The presence of a helix breaker in the hydrophobic core of signal sequences of secretory proteins prevents recognition by the signal-recognition particle in Escherichia coli
The presence of a helix breaker in the hydrophobic core of signal sequences of secretory proteins prevents recognition by the signal-recognition particle in Escherichia coli
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DOI：
10.1046/j.1432-1033.2002.03262.x
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发表时间：
2002-11-01
期刊：
影响因子：
--
通讯作者：
Tommassen, J
中科院分区：
文献类型：
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作者：
Adams, H;Scotti, PA;Tommassen, J
信号序列通常在疏水核心内含有α-螺旋去稳定化氨基酸。在大肠杆菌外膜蛋白PhoE的前体中，位置-10（Gly（-10））处的甘氨酸残基被认为是导致α-螺旋断裂的原因。以前，我们表明，取代甘氨酸（-10）的α-螺旋促进残基（丙氨酸，半胱氨酸或亮氨酸）减少质子动力依赖性的前体易位，但螺旋断路器的实际作用仍然模糊。在此，我们考虑了由Gly（-10）取代引起的信号序列中的α-螺旋结构的延伸影响前体的靶向途径的可能性。事实上，突变导致对SecB体内靶向的依赖性降低。体外交联实验表明，G-10 L和G-10 C突变体PhoE前体对信号识别颗粒（SRP）的成分之一P48具有显着增加的亲和力。此外，体外交联实验表明，G-10 L突变蛋白通过SRP途径被路由到SecYEG易位子，SRP途径是由整合内膜蛋白利用的靶向途径。总之，这些数据表明，在可切割的信号序列中的螺旋断裂防止识别SRP，从而与信号序列的疏水性，靶向途径的决定因素。
Signal sequences often contain alpha-helix-destabilizing amino acids within the hydrophobic core. In the precursor of the Escherichia coli outer-membrane protein PhoE, the glycine residue at position -10 (Gly(-10)) is thought to be responsible for the break in the alpha-helix. Previously, we showed that substitution of Gly(-10) by alpha-helix-promoting residues (Ala, Cys or Leu) reduced the proton-motive force dependency of the translocation of the precursor, but the actual role of the helix breaker remained obscure. Here, we considered the possibility that extension of the alpha-helical structure in the signal sequence resulting from the Gly(-10) substitutions affects the targeting pathway of the precursor. Indeed, the mutations resulted in reduced dependency on SecB for targeting in vivo. In vitro cross-linking experiments revealed that the G-10L and G-10C mutant PhoE precursors had a dramatically increased affinity for P48, one of the constituents of the signal-recognition particle (SRP). Furthermore, in vitro cross-linking experiments revealed that the G-10L mutant protein is routed to the SecYEG translocon via the SRP pathway, the targeting pathway that is exploited by integral inner-membrane proteins. Together, these data indicate that the helix breaker in cleavable signal sequences prevents recognition by SRP and is thereby, together with the hydrophobicity of the signal sequence, a determinant of the targeting pathway.