Tyr-326 plays a critical role in controlling SecA - preprotein interaction

被引:37
作者
Kourtz, L [1 ]
Oliver, D [1 ]
机构
[1] Wesleyan Univ, Dept Mol Biol & Biochem, Middletown, CT 06459 USA
关键词
D O I
10.1046/j.1365-2958.2000.02078.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:1342 / 1356
页数:15
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