Cline Kenneth, McCaffery Michael
Horticultural Sciences Department and Plant Molecular and Cellular Biology, University of Florida, Gainesville FL, USA.
EMBO J. 2007 Jul 11;26(13):3039-49. doi: 10.1038/sj.emboj.7601759. Epub 2007 Jun 14.
Tat systems transport completely folded proteins across ion-tight membranes. Three membrane proteins comprise the Tat machinery in most systems. In thylakoids, cpTatC and Hcf106 mediate precursor recognition, whereas Tha4 facilitates translocation. We used chimeric precursor proteins with unstructured peptides and folded domains to test predictions of competing translocation models. Two models invoke protein-conducting channels, whereas another model proposes that cpTatC pulls substrates through a patch of Tha4 on the lipid bilayer. The thylakoid system transported unstructured peptide substrates alone or when fused to folded domains. However, larger substrates stalled before completion, some with amino- and carboxyl-folded domains on opposite sides of the membrane. The length of the precursor that resulted in translocation arrest (20 to 30 nm) exceeded that expected for a single 'pull' mechanism, suggesting that a sustained driving force rather than a single pull moves the protein across the bilayer. Three different methods showed that stalled substrates were not stuck in a channel or even associated with Tat machinery. This finding favors the Tha4 patch model for translocation.
Tat系统可将完全折叠的蛋白质转运穿过离子致密膜。在大多数系统中，Tat机制由三种膜蛋白组成。在类囊体中，cpTatC和Hcf106介导前体识别，而Tha4促进转运。我们使用带有无结构肽段和折叠结构域的嵌合前体蛋白来检验竞争性转运模型的预测。两种模型涉及蛋白质传导通道，而另一种模型提出cpTatC通过脂质双层上的一片Tha4拉动底物。类囊体系统能够单独转运无结构肽底物，或者当与折叠结构域融合时也能转运。然而，较大的底物在转运完成前就停滞了，有些底物的氨基和羧基折叠结构域位于膜的两侧。导致转运停滞的前体长度（20至30纳米）超过了单一“拉动”机制预期的长度，这表明是持续的驱动力而非单次拉动使蛋白质穿过双层膜。三种不同的方法表明，停滞的底物并未被困在通道中，甚至与Tat机制也没有关联。这一发现支持了Tha4斑块模型用于转运的观点。
