Abstract
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 D-xylose by the related E. coli AraE and XylE transporters, even though the amino acid sequences of their proteins are 30-64% identical to GalP and to the mammalian GLUT family. However, unlike GLUT1, photoactivation of the [3H]forskolin-GalP complex fails to incorporate radioactivity covalently into the protein, in contrast to the effective incorporation of radioactivity from [3H]cytochalasin B into both proteins. However, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldesacetylforskol in ([125I]APS-forskolin), which labels GLUT1, is a potent labeling reagent for GalP and, to a lesser extent, for AraE. The appropriate sugar substrates of each transporter protect it against the [125I]APS-forskolin. Equilibrium binding studies using membranes from an E. coli strain that overexpresses GalP reveal a single set of high affinity binding sites for [3H]forskolin with a Kd of 1.3-1.4 microM, probably forming a 1:1 complex, compared with a value of 7.5 microM for GLUT1. Sugar substrates of GalP and cytochalasin B displace forskolin from the protein. The nonhomologous sugar-H+ symporters for L-rhamnose (RhaT), L-fucose (FucP) and lactose (LacY) in E. coli are insensitive to forskolin. Forskolin and [125I]APS-forskolin, therefore, constitute novel probes for exploring the structure-activity relationship of the bacterial GalP protein. GalP will provide an excellent model for the human glucose transporters and for elucidating the molecular basis of subtle differences in substrate and inhibitor recognition by individual members of this widespread family of transport proteins.
Highlights
Forskolin is a potent inhibitor of mammalian passive Forskolin is a potent reversible inhibitorof the mammalian glucose transporters
Port of L-arabinoseor n-xylose bythe related E. colAi raE In this studywe show that only the D-galactose-Wl symporter, and XylE transporters, even though the amino acid se- GalP, of all the known bacterial sugar-Hi symporters, is susquences of their proteins are 30-6470identical to GalP ceptible to inhibition by forskolin
This specificity is remarkaand to the mammalian GLUTfamily.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 proble, especiallybecause the D-galactose-H’ (GalP), L-arabinose-H+(AraE),and D-xylose-H+(XylE)symporters are28-64% identical in aminoacid sequence to eachother and to a family of sugar transporters, includingGLUT1-5 and 7, found in diteins
Summary
Materials-Sugars and sugar analogues (glucose-free where appro- brane of E. coli was made permeable to hydrophobic compounds by priate) were obtainedfrom Sigma. The transport inhibitors cytochala- treatment with Tns-EDTA(Leive, 1965). Cells were equilibrated i2n00 sin B, phloridzin,andphloretinwerefrom. ~-[l-~H]Galactose, ~-[U-’~Clxylose, ~-[glucose-~~CllaTcrtiso/sHeC, l, pH8.0,l mM EDTA. ~-[l-’~CJfucose, a[n4d-3Hlcytochalasin B were obtainedf romhersham 5 mM MES, pH6.5,10 mM MgSO, were added and the cells harvebsyted. A n d ~ - [ l - ~ ~ Clrham nwoesree from CEA, Fluorochem Ltd. Organisms-The E. coli strains used in this study are giveTnabinle SDS-polyacrylamide gel electrophoresis and stainingof separated pro-. Plasmids were stained gel using aMolecular Dynamics lOOA computing densitometer maintained by the addition of ampicillin (100 pg/ml) and tetracycline and expressed as percentaogfethe total membrane protein as analyzed (15pg/ml) where appropriate. The other plasmids express the transport protein constitutively
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