solution (Table 1). Variations in the sodium phosphate concentration of the extraction medium, at least within the range of 0 to 150 mM, had little influence on the reconstituted phosphate transport activity. Although glycerol and phosphate salts could be removed from the extraction buffer without significantly affecting the phosphate transport activity of the resulting proteoliposomes, this
6l
A
1986). Glycerol concentration
The reconstitution of a variety of membrane transport proteins has been shown to be greatly improved by including glycerol or another osmolyte in the solubilization buffer (Ambudkar and Maloney, 1986; Maloney and Ambudkar, 1989). In the case of the renal phosphate carrier, however, similar transport activities were observed when brush-border membrane proteins were extracted in the presence of high concentrations of glycerol (30 and 50%) or in its absence. Moreover, extracting the protein at intermediate glycerol concentrations ( 10 and 15 %) resulted in a decrease in the ability of the resulting proteoliposomes to transport phosphate in a sodium-dependent fashion. The reason for this decreased transport activity remains unknown although it was consistently observed. Ionic strength
At high ionic strength, the stability of the sodium/phosphate symporter depended on the salt used in the extraction buffer. A strong sodium-dependent phosphate transport activity was reconstituted when the protein was extracted with sodium phosphate (Table 1). Similar results were obtained when sodium
01’ 4
I 5
‘,I 6
I’ 7
I’ 8
I 9
‘: 10
PH
Fig. 4. Comparison of phosphate uptake into proteoliposomes prepared with brush-border membrane proteins extracted at different pH in buffers of high (A) and low (B) ionic strength. (A) Proteoliposomes were prepared as described in “Materials and Methods”, using an extraction buffer composed of brush-border membrane lipids (10.2 pmol of phospholipids/ml), 30% (w/v) glycerol, 1.5% (w/v) CHAPS, 1 mM dithiothreitol and 1SOmM NaH,PO,/Na,HPO,, pH 5, 6, 7, 8, or 9. (B) Proteoliposomes were prepared with an extraction buffer composed of brush-border membrane lipids (10.2 ymol of phospholipids/ml), 30% (w/v) glycerol, 1.5% (w/v) CHAPS, 1 mM dithiothreitol and 20 mM 2(N-Morpholino)ethanesulfonic acid (MES)/Tris, pH 5, MES/Tris, pH 6, Hepes/Tris, pH 7, HepeqTris, pH 8, or 2(Cyclohexylamino)ethanesulfonic acid (CHES)/Tris, pH 9. Phosphate uptake was measured at pH 7.5 in the presence of NaN4 (a) or KNO, (0). Values are means f SD of two (B) or three (A) independent experiments performed in triplicate.
316
Vincent Vachon et al. Table 1. Comparison of phosphate uptake into proteohposomes prepared with brush-border membrane proteins extracted at high ionic strength with different salts Phosphate uptake (nmol/mg protein/l5 min) Salt NaH,PO,/Na,HPO, KH2P0,/K,HP0, NaCl
Concentration (mW
NaNO,
KNO,
150 150 225
5.11 kO.41 4.76 f 0.23 2.25 + 0.21
1.94kO.12 2.01 kO.13 2.02 + 0.16
Brush-border membrane proteins were extracted with a buffer composed of the indicated salts plus 5mM Hepes/Tris, pH 7.5, I mM dithiothreitol, 30% (w/v) glycerol, 1.5% (w/v) CHAPS, and brush-border membrane lipids (10.2 pmol of phospholipids/ml), and reconstituted as described in “Materials and Methods”. Phosphate uptake was measured in the presence of NaNO, or KNO,. Values are means k SD of five independent experiments performed at least in triplicate.
activity was completely lost when both were omitted (Table 2). The fact that glycerol and phosphate can compensate for one another in stabilizing the phosphate carrier during its solubilization suggests that they may act similarly by restricting access of free water to the protein molecule and thus favor inter-subunit or intramolecular interactions which are important for stabilizing its conformation.
pH 6 and 7 (Fig. 4A). At low ionic strength, the carrier was irreversibly inactivated at pH 5, but retained its activity equally well when extracted at pH 6-9 (Fig. 4B). Dithiothreitol
PH
The effect of the pH of the extraction buffer on the stability of the sodium/phosphate symporter depended on the ionic strength. When the extraction was carried out at high ionic strength, in the presence of 150emM sodium phosphate, the phosphate transport activity was similar in the proteoliposomes prepared with protein extracted at pH 5, 8 and 9, and significantly increased in the proteoliposomes prepared with protein extracted at
The sodium/phosphate symporter also appears to be sensitive to oxidation since including 1 mM dithiothreitol in the extraction buffer resulted in a 25% increase in the reconstituted phosphate transport activity measured in the presence of a sodium gradient (6.37 + 0.38 vs 4.98 +_0.38 nmol/mg protein/l5 min; P < 0.01) without affecting the sodium-independent phosphate uptake. This effect was probably due to an increase in the activity of the transporter rather than to an increase in its stability during extraction since a similar stimulation of phosphate transport activity has been observed previously with renal brush-border membrane vesicles preincubated with dithiothreitol (Suzuki et al., 1990).
Table 2. Comparison of phosphate uptake into proteoliposomes prepared with brush-border membrane proteins extracted with or without glycerol and sodium phosphate Phosphate uptake (nmol/mg protein/l5 min) Extraction buffer Complete Without glycerol Without sodium phosphate Without glycerol nor sodium phosphate
NaNO,
KNO,
2.71 + 0.17 2.83 + 0.24 2.96 + 0.16 1.66&-0.13
1.31 kO.11 1.33_+0.12 1.35 kO.13 1.38 + 0.23
Brush-border membrane proteins were extracted with a buffer composed 5 mM Hepes/Tris, pH 7.5, 1 mM dithiothreitol, I .5% (w/v) CHAPS, brush-border membrane lipids (10.2 pmol of phosphohpids/ml), with or without 150 mM NaH,POJNa2HP0, and 30% (w/v) glycerol, and reconstituted as described in “Materials and Methods”. Phosphate uptake was measured in the presence of NaNO, or KNO1. Values are means f SD of three independent experiments performed in triplicate.
Stability of the renal sodium/phosphate symporter
6-
0-l 0
, IO
c
, 20
I 30
u
I 40
Cholesterol added (mol%) Fig. 5. Effect of added cholesterol on the uptake of phosphate into reconstituted membrane vesicles. Brush-border membrane proteins were extracted in a buffer composed of 150mM NaH,PO,/Na,HPO,, 5 mM Hepes/Tris, pH 7.5, I mM dithiothreitol, 30% (w/v) glycerol, 1.5% (w/v) CHAPS, and brush-border membrane lipids (10.2 pmol of phospholipids/ml) supplemented with different amounts of cholesterol. Because the lipid composition of the bovine brush-border membrane is unknown, the percentages indicated are based on a cholesterol-to-phospholipid ratio of 0.64 mol/mol, a value close to those reported for the renal brush-border membrane of rat (0.67) (Hise et al., 1984), dog (0.64) (Carmel et al., 1985) and rabbit (0.61) (Venien and Le Grimellec, 1988). Proteoliposomes were prepared as described in “Materials and Methods” except that the lipid suspension was supplemented with the same proportion of cholesterol as the corresponding extraction medium. Uptake of phosphate was measured in the presence of NaNO, (0) or KNO, (0). Values are means + SD of three independent experiments performed at least in triplicate.
Added cholesterol
In vitro enrichment of renal brush-border membranes with cholesterol was shown to result in a significant decrease in phosphate transport activity (Mohtoris et al., 1985; Yusufi et al., 1989; Levi et al., 1990). The sodium-dependent phosphate transport into proteoliposomes prepared with brush-border membrane lipids supplemented with different amounts of cholesterol was also decreased by such additions (Fig. 5), although to a slightly smaller extent than in brush-border membranes enriched with similar amounts of cholesterol (Levi et al., 1990). SUMMARY
AND
CONCLUSIONS
During its extraction from the brush-border membrane, the stability of the renal sodium/ phosphate cotransporter depends mainly on the concentration of the detergent, the presence of brush-border membrane lipids, and, at high ionic strength, on the presence of phosphate in the solubilization buffer. A knowledge of the importance of these parameters should be useful in designing a purification scheme for this
317
transporter which has proved to be quite readily inactivated upon extraction from its native membrane. Despite its sensitivity, purification remains, along with the recent cloning of its structural gene (Werner et al., 1990, 199i), an essential step for the identification and structural characterization of the carrier molecule, and for a detailed understanding of the molecular mechanism by which phosphate is reabsorbed by the kidney. Acknowledgements-This work was supported by grants from the Medical Research Council (MRC) of Canada to R. Beliveau, and from the Natural Sciences and Engineering Research Council (NSERC) of Canada to R. Laprade. M.-C. Delisle received a scholarship for doctoral studies from the Fonds de la recherche en Sante du Quebec (FRSQ).
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Carmel G., Rodrigue F., Carriere S. and Le Grimellec C. (1985) Composition and physical properties of lipids from plasma membranes of dog kidney. Biochim. biophys. Acta 818, 149-157. Chattopadhyay A. and London E. (1984) Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge. Analyt. Biochem. 139, 408-412. Debiec H. and Lorenc R. (1988) Identification of Nat, Pi-binding protein in kidney and intestinal brush-border membranes. Biochem. J. 255, 185-191. Debiec H., Lorenc R. and Ronco P. M. (1992) Reconstitution and characterization of a Na” /P, cotransporter protein from rabbit kidney brush-border membranes. Biochem. J. 286, 97-102. Delisle M.-C., Vachon V., Giroux S.. Potier M., Laprade R. and Beliveau R. (1992) Molecular size of the renal sodium/phosphate symporter in native and reconstituted systems. Biochim. biophys. Acta 1104, 132-136. Ducis I. and Koepsell H. (1983) A simple hposomal system to reconstitute and assay highly efficient Na+ /D-glucose cotransport from kidney brush-border membranes. Biochim. biophys. Acta 730, 119-129. Gmaj P. and Murer H. (1986) Cellular mechanisms of
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Helps C. R. and McGivan J. (1991) Adaptive regulation of Na+-dependent phosphate transport in the bovine renal epithelial cell line NBL-1. Identification of the phosphate transporter as a 55-kDa glycoprotein. Eur. J. Biochem. 200, 797-803.
Hise M. K., Mantulin W. W. and Weinman E. J. (1984) Fluidity and composition of brush border and basolateral membranes from rat kidney. Am. J. Physiol. 247, F434-F439.
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