Tyr-326 plays a critical role in controlling SecA-preprotein interaction.
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.
Kourtz
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L
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459, USA.
Oliver
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D
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D029968:Escherichia coli Proteins; D005470:Fluorometry / Q000379:methods; D026901:Membrane Transport Proteins; D016296:Mutagenesis; D017354:Point Mutation; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D014443:Tyrosine
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; D014443:Tyrosine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins