PMID:12809499
| Citation |
Satoh, Y, Matsumoto, G, Mori, H and Ito, K (2003) Nearest neighbor analysis of the SecYEG complex. 1. Identification of a SecY-SecG interface. Biochemistry 42:7434-41 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1021/bi034331a |
| Keywords |
Amino Acid Sequence; Amino Acid Substitution; Cross-Linking Reagents/chemistry; Cross-Linking Reagents/pharmacology; Cysteine/chemistry; Cysteine/genetics; Cytoplasm/metabolism; Disulfides/chemistry; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Immunoblotting; Maleimides/chemistry; Maleimides/pharmacology; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation/physiology; Plasmids/genetics; Protein Conformation; Protein Subunits; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism |
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