THE CONSTRUCTION OF A CYSTEINE-LESS MELIBIOSE CARRIER FROM ESCHERICHIA-COLI

The melibiose carrier of E. coli is a cation-sugar cotransport system. This membrane protein contains four cysteine residues and the transpo rt function is inhibited bq sulfhydryl reagents. In order to investiga te the importance of the cysteines, we have constructed a set of four melibiose transporters each of which has one cysteine replaced with se rine or valine. The sensitivity of this set of carriers to N-ethylmale imide was tested and Cys364 was identified as the target of the reagen t, 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 cys teine-less transporter was resistant to the action of N-ethylmaleimide . The cysteine-less melibiose carrier had no significant decrease in a bility to accumulate melibiose with cotransported sodium ions or proto ns. Thus, none of the 4 cysteines are necessary for the function of th e melibiose carrier. (C) 1997 Elsevier Science B.V.