The secretin-specific, chaperone-like protein of the general secretory pathway: Separation of proteolytic protection and piloting functions
The secretin-specific, chaperone-like protein of the general secretory pathway: Separation of proteolytic protection and piloting functions
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DOI：
10.1046/j.1365-2958.1996.01539.x
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发表时间：
1996-12-01
影响因子：
3.6
通讯作者：
Pugsley, AP
中科院分区：
文献类型：
--
作者：
Hardie, KR;Seydel, A;Pugsley, AP
来自产酸克雷伯氏菌的一般分泌途径的主要末端分支的伴侣样蛋白，外膜脂蛋白普尔斯，保护多聚体分泌素PulD免于降解并促进其正确定位于外膜。为了确定这些是否是可分离的功能，或是否抵抗蛋白水解的结果简单地从正确定位的PulD，我们取代了脂蛋白型信号肽的普尔斯的周质麦芽糖结合蛋白的信号肽。得到的周质普尔斯保留其保护PulD的能力，但不保留其将PulD定位于外膜和在支链淀粉酶分泌中起作用的能力。周质普尔斯与野生型普尔斯竞争以防止支链淀粉酶分泌，推测再次通过引起PulD的错误定位。一种包含普尔斯成熟部分融合到全长麦芽糖结合蛋白（MalE-普尔斯）的C-末端的杂合蛋白具有与周质普尔斯蛋白相似的性质。此外，MalE-PulS被证明与PulD通过直链淀粉亲和层析。通过用脂蛋白型信号肽取代其信号肽，MalE-普尔斯杂交体具有完全功能（即它恢复了普尔斯突变体中的普鲁兰酶分泌）。然而，这种脂肪酰化的杂合蛋白只有在它还携带有将其靶向到外膜的脂蛋白分选信号时才有功能。因此，普尔斯的两个功能是分开的，完全可以分离。在没有普尔斯的情况下，PulD的不正确定位，而不是蛋白水解，被证明是导致噬菌体休克反应高水平诱导的因素。在大肠杆菌中，欧文氏菌普尔斯同源物OutS可以替代普尔斯，并且普尔斯可以保护分泌素OutD免受蛋白水解，这表明可能存在一个类似PulS的伴侣蛋白家族。
The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca, the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion, Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE-PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE-PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE-PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fatty-acylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli, indicating the possible existence of a family of PulS-like chaperone proteins.