Fluorescence resonance energy transfer analysis of protein translocase. SecYE from Thermus thermophilus HB8 forms a constitutive oligomer in membranes.
SecY and SecE are the two principal translocase subunits that create a channel-like pathway for the transit of preprotein across the bacterial cytoplasmic membrane. Here we report the cloning, expression, and purification of the SecYE complex (TSecYE) from a thermophilic bacterium, Thermus thermophilus HB8. Purified TSecYE can be reconstituted into proteoliposomes that function in T. thermophilus SecA (TSecA) dependent preprotein translocation. After the mixing of TSecYE derivatives labeled with either a donor or an acceptor fluorophore during reconstitution, fluorescence resonance energy transfer experiments demonstrated that 2 or more units of TSecYE in the lipid bilayer associate to form a largely non-exchangeable oligomeric structure.
Mori
Hiroyuki
H
Institute for Virus Research, Kyoto University, Japan.
Tsukazaki
Tomoya
T
Masui
Ryoji
R
Kuramitsu
Seiki
S
Yokoyama
Shigeyuki
S
Johnson
Arthur E
AE
Kimura
Yoshiaki
Y
Akiyama
Yoshinori
Y
Ito
Koreaki
K
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000494:pharmacology; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011510:Proteolipids / Q000378:metabolism; D012235:Rhodamines / Q000494:pharmacology; D000069816:SEC Translocation Channels; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D016963:Thermus thermophilus / Q000201:enzymology*
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D011510:Proteolipids; D012235:Rhodamines; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C057841:SecY protein, E coli; C020485:proteoliposomes; C034996:tetramethylrhodamine iodoacetamide