Primary structure of the alanine carrier protein of thermophilic bacterium PS3.
Purified alanine carrier proteins were cleaved into peptides either chemically after solubilization in 1,1,1,3,3,3-hexafluoro-2-propanol or proteolytically with lysylendopeptidase. From the amino acid sequence analyses of these peptides, we synthesized a DNA probe and utilized it for successful cloning of a gene encoding the alanine carrier protein (acp gene). The 5'-flanking region was determined by an inverse polymerase chain reaction, and an open reading frame consisting of 1,335 nucleotides was found. The amino acid sequence deduced from the open reading frame consists of 445 amino acids, and all the partial amino acid sequences determined are included in the sequence. Although the calculated M(r) of 47,803 is significantly larger than the apparent M(r) of 42,500 as reported previously (Hirata, H., Kambe, T., and Kagawa, Y. (1984) J. Biol. Chem. 259, 10653-10656), an in vitro translation experiment revealed that the product of the acp gene migrates at a position coinciding with that of the purified alanine carrier. Hydropathy analysis suggests that the protein contains at least 8 hydrophobic segments presumably spanning membrane. A homology search on a database reveals relatively high scores of homology with either the Escherichia coli melibiose carrier or the human Na+/glucose symporter, particularly in the region from Leu246 to Glu286. Furthermore, the region also reveals low but significant similarities to other Na(+)-coupled symporters.
Kamata
H
H
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo, Japan.
Akiyama
S
S
Morosawa
H
H
Ohta
T
T
Hamamoto
T
T
Kambe
T
T
Kagawa
Y
Y
Hirata
H
H
D000595:Amino Acid Sequence; D001419:Bacteria / Q000737:chemistry*; D001426:Bacterial Proteins; D001483:Base Sequence; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D047908:Intracellular Signaling Peptides and Proteins; C042469:alanine-carrier protein, bacteria