Profiling and comprehensive expression analysis of ABC transporter solute-binding proteins of Bacillus subtilis membrane based on a proteomic approach.
We analyzed ABC transporter solute-binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) or two one-dimensional (1-D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2-D gel separation profiles of submembrane fractions solubilized by 1% n-dodecyl-beta-D-maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn(++), glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).
Bunai
Keigo
K
Institute of Biological Sciences, University of Tsukuba, Japan. kyamane@sakura.cc.tsukuba.ac.jp
Ariga
Masanori
M
Inoue
Taro
T
Nozaki
Manabu
M
Ogane
Shinya
S
Kakeshita
Hiroshi
H
Nemoto
Tadashi
T
Nakanishi
Hiroshi
H
Yamane
Kunio
K
D018528:ATP-Binding Cassette Transporters / Q000032:analysis*; D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D020869:Gene Expression Profiling / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D008565:Membrane Proteins