DeLisa Matthew P, Samuelson Patrik, Palmer Tracy, Georgiou George
Department of Chemical Engineering and the Institute for Cell and Molecular Biology, University of Texas, Austin, Texas 78712, USA.
J Biol Chem. 2002 Aug 16;277(33):29825-31. doi: 10.1074/jbc.M201956200. Epub 2002 May 20.
The twin arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates translocation of essentially folded proteins across the cytoplasmic membrane. The detailed understanding of the mechanism of protein targeting to the Tat pathway has been hampered by the lack of screening or selection systems suitable for genetic analysis. We report here the development of a highly quantitative protein reporter for genetic analysis of Tat-specific export. Specifically, export via the Tat pathway rescues green fluorescent protein (GFP) fused to an SsrA peptide from degradation by the cytoplasmic proteolytic ClpXP machinery. As a result, cellular fluorescence is determined by the amount of GFP in the periplasmic space. We used the GFP-SsrA reporter to isolate gain-of-function mutants of a Tat-specific leader peptide and for the genetic analysis of the "invariant" signature RR dipeptide motif. Flow cytometric screening of trimethylamine N-oxide reductase (TorA) leader peptide libraries resulted in isolation of six gain-of function mutants that conferred significantly higher steady-state levels of export relative to the wild-type TorA leader. All the gain-of-function mutations occurred within or near the (S/T)RRXFLK consensus motif, highlighting the significance of this region in interactions with the Tat export machinery. Randomization of the consensus RR dipeptide in the TorA leader revealed that a basic side chain (R/K) is required at the first position whereas the second position can also accept Gln and Asn in addition to basic amino acids. This result indicates that twin arginine translocation does not require the presence of an arginine dipeptide within the conserved sequence motif.
细菌和植物叶绿体的双精氨酸转运（Tat）途径介导基本折叠的蛋白质穿过细胞质膜的转运。由于缺乏适用于遗传分析的筛选或选择系统，对蛋白质靶向Tat途径机制的详细理解受到了阻碍。我们在此报告了一种用于Tat特异性输出遗传分析的高度定量蛋白质报告基因的开发。具体而言，通过Tat途径的输出可拯救与SsrA肽融合的绿色荧光蛋白（GFP），使其免受细胞质蛋白水解ClpXP机制的降解。因此，细胞荧光由周质空间中GFP的量决定。我们使用GFP-SsrA报告基因来分离Tat特异性前导肽的功能获得突变体，并对“不变”的RR二肽基序进行遗传分析。对三甲胺N-氧化物还原酶（TorA）前导肽文库进行流式细胞术筛选，结果分离出六个功能获得突变体，相对于野生型TorA前导肽，它们赋予了显著更高的输出稳态水平。所有功能获得突变均发生在（S/T）RRXFLK共有基序内或附近，突出了该区域在与Tat输出机制相互作用中的重要性。对TorA前导肽中共有的RR二肽进行随机化分析表明，第一位需要一个碱性侧链（R/K），而第二位除了碱性氨基酸外还可以接受Gln和Asn。这一结果表明，双精氨酸转运在保守序列基序内不需要精氨酸二肽的存在。
