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To test the role of cysteines in the function of GLUT1 glucose transporter, site-directed mutagenesis was used to replace all six GLUT1 cysteines with serine residues. When the individual and combined Cys-->Ser mutants were expressed in Xenopus laevis oocytes, zero-trans uptake of 3-O-methylglucose was comparable to that seen in native GLUT1. The "cysteineless" construct also retained the kinetic features of GLUT1, including an asymmetric transport mechanism and similar substrate and inhibitor affinities. Whereas GLUT1 transport was inhibited by sulfhydryl reagents, that of the "cysteineless" construct was not. These results show that cysteines are not required for GLUT1 function or oligomer formation. The "cysteineless" construct may therefore serve as a template for reintroducing cysteines back into GLUT1 at sites useful for testing transporter structure and function.