Membrane topology of the di- and tripeptide transport protein of Lactococcus lactis.
Transport of hydrophilic di- and tripeptides into Lactococcus lactis is 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 for most of the homologous proteins an alpha-helical bundle of 12 transmembrane segments, but the positions of these transmembrane segments and the location of the amino and carboxyl termini are by no means conclusive. The secondary structure of DtpT was investigated by analyzing 42 DtpT-alkaline phosphatase fusion proteins, generated by random or directed fusions of the corresponding genes. These studies confirm the presence of 12 transmembrane segments but refute several other predictions made of the secondary structure. Data obtained from the fusion proteins were substantiated by studying the accessibility of single cysteine mutants in putative cytoplasmic or extracellular loops by membrane (im)permeant sulfhydryl reagents. The deduced topology model of DtpT consists of a bundle of 12 alpha-helixes with a short amino and a large carboxyl 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 half is largely different.
Hagting
A
A
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, Haren, The Netherlands.
vd Velde
J
J
Poolman
B
B
Konings
W N
WN
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001710:Biotin; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry*; D003545:Cysteine / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D013294:Lactococcus lactis / Q000737:chemistry*; D026901:Membrane Transport Proteins; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016415:Sequence Alignment
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004151:Dipeptides; D026901:Membrane Transport Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C086845:dtpT protein, Lactococcus lactis; D001710:Biotin; D000469:Alkaline Phosphatase; D003545:Cysteine