Differential effects of ionic strength, divalent cations and pH on the pore-forming activity of Bacillus thuringiensis insecticidal toxins.

The combined effects of ionic strength, divalent cations, pH and toxin concentration on the pore-forming activity of Cry1Ac and Cry1Ca were studied using membrane potential measurements in isolated midguts of Manduca sexta and a brush border membrane vesicle osmotic swelling assay. The effects of ionic strength and divalent cations were more pronounced at pH 10.5 than at pH 7.5. At the higher pH, lowering ionic strength in isolated midguts enhanced Cry1Ac activity but decreased considerably that of Cry1Ca. In vesicles, Cry1Ac had a stronger pore-forming ability than Cry1Ca at a relatively low ionic strength. Increasing ionic strength, however, decreased the rate of pore formation of Cry1Ac relative to that of Cry1Ca. The activity of Cry1Ca, which was small at the higher pH, was greatly increased by adding calcium or by increasing ionic strength. EDTA inhibited Cry1Ac activity at pH 10.5, but not at pH 7.5, indicating that trace amounts of divalent cations are necessary for Cry1Ac activity at the higher pH. These results, which clearly demonstrate a strong effect of ionic strength, divalent cations and pH on the pore-forming activity of Cry1Ac and Cry1Ca, stress the importance of electrostatic interactions in the mechanism of pore formation by B. thuringiensis toxins.

Analysis of the properties of Bacillus thuringiensis insecticidal toxins using a potential-sensitive fluorescent probe.

A potential-sensitive fluorescent probe, 3,3'-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K(+) ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta. Fluorescence levels were strongly influenced by the pH and ionic strength of the media. Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength. Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane. Valinomycin greatly increased the potential generated by the diffusion of K(+) ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels. In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.

Ion channels induced in planar lipid bilayers by the Bacillus thuringiensis toxin Cry1Aa in the presence of gypsy moth (Lymantria dispar) brush border membrane.

The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm cis/150 mm trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much lower concentrations (1.1-2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to 420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P(Cl)/P(NMDG) permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of the pores formed by 100-500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P(Cl)/P(NMDG) permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties.

Differential effects of pH on the pore-forming properties of Bacillus thuringiensis insecticidal crystal toxins.

The effect of pH on the pore-forming ability of two Bacillus thuringiensis toxins, Cry1Ac and Cry1C, was examined with midgut brush border membrane vesicles isolated from the tobacco hornworm, Manduca sexta, and a light-scattering assay. In the presence of Cry1Ac, membrane permeability remained high over the entire pH range tested (6.5 to 10.5) for KCl and tetramethylammonium chloride, but was much lower at pH 6.5 than at higher pHs for potassium gluconate, sucrose, and raffinose. On the other hand, the Cry1C-induced permeability to all substrates tested was much higher at pH 6.5, 7.5, and 8.5 than at pH 9.5 and 10.5. These results indicate that the pores formed by Cry1Ac are significantly smaller at pH 6.5 than under alkaline conditions, whereas the pore-forming ability of Cry1C decreases sharply above pH 8.5. The reduced activity of Cry1C at high pH correlates well with the fact that its toxicity for M. sexta is considerably weaker than that of Cry1Aa, Cry1Ab, and Cry1Ac. However, Cry1E, despite having a toxicity comparable to that of Cry1C, formed channels as efficiently as the Cry1A toxins at pH 10.5. These results strongly suggest that although pH can influence toxin activity, additional factors also modulate toxin potency in the insect midgut.

Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac.

The four salt bridges (Asp(222)-Arg(281), Arg(233)-Glu(288), Arg(234)-Glu(274), and Asp(242)-Arg(265)) linking domains I and II in Cry1Aa were abolished individually in alpha-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp(242)-Arg(265) bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.

Exchange of domain I from Bacillus thuringiensis Cry1 Toxins Influences protoxin stability and crystal formation.

Influence of domain I exchange on the stability and production of Bacillus thuringiensis Cry1 protoxins as well as on the shape of inclusion and toxicity to Spodoptera exigua and Plutella xylostella larvae was investigated. Chimeric genes were prepared by exchanging the regions coding for domain I between Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The AcCC chimera accumulated into bipyramidal inclusion bodies, whereas CEE produced round-shaped inclusion bodies, and ECC and AaEE protoxins produced small granules. AbEE and EAaAa did not produce any inclusion body and were visualized by immunodetection only. AcCC, CEE, ECC, and AaEE were stable to trypsin, whereas AbEE and EAaAa were not. Bioassays showed that the chimeras were not toxic in vivo. However, S. exigua larvae fed with the activated AcCC toxin displayed a lower growth rate.

Ion channel activity from the midgut brush-border membrane of gypsy moth (Lymantria dispar) larvae.

Ion channels from the midgut apical membrane of gypsy moth (Lymantria dispar) larvae were studied following mechanical fusion of brush-border membrane vesicles with planar phospholipid bilayer membranes. In symmetrical 300 mmol l(-)(1) KCl (pH 9.0), nine different channels with conductances ranging from 27 to 795 pS and linear current/voltage relationships were resolved. In the presence of a KCl gradient across the bilayer (450 mmol l(-)(1 )cis/150 mmol l(-)(1 )trans), 11 different conductance levels ranging from 16 to 850 pS were detected. The channels were slightly cationic: the zero-current reversal potential was shifted by -5 mV to -21 mV compared with symmetrical KCl conditions, corresponding to p(K)/p(Cl) permeability ratios of 1.5-8.0. Most channels were neither voltage-dependent nor Ca(2+)-sensitive and displayed complex gating kinetics. Addition of Ba(2+) or Cs(+) to both sides of the bilayer had little effect on channel activity, but fewer distinct channels were observed when KCl was replaced by potassium gluconate, suggesting an effect of Cl(-) on channel activity. A reduced number of channels was also detected when KCl was replaced by N-methyl-d-glucamine-HCl. Under asymmetrical N-methyl-d-glucamine-HCl conditions, only anionic channels were observed. They exhibited current rectification (35 pS at negative voltages and 81 pS at positive voltages) and were strongly voltage-dependent.

Kinetic properties of the channels formed by the bacillus thuringiensis insecticidal crystal protein Cry1C in the plasma membrane of Sf9 cells.

Spectrofluorimetric measurements were conducted to quantify, in real-time, membrane permeability changes resulting from the treatment of Sf9 insect cells (Spodoptera frugiperda, Lepidoptera) with different Bacillus thuringiensis Cry insecticidal proteins. Coumarin-derived CD222 and Merocyanin-540 probes were respectively used to monitor extracellular K(+) and membrane potential variations upon Sf9 cells incubation with Cry toxins. Our results establish that Cry1C induces, after a delay, the depolarization of the cell membrane and the full depletion of intracellular K(+). These changes were not observed upon Sf9 cells treated with Cry1A family toxins. Both the rate of the K(+) efflux and the delay before its onset were dependent on toxin concentration. Both parameters were sensitive to temperature but only the delay was affected by pH. Cry1C-induced K(+) efflux was inhibited by lanthanum ions in a dose-dependent manner. This study provides the first kinetic and quantitative characterization of the ion fluxes through the channels formed by a Cry toxin in the plasma membrane of a susceptible insect cell line.

Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins.

Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C.

Real-time fluorimetric analysis of gramicidin D- and alamethicin-induced K+ efflux from Sf9 and Cf1 insect cells.

Gramicidin D and alamethicin are pore-forming peptides which exhibit lethal properties against a large spectrum of cells. Despite a wealth of experimental data from artificial membranes, the time course and quantitative analysis of the activity of these ionophores are not well described in living cells. In the present study, the newly described fluorescent dye CD-222 was used to monitor extracellular potassium ion concentration and report the effects of these antibiotics on the K+ permeability of the plasma membrane of Spodoptera frugiperda (Sf9) and Choristoneura fumiferana (Cf1) insect cells. Both peptides induced a rapid efflux of intracellular K+ as a consequence of ion channel formation in the cell membrane. K+ efflux began without any measurable delay. While the final extracellular K+ concentration was unaffected by ionophore concentration, the rate of K+ efflux was dose dependent. Using a model describing the partition of the peptides in lipid membranes, the K+ efflux kinetic parameters were determined for both cell types and both pore formers. The proposed stoichiometry for the channel formed by gramicidin in living cells is in good agreement with the two-monomers model based on data from artificial membrane systems. The K+-permeable channel formed by alamethicin in insect cells appears to involve three monomers.

Video imaging analysis of the plasma membrane permeabilizing effects of Bacillus thuringiensis insecticidal toxins in Sf9 cells.

The size and ionic selectivity of the pores formed by the insecticidal crystal protein Cry1C from Bacillus thuringiensis in the plasma membrane of Sf9 cells, an established cell line derived from the fall armyworm Spodoptera frugiperda, were analyzed with a video imaging technique. Changes in the permeability of the membrane were estimated from the rate of osmotic swelling of the cells. In the presence of Cry1C, which is toxic to Sf9 cells, the permeability of the cell membrane to KCl and glucose increased in a dose-dependent manner. In contrast, Cry1Aa, Cry1Ab and Cry1Ac, toxins to which Sf9 cells are not susceptible, had no detectable effect. Pores formed by Cry1C allowed the diffusion of sucrose, but were impermeable to the trisaccharide raffinose. On the basis of the hydrodynamic radii of these substances, the diameter of the pores was estimated to be 1.0-1.2 nm. In the presence of salts, the rate of swelling of cells exposed to Cry1C was about equally influenced by the size of the anion as by that of the cation, indicating that the ionic selectivity of the pores is low.

Radiation-inactivation analysis of the oligomeric structure of the renal sodium/D-glucose symporter.

The radiation-inactivation size (RIS) of the rat renal brush-border membrane sodium/D-glucose cotransporter was estimated from the loss of transport activity in irradiated membrane vesicles. The RIS depended on the electrochemical conditions present when measuring transport activity. A RIS of 294 +/- 40 kDa was obtained when transport was measured in the presence of a sodium electrochemical gradient. Under sodium equilibrium conditions, the RIS was 84 +/- 25 kDa in the presence of a glucose gradient, and 92 +/- 20 kDa in its absence. In the absence of a sodium gradient, but in the presence of an electrical potential gradient, the RIS increased to 225 +/- 49 kDa. The 294 kDa result supports earlier suggestions that the Na+ gradient-dependent glucose transport activity is mediated by a tetramer. Individual monomers appear, however, to carry out glucose transport under equilibrium exchange conditions or when a glucose gradient serves as the only driving force. The electrical potential gradient-driven glucose transport RIS appears to involve three functional subunits.

Effect of Bacillus thuringiensis toxins on the membrane potential of lepidopteran insect midgut cells.

To test whether the ability of Bacillus thuringiensis toxins to form pores in the midgut epithelial cell membrane of susceptible insects correlates with their in vivo toxicity, we measured the effects of different toxins on the electrical potential of the apical membrane of freshly isolated midguts from gypsy moth (Lymantria dispar) and silkworm (Bombyx mori) larvae. In the absence of toxin, the membrane potential, measured with a conventional glass microelectrode, was stable for up to 30 min. It was sensitive to the K+ concentration and the oxygenation of the external medium. Addition of toxins to which L. dispar is highly [CryIA(a) and CryIA(b)] or only slightly [CryIA(c) and CryIC] sensitive caused a rapid, irreversible, and dose-dependent depolarization of the membrane. CryIF, whose toxicity towards L. dispar is unknown, and CryIE, which is at best poorly active in vivo, were also active in vitro. In contrast, CryIB and CryIIIA, a coleopteran-specific toxin, had no significant effect. The basolateral-membrane potential was unaffected by CryIA(a) or CryIC when the toxin was applied to the basal side of the epithelium. In B. mori midguts, the apical-membrane potential was abolished by CryIA(a), to which silkworm larvae are susceptible, but CryIA(b) and CryIA(c); to which they are resistant, had no detectable effect. Although the technique discriminated between active and inactive toxins, the concentration required to produce a given effect varied much less extensively than the sensitivity of gypsy moth larvae, suggesting that additional factors influence the toxins' level of toxicity in vivo.

Involvement of disulphide bonds in the renal sodium/phosphate co-transporter NaPi-2.

The rat renal brush border membrane sodium/phosphate co-transporter NaPi-2 was analysed in Western blots with polyclonal antibodies raised against its N-terminal and C-terminal segments. Under reducing conditions, proteins of 45-49 and 70-90 kDa (p45 and p70) were detected with N-terminal antibodies, and proteins of 40 and 70-90 kDa (p40 and p70) were detected with C-terminal antibodies. p40 and p45 apparently result from a post-translational cleavage of NaPi-2 but remain linked through one or more disulphide bonds. Glycosidase digestion showed that both polypeptides are glycosylated; the cleavage site could thus be located between Asn-298 and Asn-328, which have been shown to constitute the only two N-glycosylated residues in NaPi-2. In the absence of reducing agents, both N-terminal and C-terminal antibodies detected p70 and a protein of 180 kDa (p180), suggesting the presence of p70 dimers. Much higher concentrations of beta-mercaptoethanol were required to produce a given effect in intact membrane vesicles than in solubilized proteins, indicating that the affected disulphide bonds are not exposed at the surface of the co-transporter. Phosphate transport activity decreased with increasing concentrations of reducing agents [beta-mercaptoethanol, dithiothreitol and tris-(2-carboxyethyl)phosphine] and was linearly correlated with the amount of p180 detected. The target sizes estimated from the radiation-induced loss of intensity of p40, p70 and p180 were all approx. 190 kDa, suggesting that NaPi-2 exists as an oligomeric protein in which the subunits are sufficiently close to one another to allow substantial energy transfer between the monomers. When protein samples were pretreated with beta-mercaptoethanol [2.5% and 5% (v/v) to optimize the detection of p40 and p70] before irradiation, target sizes estimated from the radiation-induced loss of intensity of p40 and p70 were 74 and 92 kDa respectively, showing the presence of disulphide bridges in the molecular structure of NaPi-2.

The renal sodium/phosphate symporters: evidence for different functional oligomeric states.

The oligomeric size of the rat renal sodium/phosphate symporters was estimated in brush-border membrane vesicles submitted to radiation inactivation. Altering the electrochemical conditions under which phosphate transport was measured resulted in different molecular size determinations. The radiation inactivation size (RIS) obtained from the radiation-induced loss of transport activity measured in the presence of a sodium gradient was 200 kDa. Under sodium equilibrium conditions, in the presence of a phosphate gradient as the only driving force, transport fell to 13% of the activity measured in the presence of a sodium gradient, and the RIS was 62 kDa. Addition of an outwardly-directed proton gradient increased the transport activity to 29% of that measured in the presence of a sodium gradient. The RIS measured under these conditions was 124 kDa. Under all conditions tested, phosphate uptake by irradiated vesicles was significantly reduced but remained linear during the first 5 s of incubation. The radiation-induced loss of transport activity was thus attributable to a direct inactivation of the transporter rather than to a decrease in the physical integrity of the vesicles. These results are consistent with a tetrameric structure composed of subunits of about 62 kDa and suggest that phosphate transport involves both monomers and tetramers.

Endogenous K+/H+ exchange activity in the Sf9 insect cell line.

The fluorescent pH indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to investigate changes in the intracellular pH (pHi) of individual Sf9 cells in response to changes in the composition of the external medium. Under standard conditions, the resting pHi was 0.2-0.3 unit lower than the extracellular pH (6.5). The extracellular concentration of K+ had a major influence on pHi. Removal of K+ from the medium resulted in a rapid but reversible acidification of the cells. The buffer capacity of the cells was a U-shaped function of pHi with a minimum at about pHi = 6.2. In the presence of K+, a change in the pH of the medium was followed by an equivalent change in pHi. In its absence, however, changes in the external pH had little effect on the pH of the cells. Following removal of K+ from the medium, the cells realkalinized at an initial rate which increased with increasing concentration of added K+. This cation was about 30 times more effective in promoting realkalinization of the cells than Li+ Na+, Rb+, and Cs+. The apparent Km for K(+)-dependent H+ efflux was about 12 mM and was slightly modulated by extracellular pH. These results strongly suggest that, in Sf9 cells, a K+/H+ antiporter plays a key role in the movement of protons across the cell membrane.

Ionic permeabilities induced by Bacillus thuringiensis in Sf9 cells.

The effect of Bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular pH (pHi) of individual Sf9 cells of the lepidopteran species Spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (PBFI) and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The sequential removal of K+ and Na+ from the medium, in the presence of CryIC, a toxin which is highly active against Sf9 cells, caused sharp shifts in the fluorescence ratio of PBFI, demonstrating a rapid efflux of these ions. In Sf9 cells, pHi depends strongly on the activity of a K+/H+ exchanger. In the absence of toxin, removal of K+ from the external medium resulted in a reversible acidification of the cells. In the presence of CryIC, pHi equilibrated rapidly with that of the bathing solution. This effect was both time- and concentration-dependent. In contrast with CryIC, CryIIIA, a coleopteran-specific toxin, and CryIA(a), CryIA(b) and CryIA(c), toxins which are either inactive or poorly active against Sf9 cells, had no detectable effect on pHi. B. thuringiensis endotoxins thus appear to act specifically by increasing the permeability of the cytoplasmic membrane of susceptible cells to at least H+, K+ and Na+.

Molecular study of P-glycoprotein in multidrug resistance using surface plasmon resonance.

P-Glycoprotein is an integral membrane protein which mediates the energy-dependent efflux of various antitumor agents from multidrug-resistant cancer cells. Surface plasmon resonance was used for the detection of P-glycoprotein after solubilization from drug-resistant and drug-sensitive Chinese hamster ovary cells and for the analysis of its interaction with cyclosporin A, a competitive inhibitor of drug efflux. Detection of P-glycoprotein relied on its binding to the monoclonal antibody C219 which was immobilized on a sensor chip. Binding of Zwittergent 3-14-solubilized P-glycoprotein to the antibody was concentration-dependent and reflected the relative abundance of P-glycoprotein in both cell lines. It was abolished when C219 was omitted or replaced by a rabbit anti-mouse IgG antibody and considerably reduced after precipitation of P-glycoprotein with wheat germ agglutinin. Preincubation of solubilized proteins with cyclosporin A increased the amount of protein bound to the antibody by approximately 30%. These results indicate that surface plasmon resonance is well suited to the detection of P-glycoprotein from biological samples and shows promise as a tool for the study of its interaction with different drugs.

Factors affecting the stability of the renal sodium/phosphate symporter during its solubilization and reconstitution.

Phosphate is reabsorbed across the brush-border membrane of the proximal tubule by a specific sodium-dependent symporter. Like the other brush-border membrane transport proteins of the kidney, the phosphate carrier remains to be isolated in a functional state. To establish a set of parameters that allow to preserve its biological activity, the phosphate carrier was solubilized under systematically varied conditions and reconstituted into proteoliposomes. Successful reconstitution was achieved only when the extraction buffer contained lipids extracted from the renal brush-border membrane. Glycerol, an osmolyte which reduces the water activity of the solution, was also required. It could however be replaced by 150 mM sodium or potassium phosphate. Below this concentration and in the presence of glycerol, the ionic strength of the solution had little effect on the stability of the transporter, but sodium phosphate could not be replaced by sodium chloride. Phosphate transport in reconstituted vesicles depended on the concentration of detergent and pH of the extraction buffer. Finally, transport activity was increased when solubilization was carried out in the presence of a reducing agent, dithiothreitol. These results should be helpful during the purification and further characterization of the renal phosphate symporter.

Molecular size of the functional complex and protein subunits of the renal phosphate symporter.

The oligomeric structure of the rabbit renal brush-border membrane sodium/phosphate cotransporter was examined with the radiation inactivation and fragmentation technique. The size of its functional complex (its "radiation inactivation size") was estimated from the rate of decay of its sodium-dependent transport activity as a function of the radiation dose. A radiation inactivation size of 223 +/- 42 kDa was obtained. The polypeptide constituting the monomeric unit of the Na1+/Pi symporter was detected by immunoblotting with polyclonal anti-peptide antibodies directed against the 14 amino acid C-terminal portion of the symporter molecule. Its apparent molecular size estimated by comparison with standards following SDS-polyacrylamide gel electrophoresis was 64,000. This value is in good agreement with its known molecular mass of 51,797 Da calculated from the amino acid sequence deducted from the nucleotide sequence of its gene since this protein is probably glycosylated. The loss of labeling intensity of the polypeptide of M(r) = 64,000 was also measured as a function of radiation dose. The molecular size calculated from these data (its "target size") was 165 +/- 20 kDa. The target size estimated for the rat phosphate cotransporter was 184 +/- 46 kDa, and its previously reported radiation inactivation size was 234 +/- 14 kDa. These results strongly suggest that the renal Na1+/Pi cotransporter exists as an oligomeric protein, probably a homotetramer. The fact that the values obtained for the target size are about 3/4 those obtained for the radiation inactivation size of these cotransport proteins indicates that their subunits are closely associated since most of their subunits appear to be fragmented by a single ionizing radiation hit.