The construction of a cysteine-less melibiose carrier from E. coli.
The melibiose carrier of E. coli is a cation-sugar cotransport system. This membrane protein contains four cysteine residues and the transport function is inhibited by sulfhydryl reagents. In order to investigate the importance of the cysteines, we have constructed a set of four melibiose transporters each of which has one cysteine replaced with serine or valine. The sensitivity of this set of carriers to N-ethylmaleimide was tested and Cys364 was identified as the target of the reagent. In addition, we constructed a melibiose transporter in which all 4 cysteines were replaced with either serine (Cys110, Cys310, and Cys364) or valine (Cys235) and we found that, as expected, the resulting cysteine-less transporter was resistant to the action of N-ethylmaleimide. The cysteine-less melibiose carrier had no significant decrease in ability to accumulate melibiose with cotransported sodium ions or protons. Thus, none of the 4 cysteines are necessary for the function of the melibiose carrier.
Weissborn
A C
AC
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Botfield
M C
MC
Kuroda
M
M
Tsuchiya
T
T
Wilson
T H
TH
D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003545:Cysteine; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D027981:Symporters; D014633:Valine
D017931:DNA Primers; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D027981:Symporters; D012694:Serine; C021548:melibiose permease; D008553:Melibiose; D014633:Valine; D003545:Cysteine; D005033:Ethylmaleimide