PMID:10998167
| Citation |
Kourtz, L and Oliver, D (2000) Tyr-326 plays a critical role in controlling SecA-preprotein interaction. Mol. Microbiol. 37:1342-56 |
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| Abstract |
SecA is an essential ATP-dependent motor protein that interacts with the preprotein and translocon to drive protein translocation across the eubacterial plasma membrane. A region containing residues 267-340 has been proposed to comprise the preprotein binding site of Escherichia coli SecA. To elucidate the function of this region further, we isolated mutants using a combination of region-specific polymerase chain reaction (PCR) mutagenesis and a genetic and biochemical screening procedure. Although this region displayed considerable plasticity based on phylogenetic and genetic analysis, Tyr-326 was found to be critical for SecA function. secA mutants with non-conservative substitutions at Tyr-326 showed strong protein secretion defects in vivo and were completely defective for SecA-dependent translocation ATPase activity in vitro. The SecA-Y326 mutant proteins were normal in their membrane, SecYE and nucleotide-binding properties. However, they exhibited a reduced affinity for preprotein and were defective in preprotein release, as assessed by several biochemical assays. Our results indicate that the region containing Tyr-326 functions as a conformational response element to regulate the preprotein binding and release cycle of SecA. |
| Links | |
| Keywords |
Adenosine Diphosphate/metabolism; Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/metabolism; Bacterial Proteins; Binding Sites; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/metabolism; Cell Membrane/metabolism; Escherichia coli Proteins; Fluorometry/methods; Membrane Transport Proteins; Mutagenesis; Point Mutation; Protein Conformation; Protein Precursors/metabolism; Tyrosine |
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