Cysteine-scanning Mutagenesis and Substituted Cysteine Accessibility Analysis of Transmembrane Segment 4 of the Glut1 Glucose Transporter

Abstract

A low resolution model has been proposed for the exofacial conformation of the Glut1 glucose transporter in which eight transmembrane segments form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 4, predicted to be an inner helix in this structural model, was investigated by cysteinescanning mutagenesis in conjunction with the substituted cysteine accessibility method using the membrane-impermeant, sulfhydrylspecific reagent, p-chloromercuribenzenesulfonate (pCMBS). A functional, cysteine-less, parental Glut1 molecule was used to produce 21 Glut1 point mutants by individually changing each residue along transmembrane helix 4 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. In striking contrast to all of the other seven predicted inner helices, none of the 21 helix 4 single-cysteine mutants was demonstrably inhibited by pCMBS. However, cysteine substitution within helix 4 resulted in an unusually high number of severely transport-defective mutants. The low absolute transport activities of two of these mutants (G130C and G134C) were due to their extremely low levels of expression, presumably a result of structural instability and consequent degradation in oocytes, suggesting that these two residues play an important role in maintaining the native structure of Glut1. The other two transport-defective mutants (Y143C and E146C) exhibited low specific transport activities, implying that these two residues play an important role in the transport cycle. Based on these data, we conclude that the exoplasmic end of helix 4 lies outside the inner helical bundle in the exofacial configuration of Glut1.

Transmembrane Segment 6 of the Glut1 Glucose Transporter Is an Outer Helix and Contains Amino Acid Side Chains Essential for Transport Activity

“…Note that the orientations of helices 4 and 12 are arbitrary because of the absence of reactivity in the former case or lack of periodicity in pCMBS sensitivity in the latter. Also, although helix 4 is shown as an inner helix in this diagram, its lack of pCMBS sensitivity suggests that its exoplasmic end may lie outside of the inner bundle in the exofacial orientation (25). It should also be emphasized that it is uncertain as to whether the structural and biophysical properties of Glut proteins are identical in Xenopus oocytes and in native mammalian cells.…”

“…Homology modeling of the cytoplasmic conformation of Glut1 based on these structures suggests that helices 1, 2, 4, 5, 7, 8, 10, and 11 comprise an inner bundle of transmembrane helices that form a substrate-binding site near the center of the bilayer, whereas helices 3, 6, 9, and 12 are predicted to encircle this inner helical bundle (24). The available experimental evidence suggests that the exofacial half of helix 4 may not be exposed to the external aqueous compartment in the exofacial configuration (25), although it comprises an inner helix in the endofacial confor-mations of the bacterial transporters. It is thus possible that the cytoplasmic and exoplasmic arrangements of the inner helical bundle of MFS transporters are not identical.…”

Structure of, and functional insight into the GLUT family of membrane transporters

“…45,62,63 Cysteine scanning mutagenesis was subsequently employed to probe which residues lined this proposed central pore. [64][65][66][67][68][69][70][71][72][73][74] The resulting model indicated that TMs 2, 4, 5, 7, 8, 11, and possibly 1 and 10 formed a central aqueous transport channel for glucose, whereas TMs 3, 6, 9, and 12 formed a structural scaffold on the outside of the protein. 70 A possible substrate binding site was also proposed, involving Q161, Q282, and W412.…”

Transport and signaling through the phosphate-binding site of the yeast Pho84 phosphate transceptor

“…SCAM analysis is a well-established methodology to identify amino acid residues of which the side chain is exposed into the ligand-binding site of receptors or the substrate-binding site of transporters (23,24). We have replaced different residues of TMD IV and TMD VIII in Pho84 individually by cysteine using site-directed mutagenesis.…”