Pérez-Rodríguez Ritsdeliz, Fisher Adam C, Perlmutter Jason D, Hicks Matthew G, Chanal Angélique, Santini Claire-Lise, Wu Long-Fei, Palmer Tracy, DeLisa Matthew P
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
J Mol Biol. 2007 Mar 30;367(3):715-30. doi: 10.1016/j.jmb.2007.01.027. Epub 2007 Jan 12.
All secreted proteins in Escherichia coli must be maintained in an export-competent state before translocation across the inner membrane. In the case of the Sec pathway, this function is carried out by the dedicated SecB chaperone and the general chaperones DnaK-DnaJ-GrpE and GroEL-GroES, whose job collectively is to render substrate proteins partially or entirely unfolded before engagement of the translocon. To determine whether these or other general molecular chaperones are similarly involved in the translocation of folded proteins through the twin-arginine translocation (Tat) system, we screened a collection of E. coli mutant strains for their ability to transport a green fluorescent protein (GFP) reporter through the Tat pathway. We found that the molecular chaperone DnaK was essential for cytoplasmic stability of GFP bearing an N-terminal Tat signal peptide, as well as for numerous other recombinantly expressed endogenous and heterologous Tat substrates. Interestingly, the stability conferred by DnaK did not require a fully functional Tat signal as substrates bearing translocation defective twin lysine substitutions in the consensus Tat motif were equally unstable in the absence of DnaK. These findings were corroborated by crosslinking experiments that revealed an in vivo association between DnaK and a truncated version of the Tat substrate trimethylamine N-oxide reductase (TorA502) bearing an RR or a KK signal peptide. Since TorA502 lacks nine molybdo-cofactor ligands essential for cofactor attachment, the involvement of DnaK is apparently independent of cofactor acquisition. Finally, we show that the stabilizing effects of DnaK can be exploited to increase the expression and translocation of Tat substrates under conditions where the substrate production level exceeds the capacity of the Tat translocase. This latter observation is expected to have important consequences for the use of the Tat system in biotechnology applications where high levels of periplasmic expression are desirable.
大肠杆菌中所有分泌蛋白在穿过内膜进行转运之前，必须保持一种具备转运能力的状态。就Sec途径而言，这一功能由专门的SecB伴侣蛋白以及通用伴侣蛋白DnaK-DnaJ-GrpE和GroEL-GroES来执行，它们共同的作用是在转运体结合之前使底物蛋白部分或完全展开。为了确定这些或其他通用分子伴侣是否同样参与折叠蛋白通过双精氨酸转运（Tat）系统的转运过程，我们筛选了一系列大肠杆菌突变株，检测它们通过Tat途径转运绿色荧光蛋白（GFP）报告基因的能力。我们发现分子伴侣DnaK对于带有N端Tat信号肽的GFP在细胞质中的稳定性至关重要，对于许多其他重组表达的内源性和异源性Tat底物也是如此。有趣的是，DnaK赋予的稳定性并不需要完全功能性的Tat信号，因为在共有Tat基序中带有转运缺陷型双赖氨酸替代的底物在没有DnaK的情况下同样不稳定。交联实验证实了这些发现，该实验揭示了DnaK与带有RR或KK信号肽的Tat底物三甲胺N-氧化物还原酶（TorA502）的截短版本之间在体内的关联。由于TorA502缺乏九个对于辅因子附着至关重要的钼辅因子配体，DnaK的参与显然与辅因子的获取无关。最后，我们表明，在底物产生水平超过Tat转运酶能力的条件下，可以利用DnaK的稳定作用来提高Tat底物的表达和转运。后一观察结果预计将对Tat系统在需要高水平周质表达的生物技术应用中的使用产生重要影响。
