Nearest neighbor analysis of the SecYEG complex. 1. Identification of a SecY-SecG interface.
Integral membrane components SecY, SecE, and SecG of protein translocase form a complex in the Escherichia coli plasma membrane. To characterize subunit interactions of the SecYEG complex, a series of SecY variants having a single cysteine in its cytoplasmic (C1-C6) or periplasmic (P1-P5) domain were subjected to site-specific cross-linking experiments using bifunctional agents with thiol-amine reactivity. Experiments using inverted membrane vesicles revealed specific cross-linkings between a cysteine residue placed in the C2 or C3 domain of SecY and the cytosolic lysine (Lys26) near the first transmembrane segment of SecG. These SecY Cys residues also formed a disulfide bond with an engineered cytosolic cysteine at position 28 of SecG. Thus, the C2-C3 region of SecY is in the proximity of the N-terminal half of the SecG cytoplasmic loop. Experiments using spheroplasts revealed the physical proximity of P2 (SecY) and the C-terminal periplasmic region of SecG. In addition, mutations in secG were isolated as suppressors against a cold-sensitive mutation (secY104) affecting the TM4-C3 boundary of SecY. These results collectively suggest that a C2-TM3-P2-TM4-C3 region of SecY serves as an interface with SecG.
Satoh
Yasunari
Y
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Matsumoto
Gen
G
Mori
Hiroyuki
H
Ito
Koreaki
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D008301:Maleimides / Q000737:chemistry / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000069816:SEC Translocation Channels
D003432:Cross-Linking Reagents; D004220:Disulfides; D029968:Escherichia coli Proteins; D008301:Maleimides; D008565:Membrane Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; C088616:SecG protein, E coli; C057841:SecY protein, E coli; D003545:Cysteine