MEMBRANE TOPOLOGY OF THE DI-PEPTIDE AND TRIPEPTIDE TRANSPORT PROTEIN OF LACTOCOCCUS-LACTIS

Transport of hydrophilic di- and tripeptides into Lactococcus lactis i s mediated by a proton motive force-driven peptide transport protein ( DtpT) that shares similarity with eukaryotic peptide transporters, e.g ., from kidney and small intestine of rabbit, man, and rat. Hydropathy profiling in combination with the ''positive inside rule'' predicts f or most of the homologous proteins an a-helical bundle of 12 transmemb rane segments, but the positions of these transmembrane segments and t he location of the amino and carboxyl termini are by no means conclusi ve. The secondary structure of DtpT was investigated by analysing 42 D tpT-alkaline phosphatase fusion proteins, generated by random or direc ted fusions of the corresponding genes. These studies confirm the pres ence of 12 transmembrane segments but refute several other predictions made of the secondary structure. Data obtained from the fusion protei ns were substantiated by studying the accessibility of single cysteine mutants in putative cytoplasmic or extracellular loops by membrane (i m)permeant sulfhydryl reagents. The deduced topology model of DtpT con sists of a bundle of 12 alpha-helixes with a short amino and a large c arboxyl terminus, both located at the cytoplasmic site of the membrane . On the basis of sequence comparisons with DtpT, it seems likely that the structure model of the amino-terminal half of DtpT also holds for the eukaryotic peptide transporters, whereas the carboxyl-terminal ha lf is largely different.