PMID:9287332
| Citation |
Ramamurthy, V and Oliver, D (1997) Topology of the integral membrane form of Escherichia coli SecA protein reveals multiple periplasmically exposed regions and modulation by ATP binding. J. Biol. Chem. 272:23239-46 |
|---|---|
| Abstract |
SecA insertion and integration into the Escherichia coli inner membrane is a critical step for the catalysis of protein translocation across this layer. To understand this step further, SecA topology was investigated. To determine which regions of SecA are periplasmically exposed, right-side out membrane vesicles were prepared from strains synthesizing monocysteine SecA variants produced by mutagenesis and probed with a membrane-impermeant sulfhydryl-labeling reagent. To determine which regions of SecA contain membrane-integration determinants, inverted inner membrane vesicles were subjected to proteolysis, and integral-membrane fragments of SecA were identified with region-specific antibodies. The membrane association properties of various truncated SecA species produced in vivo were also determined. Our analysis indicates that the membrane topology of SecA is complex with amino-terminal, central, and carboxyl-terminal regions of SecA integrated into the membrane where portions are periplasmically accessible. Furthermore, the insertion and penetration of the amino-terminal third of SecA, which includes the proposed preprotein-binding domain, is subject to modulation by ATP binding. The importance of these studies to the cycle of membrane insertion and de-insertion of SecA that promotes protein translocation and SecA's proximity to the preprotein channel are discussed. |
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| Keywords |
ATP-Binding Cassette Transporters; Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/metabolism; Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/metabolism; Bacterial Proteins/secretion; Biological Transport; Carrier Proteins/chemistry; Cell Fractionation; Cell Membrane/chemistry; Cell Membrane/metabolism; Cysteine/genetics; Escherichia coli/chemistry; Escherichia coli/metabolism; Escherichia coli Proteins; Maltose-Binding Proteins; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Membrane Transport Proteins; Models, Molecular; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Peptide Fragments/chemistry; Peptide Mapping; Protein Conformation; Protein Precursors/metabolism; Sequence Homology, Amino Acid; Species Specificity |
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