Rusch S L, Kendall D A
Department of Molecular & Cell Biology, University of Connecticut, Storrs 06269.
J Biol Chem. 1994 Jan 14;269(2):1243-8.
We have examined the sequence constraints on the amino-terminal region of the mature portion of alkaline phosphatase that are important for its efficient transport in Escherichia coli. Using a homopolymeric sequence of serines to replace 6 residues in this region, a transport-incompetent mutant was produced. Reintroduction of residues from the native sequence which restore charge and beta-turn potential resulted in little improvement. However, by replacing the hydrophobic core of the signal peptide with a homopolymeric series of leucines, not only was transport restored but precursor processing was more efficient than for the wild type and was insensitive to disruption of the protonmotive force. Moreover, we have titrated the signal peptide with leucine to alanine substitutions (Doud, S. K., Chou, M. M., and Kendall, D. A. (1993) Biochemistry 32, 1251-1256) and determined the minimum level of hydrophobicity necessary to achieve transport of the mutant protein. The results indicate that signal peptide hydrophobicity can completely override possible requirements for negatively charged residues and strong beta-turn forming potential in the mature protein and that the polyleucine-containing signal peptide may act as a generic signal sequence for the transport of non-native proteins in E. coli.
我们研究了碱性磷酸酶成熟部分氨基末端区域的序列限制，这些限制对其在大肠杆菌中的有效转运很重要。使用丝氨酸的同聚物序列取代该区域的6个残基，产生了一个无转运能力的突变体。重新引入来自天然序列的残基，恢复电荷和β-转角潜力，改善甚微。然而，通过用一系列亮氨酸同聚物取代信号肽的疏水核心，不仅恢复了转运，而且前体加工比野生型更有效，并且对质子动力的破坏不敏感。此外，我们用亮氨酸到丙氨酸的取代对信号肽进行了滴定（杜德，S.K.，周，M.M.，和肯德尔，D.A.（1993年）《生物化学》32卷，1251 - 1256页），并确定了实现突变蛋白转运所需的最低疏水性水平。结果表明，信号肽疏水性可以完全超越成熟蛋白中带负电荷残基和强β-转角形成潜力的可能要求，并且含多聚亮氨酸的信号肽可能作为大肠杆菌中非天然蛋白转运的通用信号序列。
