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 combin...

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Bibliographic Details
Published in:	Biochemistry (Easton) Vol. 36; no. 22; pp. 6777 - 6785
Main Authors:	Hagting, Anja, Velde, Joeke v. d, Poolman, Bert, Konings, Wil N
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 03-06-1997
Subjects:	Alkaline Phosphatase - genetics Amino Acid Sequence Animals Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism Base Sequence Biotin Carrier Proteins - chemistry Carrier Proteins - genetics Carrier Proteins - metabolism Cell Membrane - chemistry Cysteine - metabolism Dipeptides - metabolism Escherichia coli - genetics Humans Lactococcus lactis Lactococcus lactis - chemistry Membrane Transport Proteins Models, Chemical Molecular Sequence Data Mutagenesis, Site-Directed Peptides - metabolism Plasmids Polymerase Chain Reaction Protein Structure, Secondary Recombinant Fusion Proteins - chemistry Sequence Alignment
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Summary:	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 α-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 α-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.
Bibliography:	This work was supported by a grant from the BRIDGE-T project of the EC-Science Foundation Programme (Contract BIOT-CT91-0263). Abstract published in Advance ACS Abstracts, May 15, 1997. istex:92EB77D5A331FD37186D6CF44012824858257781 ark:/67375/TPS-2PHFJCNX-B ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23
ISSN:	0006-2960 1520-4995
DOI:	10.1021/bi963068t