Functional characterization of amphipathic α-helix in the osmoregulatory ABC transporter OpuA.
The ATP-binding-cassette transporter OpuA from Lactococcus lactis is composed of two ATPase subunits (OpuAA) and two subunits (OpuABC) with the transmembrane domain fused to an extracellular substrate-binding protein. Of the almost 1900 homologues of OpuA known to date, a subset has an amino-terminal amphipathic helix (plus extra transmembrane segment) fused to the core of the transmembrane domain of the OpuABC subunit. FRET measurements indicate that the amphipathic α-helix is located close to the membrane surface, where its hydrophobic face interacts with the transport protein rather than the membrane lipids. Next, we determined the functional role of this accessory region by engineering the amphipathic α-helix. We analyzed the consequence of the mutations in intact cells by monitoring growth and transport of glycine betaine under normal and osmotic stress conditions. More detailed studies were performed in hybrid membrane vesicles, proteoliposomes, and bilayer nanodisks. We show that the amphipathic α-helix of OpuA is necessary for high activity of OpuA but is not critical for the biogenesis of the protein or the ionic regulation of transport.
Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, Netherlands Proteomics Centre and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Nadia
N
Gul
Gea
G
Schuurman-Wolters
Akira
A
Karasawa
Bert
B
Poolman
D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D015640:Ion Channel Gating / Q000502:physiology; D013294:Lactococcus lactis / Q000201:enzymology*; D050356:Lipid Metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D009995:Osmosis / Q000502:physiology*; D017433:Protein Structure, Secondary / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism / Q000502:physiology
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C122133:BusAA protein, Lactococcus lactis; D021122:Protein Subunits; D011510:Proteolipids; C020485:proteoliposomes; D000251:Adenosine Triphosphatases; C412743:OpuA protein, Lactococcus lactis