Quantitation of de novo localized (15)N-labeled lipoproteins and membrane proteins having one and two transmembrane segments in a Bacillus subtilis secA temperature-sensitive mutant using 2D-PAGE and MALDI-TOF MS.
We developed a means of quantifying proteins that have just localized in the cytoplasmic membrane using 15N-whole cell labeling together with 2D-PAGE and MALDI-TOF MS. The localization of 18 among 20 proteins consisting of 8 lipoproteins, 11 integral membrane proteins having one or two transmembrane segments and one secretory protein in the membrane fractions of Bacillus subtilis, was inhibited by the absence of SecA in a temperature-sensitive mutant. The time course of inhibition indicated that SecA participates in the localization of those proteins through immediately dependent, delayed dependent, and independent ways.
Bunai
Keigo
K
Cellular Dynamics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. bunaik@riken.jp
Nozaki
Manabu
M
Kakeshita
Hiroshi
H
Nemoto
Tadashi
T
Yamane
Kunio
K
D000251:Adenosine Triphosphatases; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D008074:Lipoproteins / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D026901:Membrane Transport Proteins; D009154:Mutation; D009587:Nitrogen Isotopes; D021381:Protein Transport; D040901:Proteomics / Q000379:methods*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D001426:Bacterial Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009587:Nitrogen Isotopes; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins