Forskolin Specifically Inhibits the Bacterial Galactose-H+ Transport Protein, GalP*

Forskolin is a potent inhibitor of mammalian passive glucose transporters. Here we show that forskolin is a remarkably specific inhibitor of energized D-galactose transport by the GalP sugar-H+ symport protein of Escherichia coli. Surprisingly, it does not inhibit transport of L-arabinose or n-xylose by the related E. coli AraE and XylE transporters, even though the amino acid sequences of their proteins are 30-6470 identical to GalP and to the mammalian GLUT family. However, unlike GLUT1, photoactivation of the [SH]forskolin-GalP complex fails to incorporate radioactivity covalently into the protein, in contrast to the effective incorporation of radioactivity from [SHlcytochalasin B into both pro- teins. However, 3-[’2s1]iodo-4-azidophenethylamido-7-O-succinyldesacetylforskolin (~’261]APS-forskolin), which labels

5 Recipient of a studentship from the Science and Engineering Research Council, with additional support from the Cambridge Philosophical Society. ** Supported by National Institutes of Health Grant DK 36855 and a grant from the American Heart Association, Illinois Affiliate .
Sf Recipient of a studentship from the Medical Research Council. Present address: Dept. of Biochemistry, University of Southampton, Southampton, SO9 3TU UK.
Forskolin is a potent reversible inhibitor of the mammalian GLUTl and possibly other GLUT transporters (Laurenza et al., 1989;Sergeant and Kim, 1985;Joost et al., 1988). From its chemical formula, it can be visualized in a conformation where part of the molecule is strikingly similar to n-galactose ( Fig. 1).
In this study we show that only the D-galactose-Wl symporter, GalP, of all the known bacterial sugar-Hi symporters, is susceptible to inhibition by forskolin. This specificity is remarkable, especially because the D-galactose-H' (GalP), L-arabinose-H+ (AraE), and D-xylose-H+ (XylE) symporters are 28-64% identical in amino acid sequence to each other and to a family of sugar transporters, including GLUT1-5 and 7, found in diverse organisms from cyanobacteria to man (Henderson et al., 1992;Baldwin, 1993). In Escherichia coli, there are also sugar-H+ symport proteins for the uptake of L-fucose (FucP), Lrhamnose (RhaT), and lactose (Lacy). The parentheses contain their designated phenotype names; their properties have been reviewed recently (Henderson et al., 1992). FucP, RhaT, and Lacy have amino acid sequences that are not recognizably similar to GalP, AraE, or XylE or to each other (Henderson et al., 1992;Tate et al., 19921, and so the observed insensitivity to forskolin is not unexpected. However, all are predicted to adopt a 12-helix configuration in the membrane (Henderson, 1993) except for RhaT, which is expected to be a 10-helix protein . In order to demonstrate sensitivity to forskolin, the outer membrane of the bacterial cell must be permeabilized, or rightside-out subcellular vesicles must be used, presumably to allow the antibiotic to reach the inner membrane where the sugar-H' symport proteins are located.

4-azidophenethylamido-7-O-succinyldesacetylforskolin (['2511-
APS-forskolin)2 (Wadzinski et al., 19871, are potent labeling reagents €or the human GLUTl protein (Shanahan et ai., 1987;Wadzinski et al., 1988). Surprisingly, [3Hlforskolin did not label GalP when irradiated, but both GalP and AraE were labeled by [1251]APS-forskolin. The appropriate sugar substrates protected the proteins against the photolabeling. The binding of L3H1forskolin to the overexpressed GalP protein could be measured directly in equilibrium dialysis experiments, which enabled calculation of the dissociation constant for forskolin and of the number of binding sites; the appropriate sugar substrates or cytochalasin B displaced forskolin from the protein. The potencies of forskolin, cytochalasin B, phloridzin, and phloretin as The D or L form of a sugar is distinguished on first mention, but it is * The abbreviations used are: [12511AF'S-forskolin, 3-112511iodo-4-azido-omitted thereafter unless essential for clarity. inhibitors of the GalP, AraE, and XylE proteins are compared with their effectiveness against the mammalian sugar transport proteins. The GalP and AraE proteins are both susceptible to cytochalasin B (Cairns et al., 1991), which is a well characterized inhibitor of the GLUT proteins (Bloch, 1973;Jung and Rampal, 19771, and irradiation of either GalP or AraE with UV light in the presence of [3Hlcytochalasin B leads to the covalent attachment of radioactivity to the protein, which also occurs with GLUTl (Carter-Su et al., 1982;Charalambous et al., 1989;Cairns et al., 1991).
These novel observations first reinforce the conclusion that the structure-activity relationship of the bacterial GalP protein is very similar to that of the human GLUTl protein. GalP is, therefore, an excellent model with which to exploit the convenience of investigations in E. coli (e.g. ease of genetical manipulation and cheap production of large quantities of active protein) to elucidate the molecular features of the binding sites for inhibitors and for substrates and the mechanism of the translocation process for a most important family of nutrient transport proteins. Secondly, forskolin and its chemical derivatives provide novel tools with which to dissect the subtle molecular differences between GalP and GLUTl and between these proteins and AraE or XylE, which will enable the structural parameters critical for discrimination between substrates and inhibitors to be determined.

EXPERIMENTAL PROCEDURES
Materials-Sugars and sugar analogues (glucose-free where appropriate) were obtained from Sigma. The transport inhibitors cytochalasin B, phloridzin, and phloretin were from Aldrich; forskolin was from Sigma. ~-[l-~H]Galactose, ~-  were obtained f r o m h e r s h a m Corp.; [12-3Hlforskolin was from DuPont NEN; and ~-[U-l~Clarabinose and ~-[l-~~Clrhamnose were from CEA, Fluorochem Ltd.
Organisms-The E. coli strains used in this study are given in Table   I. The strains were grown on either TY medium (10 g of tryptone, 10 g of yeast extract, and 5 g of NaCl in 1 liter) or minimal salts  containing 20 mM glycerol and supplemented with the appropriate growth requirements at 80 pg/ml (Table I). Plasmids were maintained by the addition of ampicillin (100 pg/ml) and tetracycline (15 pg/ml) where appropriate. Gene expression from the lambda O,P, promoter was induced by the addition of nalidixic acid to the culture (Maiden et dl., 1988), which inactivates the CI A repressor encoded on the chromosome of the lysogenic host strain, E. coli AR120. The other plasmids express the transport protein constitutively.
Preparation of Subcellular Vesicles-Subcellular vesicles were either prepared from intact cells by explosive decompression in a French press (Futai, 1978) or from spheroplasts (Witholt et al., 1976 Witholt andBoekhout, 1978) by the method of Kaback (Kaback, 1971). The former procedure yields predominantly inside-out vesicles, whereas the latter yields predominantly right-side-out vesicles.
Preparation of Inner Membranes-Inner membranes were purified from French press vesicles by sucrose density gradient centrifugation (Osborn et al., 1972). The "golden" inner membrane fraction was isolated from a zone between 30 and 40% sucrose.
Equilibrium Binding of Forskolin-Equilibrium dialysis was carried out as described by Walmsley et al. (1993). The binding of [3Hlforskolin to inner membranes (1 mg/ml) containing GalP was measured over the range of 0.05-30 p~ forskolin a t 8 "C in the presence or absence of unlabeled sugar. Ratios of bound to free forskolin were calculated from the equilibrium distribution of the radiolabeled ligand and used to determine both the Kd and the number of forskolin binding sites by an unweighted nonlinear least squares fit of the data to an hyperbola using the Biosoft program Ultrafit 2.1.
Photoaffinity Labeling-Photoactivatable ligands were incorporated into membrane proteins by irradiation with ultraviolet light (Carter-Su et al., 1982;Cairns et al., 1991). Inner membrane vesicles (2 mg/ml) were preincubated with or without 500 mM sugar at 4 "C in photolabeling buffer (50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH7.4) and transferred to quartz cuvettes containing 0.4 nM [12511APS-forskolin, 0.5 [3HJcytochalasin B or 0.5 p~ [3Hlforskolin. The samples were flushed with argon to reduce free radical production and irradiated with ultraviolet light (R52G lamp, U.V. Products Inc., San Gabriel, CA) for 10 min. After irradiation, noncovalently bound ligand was removed by diluting the sample in photolabeling buffer, containing 1% mercaptoethanol (for ['2511APS-forskolin labeling) or 50 p unlabeled cytochalasin B or forskolin (depending on the radioactive ligand used), and centrifuging (130,000 x g for 2 h a t 4 "C). The resulting membrane pellet was resuspended in 15 mM TridHC1, pH 7.5 and assayed for protein, and 25-pg samples were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.
Detection of Radiolabeled Proteins-An SDS-polyacrylamide gel was dried under vacuum a t 80 "C onto Whatman No. 3" chromatography paper using a Bio-Rad slab gel dryer, model 1125B. Detection of tritium was achieved by presoaking the gel in a fluorography reagent (Amplifym, Amersham Corp.). Dried gels containing 1251-radiolabeled protein were examined by autoradiography. For quantitative counting, each track of the gel was sliced into 1-mm strips, and the y emissions were measured using the Packard Multiplar 4 Gamma counter. To quantitate the incorporation of [3Hlcytochalasin B, the region of the SDS-polyacrylamide gel containing the radioactive protein of interest was cut out of the gel and dissolved in 100 p1 of hydrogen peroxide overnight; the incorporated tritium was then measured by liquid scintillation counting.
Permeabilization of the Outer Membrane o f E . coli-The outer membrane of E. coli was made permeable to hydrophobic compounds by treatment with Tns-EDTA (Leive, 1965). Cells were equilibrated in 200 mM TridHCl, pH 8.0, before the addition of an equal volume of 200 mM Tris/HCl, pH 8.0,l mM EDTA. After 30 min, 100 volumes of 150 mM KCl, 5 mM MES, pH 6.5,10 mM MgSO, were added and the cells harvested by centrifugation.
Separation of Proteins by Gel Electrophoresis and Densitometry-SDS-polyacrylamide gel electrophoresis and staining of separated proteins with Coomassie Blue were performed by the procedures of Henderson and Macpherson (1986). The amounts of overexpressed GalP protein were measured by quantitative densitometry of the Coomassiestained gel using a Molecular Dynamics lOOA computing densitometer and expressed as percentage of the total membrane protein as analyzed by the Imagequant program.
Determination of Protein Concentration-Protein concentrations were determined by the method of Schaffner and Weissman (1973).    Roberts (1992) 19831, while the other route for galactose transport into the cells, designated Mgl (Robbins, 1975;Robbins and Rotman, 1975;Henderson and Giddens, 19771, is inactive. When cells were exposed to 80 p~ forskolin for periods of up to 30 min, there was very little effect of the forskolin on the time course of galactose accumulation (Fig. 2a). However, when the cell wall was permeabilized by treatment with Tris-EDTA, pH 8 (see "Experimental Procedures") galactose transport was almost completely inhibited by the forskolin within 3 min (Fig. 2b). The permeabilization process itself reduced transport by 15-40%. In separate experiments (data not shown), the permeabilized forskolin-treated cells were washed after inhibition of transport had been demonstrated, and the transport activity of the cells was found to be restored by a t least 90%; this indicates that the binding of forskolin is reversible.

Sensitivity to Forskolin Requires Permeabilization
Subsequent investigations of the effects of forskolin were, therefore, performed on right-side-out subcellular vesicles made by the method of Kaback (19711, in which access of antibiotics to the cytoplasmic membrane is unimpeded by cell wall components. An additional advantage of using vesicles is that alternative routes of entry via binding protein-dependent or phosphotransferase transport systems is eliminated. Sensitivity of E. coli Sugar-H+ Symport Systems to Forsko-1in"Subcellular vesicles were prepared from appropriate E. coli strains (Table I) induced for the presence of either the GalP, AraE, XylE, RhaT, FucP, or Lacy transport systems. Measurements of galactose transport at increasing concentrations of forskolin showed that 80 p~ forskolin gave nearly 90% inhibition of the GalP transport system, with an Io,5 value of about 6 J~M (Fig. 3). However, arabinose transport by AraE and xylose transport by XylE were insensitive to forskolin (Fig. 31, despite their 64 and 34% identities in amino acid sequence to GalP (Henderson, 1990;Roberts, 1992).
The differential sensitivities of the three homologous transporters were confirmed by repeating the measurements using a fixed high concentration of 80 J~M forskolin (Fig. 4). Furthermore, the transport of rhamnose by RhaT, of fucose by FucP, and of lactose by Lacy were insensitive to 80 p~ forskolin (Fig. 4).
Despite the functional similarity of all of these transporters and the high degree of structural similarity of GalP, AraE, and XylE inferred from the homology of their sequences, only GalP is susceptible to forskolin.
Sensitivity of GalP to Different Inhibitors of Mammalian GLUT ll-ansporters-The mammalian glucose transporter, GLUT1, of erythrocytes is inhibited by cytochalasin B and phloretin as well as by forskolin (reviewed by Baldwin (1993)). The glucose-Na' symporter, SGLT, of intestine and kidney is insensitive to all of these, but it is powerfully inhibited by the glucosidic phloretin derivative, phloridzin (Aronson and Sacktor, 1975). Phloridzin does inhibit GLUT1, but it does so with such a low affmity that it is thought to be nonspecific (Krupka, 1971). Cytochalasin B is already known to inhibit the GalP and AraE sugar-H' symporters of E. coli (Petro, 1988;Cairns et al., 1991;Walmsley et al., 1993), and the relative effectiveness of all these inhibitors against GalP, AraE, and XylE was examined using a fixed concentration of 80 p~ inhibitor.
The results (Fig. 5a) show that GalP, like the mammalian GLUT1 transporter, is inhibited by both forskolin and cytochalasin B. Despite its 64% identity to GalP, AraE is sensitive only to cytochalasin B and not to forskolin. XylE is 34% identical to GalP and 29% identical to AraE, but it is not sensitive to either Only transport by the GalP protein is sensitive to forskolin in E. coli. The effect of forskolin (80 p~) on the rate of sugar uptake by sugar transport proteins of E. coli was measured. Transport assays were carried out using right-side-out vesicles prepared from strains JM1576 (expressing GalP), SB5314 (AraE), EJ68 (XyIE), JM2418 (FucP), and JM2513 (RhaT, Lacy). Uptake was assayed after 15 s to calculate the initial rate of transport, which is expressed as a percentage of the control value. The points represent the mean S.D. of at least five uptakes. cytochalasin B or to forskolin. None of the bacterial transporters is affected by phloridzin, which is consistent with the absence of sequence homologies between any of these sugar-H+ symporters and the mammalian glucose-Na' symporter; it also fits with the insensitivity of GLUT1, with which GalP, AraE, and XylE are homologous, to phloridzin.
With 80 VM phloretin, AraE appeared to be more sensitive than XylE or GalP (Fig. 5a). However, when the concentration of phloretin was increased, all three were progressively inhibited, to over 80% a t 240 PM phloretin (Fig. 56). Phloretin, therefore, fails to exhibit the discrimination between the homologous transporters that is so striking for the other inhibitors.
Forskolin and cytochalasin B, but not phloridzin or phloretin, will be invaluable tools for elucidating the subtle differences between the ligand binding sites of the GalP and the AraE proteins since the two proteins are 64% identical in amino acid sequence, yet GalP binds both forskolin and cy- calculate the initial rate of transport, which is expressed as a percentage of the control value. Each point represents the mean of at least three separate uptakes.
(pPER3) (Roberts, 1992). The membranes were mixed with ['2sIlAPS-forskolin and irradiated. To confirm that ['2sI]APSforskolin could label GalP specifically, the effect of sugars on the incorporation of radioactivity was also investigated. Membranes containing overexpressed levels of GalP were photolabeled in the presence of D-or L-galactose, Dor L-glucose, or cytochalasin B to show that ligands recognized by GalP could reduce the incorporation of inhibitor. The hexose D-galactose is the physiological substrate for the D-galactose-H' transporter, whereas D-glucose is the tightest binding substrate ligand under nonphysiological conditions (Horne and Cairns et al., 1991;Walmsley et al., 1994b). Incorporation of label was also compared with that into membranes prepared from the GalP-deleted host strain (E. coli strain JM1100) carrying the control plasmid pBR322. The majority of the photolabel was incorporated into a single protein having an equivalent mobility to that of the Coomassie Blue-stained GalP protein (35 kDa) in the membrane preparation (Fig. 6). D-Galactose significantly reduced incorporation of radioactivity compared with L-galactose, suggesting that ['2sIlAF'S-forskolin is specifically labeling the GalP protein. The major labeled species predicted to be the GalP protein is only seen for membranes containing overexpressed GalP, and is not present in the control membranes (data not shown). Furthermore, incorporation of radioactivity is substantially reduced in the presence of D-galactose and D-glucose, and less so with the inhibitor cytochalasin B; D-glucose gave the highest level of (pPER3) (pMM27) (pBJM1) lM1100 AR120 AR120 protection (83%) compared with D-galactose (67%), reflecting that D-glucose is the better substrate. In both cases, the Lsugars afforded much less protection (23-31%), indicative of the expected stereospecificity of sugar recognition. Cytochalasin B also displaced the ligand (37%), but was not very effective, perhaps because the concentration of 80 PM is insufficient to displace the much tighter binding ['2511APS-forskolin (Wadzinski et al., 1988).
The minor labeled proteins in the membrane most likely represent either oligomers or breakdown products of the sugar transporter, since incorporation of the radioactivity into these proteins is also reduced by substrate (Fig. 6).
Photoaffinity Labeling of the AraE and XylE Proteins with ['2~511APS-forskolin-Inner membrane vesicles containing overexpressed levels of either AraE or XylE, E. coli strains AR120 (pMM25) and AR120 (pBJMl), respectively (Table I), were also irradiated in the presence of ['2sIlAPS-forskolin. A protein of equivalent electrophoretic mobility to AraE (35 kDa) was labeled by ['251]APS-forskolin; however, a band of identical mobility was also labeled in XylE membranes but migrating a t a lower mobility than that expected for XylE, 38 kDa (Fig. 6). It was, therefore, unclear whether the labeled protein was actually AraE or GalP.
To see whether ['251]APS-forskolin was being incorporated into the AraE protein, inner membranes from strain AR120 (pMM27) containing the overexpressed AraE protein were labeled with either ['251]APS-forskolin or with [SHlcytochalasin B in the presence of L-arabinose, D-glucose, D-galactose, or D-arabinose (Fig. 7). [3H]Cytochalasin B was displaced from the membranes specifically by the pentose L-arabinose (77%), while the hexose sugars D-glucose (20%) and o-galactose (24%) afforded very little protection, which is consistent with labeling ofAraE and not GalP. However, ['251]APS-forskolin was displaced most strongly by the hexoses, D-glucose (74%) and D-galactose (63%), and very poorly by the pentose, L-arabinose (19%), consistent with labeling of GalP and not AraE. Since the L-arabinose-H+ transporter discriminates against sugars carrying a C-6 hydroxyl group (Petro, 1988;Walmsley et al., 19931, i t appears likely that the major species labeled by ['2sIlAPS-forskolin is not the overexpressed AraE but instead a low level of GalP protein, which has an equivalent electrophoretic mobility. This interpretation requires that ['2sI]APS-forskolin has a much higher affinity for GalP than AraE, so that most of it interacts preferentially with the low level of GalP rather than the overexpressed AraE protein. In contrast, the majority of L3H1cytochalasin B interacts with the AraE protein because it has a moderately higher affinity for AraE than GalP (Cairns et al., 1991;Walmsley et al., 1993), and because AraE is much more abundant than GalP in these membrane preparations. This would also explain the findings of Wadzinski et al. (19901, who reported the apparently anomalous result that a [12511APS-forskolin-labeled protein was protected by D-glucose in L-arabinose-induced strains of E. coli. It would appear that this labeled species was most likely the GalP protein rather than an arabinose transporter. To clarify whether GalP, AraE, or both proteins were being labeled by ['251]AF'S-forskolin the AraE protein was expressed in the g a l P strain, E. coli strain JM1100, using the expression construct plasmid pMTC2O (Table I), which produces AraE constitutively, albeit a t low levels (below that of the sensitivity of detection by Coomassie Blue stain). Inner membrane vesicles were irradiated with ['251]APS-forskolin in the presence of Larabinose or D-fucose (substrates for AraE) or the hexoses Dglucose or D-galactose (substrates for GalP) in addition to cytochalasin B and forskolin. D-Arabinose was included as a negative control (Fig. 8) The major labeled species had a mobility equivalent to that expected for AraE (35 kDa). Furthermore, high levels of pro- tection were given by L-arabinose (59%) and D-fucose (55961, compared with low levels of protection afforded by D-galactose (14%) and D-glucose (6%). It, therefore, appears that the glucose/galactose-sensitive labeling in the membranes of E. coli strain AR120 was most likely due to preferential incorporation into GalP and that [1251]APS-forskolin does label the AraE protein, albeit with a lower affinity than for GalP. Thus, ['251]APS-forskolin is a specific photolabel for both the D-galactose-H+ and the L-arabinose-H+ transporters but not for the D-xylose-H' transporter.
Equilibrium Binding of Forskolin to the GalP Protein-Binding of [3H]forskolin to membranes containing overexpressed GalP protein (Roberts, 1992) was investigated using equilibrium dialysis, and the data were fitted to a single hyperbolic function (Cleland, 1979) (Fig. 9a) (Roberts, 1992;Dent, 1993),3 if a ratio of about 1:l for the binding of forskolin to GalP is assumed. The binding of forskolin was prevented by sugars (Table  11) in the order D-glucose > D-galactose >> L-arabinose > Dxylose > L-glucose > L-galactose, which is consistent with the N. G. Rutherford, unpublished data. known specificity of the GalP protein for sugar binding (Walmsley et al., 1994b); forskolin was also displaced competitively by cytochalasin B.4 Forskolin is, therefore, an excellent probe for future investigations of the nature of the ligand binding sites in wild type and mutated GalP proteins, CONCLUSIONS All of the results show that only GalP out of the six sugar-H' symport systems in E. coli is inhibited by forskolin. It is particularly surprising that the E. coli protein for arabinose transport, AraE, which is 64% identicar in amino acid sequence to GalP, is not affected by forskolin. This remarkable specificity, together with the susceptibility of GalP to cytochalasin B and its substrate specificity (Cairns et al., 1991), confirms that it is a very close functional homologue of the mammalian GLUTl glucose transporter, consistent with the similarity of their amino acid sequences (33% identity and additional conservative substitutions). However, there are differences between GalP and GLUTl. GalP transports H' ions and GLUTl does not; the Kd of GalP for forskolin is about 1.4 p~, whereas that of GLUTl is 7.5 p~ (Sergeant and Kim, 1985); [3Hlforskolin covalently modifies the GLUTl protein (Shanahan et al., 19871, but does not modify Gale upon photoactivation of the ligandprotein complex. The Kd of GalP for cytochalasin B is 2-6 p~, whereas that of GLUTl is 0.1 PM (Deves and Krupka, 1978;Cairns et al., 1991;Walmsley et al., 1994a); also, there are minor differences in the affinities for different carbohydrate ligands (Henderson and Maiden, 1990;Walmsley et al., 1994b). GalP binds forskolin, or cytochalasin B, much more tightly than sugars, for which Kd values are in the 0.2-200 m~ range (Walmsley et al., 1994b), although the K, values for sugars under conditions of energized transport can be as low as 0.01 mM for glucose (Cairns et al., 1991). Forskolin will be a powerful tool to aid the elucidation of the molecular features that determine recognition of ligands by determining Kd values for forskolin of the whole family of glucose transporter proteins of varying sequences (usually about 30% identical) in diverse organisms, and by a judicious program of mutagenesis of the GalP protein itself.
It is not known how widespread GalP is amongst bacterial species, but it does at least occur in Enterobacteria other than E. coli, including Salmonella typhimurium, Klebsiella pneumoniae, and Erwinia c a r o t o~o r a .~ The ability to bind forskolin

Sugar-H+ Dansporters
and [12511APS-forskolin will be a convenient method for establishing this in the future using membrane preparations from the bacterial strains. Wadzinski et al. (1990) concluded from some preliminary evidence that [12511APS-forskolin labeled an arabinose transport protein ofE. coli, although the best protection was given by glucose, rather than arabinose. This apparent discrepancy with our initial results is resolved by our observation that the low levels of I'Z51]APS-forskolin preferentially label the GalP protein when GalP and AraE are expressed together, but that if GalP is completely eliminated by genetic deletion then ['2511APS-forskolin will label the AraE protein specifically, as indicated by the competition given by substrates of AraE but not GalP, in the appropriate mutant strain. The hydrophobic substituent group in ['251]APS-forskolin, compared with forskolin itself, clearly increases the affinity of the molecule for both GalP and AraE. It will be of interest to determine which part of the GalP and AraE proteins is actually labeled by the photoaffinity reagent using partial proteolytic digestions and N-terminal sequencing of radioactivity-containing peptides. The future isolation of mutants impaired or improved in forskolin binding should also be illuminating in this regard.
With the overexpressed GalP protein, the binding is SUEciently tight to provide reliable estimates of the concentration of the protein present (assuming a 1:l stoichiometry), which is consistent with the data obtained so far by quantitative densitometry. This will be valuable for measuring the stability of the protein during purification and crystallization trials. Perhaps the very tight binding of ['251]APS-forskolin will actually stabilize the solubilized protein.
This paper establishes that forskolin and its chemical derivatives will provide invaluable tools for the elucidation of the structure-activity relationships of a series of homologous sugar transport proteins. These proteins are important, not only for their key role in nutrient acquisition by diverse organisms ranging from bacteria to man, but because they have a n essentially common structure, variations on which give rise to different substrate specificities and kinetic parameters vital to the performance of their different roles in the physiology of the host organism.